Methods and systems for converting progenitor cells into gastric tissue by directed differentiation

Abstract

To provide methods and systems for creating gastric tissues that can be used to construct robust in vitro systems for elucidating mechanisms underlying human stomach development and disease and for identifying novel treatments.SOLUTION: Disclosed are methods of inducing formation of gastric cells and / or a gastric tissue, such as in the form of a gastric organoid. The formation of gastric cells and / or tissue may be carried out by the activating and / or inhibiting one or more signaling pathways within a precursor cell. Also disclosed are methods for using the disclosed gastric cells, gastric tissues and / or gastric organoids derived from the precursor cells.SELECTED DRAWING: None

Description

Technical Field

[0001] Description of Federal Support Research This invention was made with government support under grants DK080823, DK092456, and GM063483 from the National Institutes of Health. The government has certain rights in this invention.

[0002] Claims of Priority This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 003,719, filed May 28, 2014, to Wells et al., entitled "Methods and Systems for Converting Precursor Cells into Gastric Tissues through Directed Differentiation," for all purposes.

[0003] Disclosed herein are methods and systems for converting stem cells into specific tissues or organs by directed differentiation. Specifically, methods and systems for promoting formation of definitive endoderm from human pluripotent stem cells are disclosed. Also disclosed are methods and systems for promoting formation of gastric organoids or gastric tissues from differentiated definitive endoderm.

Background Art

[0004] The function and structure of the stomach vary widely among mammalian species in adaptation to diverse habitats and diets. As a result, there are significant limitations to non-human gastric development and disease models. For example, the bacterium Helicobacter pylori infects 50% of the world's population, 10% develop peptic ulcer disease, and 1-2% 1~3develops gastric cancer. Gastric diseases, including peptic ulcer disease and gastric cancer, affect 10% of the world's population and are generally caused by chronic Helicobacter pylori (H. pylori) infection. The current models of H. pylori-induced diseases rely on animal models that do not exhibit the same pathophysiological characteristics as the human response to infection 4 , and gastric cell lines lack the cellular and structural complexity of the gastric epithelium in vivo. Therefore, there is no model suitable for studying the effects of H. pylori infection as it occurs in humans. Recent advances using adult gastric stem cells have made it possible to grow rodent gastric epithelium in vitro 5 , but obtaining these cells from human patients may require surgery. Furthermore, such methods cannot be used to model human gastric embryogenesis or stromal-epithelial interactions. Due to differences in embryonic development and adult gastric structure among species, the mouse model is suboptimal for studying organogenesis and pathogenesis of this organ. Therefore, a robust in vitro system is needed to elucidate the mechanisms underlying human gastric development and disease and to identify novel therapeutics useful for the treatment of such diseases in humans.

Summary of the Invention

Problems to be Solved by the Invention

[0005] What is needed in the art is a method and system for precisely controlling the end points of progenitor cells, such as human pluripotent stem cells, to create desired specific types of tissues or organisms, specifically gastric tissue that can be used for one or more of the aforementioned purposes.

Means for Solving the Problems

[0006] Methods are disclosed for inducing the formation of gastric cells and / or gastric tissue, such as the morphology of gastric organoids. The formation of gastric cells and / or tissue can be achieved by activating and / or inhibiting one or more signaling pathways within progenitor cells. Also disclosed are methods of using the disclosed gastric cells, gastric tissue, and / or gastric organoids derived from progenitor cells.

[0007] Those skilled in the art will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0008] This patent or application file contains at least one drawing created in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fees.

Brief Description of the Drawings

[0009]

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Mode for Carrying Out the Invention

[0010] Unless otherwise noted, terms are to be understood according to their conventional usage by those of ordinary skill in the relevant art.

[0011] As used herein, the term "totipotent stem cell" (also known as an omnipotent stem cell) is a stem cell that can differentiate into embryonic cell types and extraembryonic cell types. Such cells can construct a viable complete organism. These cells are created by the fusion of an egg cell and a sperm cell. Cells created by the first few divisions of a fertilized egg are also totipotent.

[0012] As used herein, the term "pluripotent stem cell (PSC)" refers to any cell that can differentiate into almost any cell type of a living body, i.e., cells derived from any of the three germ layers (germ epithelium) including the endoderm (inner lining of the stomach, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital organs), and ectoderm (epidermal tissue and nervous system). PSCs may be descendants of the inner cell mass cells of a pre-implantation blastocyst, or may be obtained through the induction of non-pluripotent cells, such as adult somatic cells, by forcibly expressing certain genes. Pluripotent stem cells can be derived from any suitable source, as will be readily understood by those of ordinary skill in the art. Examples of sources of pluripotent stem cells include, but are not limited to, mammalian sources including humans, rodents, pigs, and cows.

[0013] As used herein, the term "induced pluripotent stem cell (iPSC)", often abbreviated as iPS cell, refers to a type of pluripotent stem cell artificially obtained from normally non-pluripotent cells, such as adult somatic cells, by inducing the "forced" expression of certain genes.

[0014] As used herein, the term "embryonic stem cell (ESC)", often abbreviated as ES cell, refers to pluripotent cells derived from the inner cell mass of the blastocyst, which is an early embryo. For the purposes of the present invention, the term "ESC" is sometimes used in a broader sense to further include embryonic germ cells.

[0015] As used herein, the term "progenitor cell" includes any cell that can be used in the methods described herein, in which one or more progenitor cells acquire the ability to self-renew or to differentiate into one or more specialized cell types. In some embodiments, the progenitor cells are pluripotent or have the potential to become pluripotent. In some embodiments, the progenitor cells are subjected to treatment with external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, the progenitor cells can be totipotent (or omnipotent) stem cells; pluripotent stem cells (artificial or non-artificial); multipotent stem cells; oligopotent stem cells and unipotent stem cells. In some embodiments, the progenitor cells can be derived from an embryo, fetus, infant, child, or adult. In some embodiments, the progenitor cells can be somatic cells that have been subjected to treatment such that pluripotency is conferred by genetic manipulation or protein / peptide treatment.

[0016] 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" refers to the process by which less specialized cells become a particular specialized target cell type. The particularity of the specialized target cell type can be determined by any applicable method that can be used to define or modify the fate of the initial cells. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0017] As used herein, the term "cellular constituent" refers to individual genes, proteins, mRNAs that express genes, and / or any other variable cellular component or protein activity, e.g., the degree of protein modification (e.g., phosphorylation) typically measured by those of ordinary skill in the art in biological experiments (e.g., by microarray or immunohistochemistry). Important discoveries regarding biological systems, the complex network of biochemical processes underlying common human diseases, as well as gene discovery and structure determination can now be due to the application of cellular constituent abundance data as part of the research process. Cellular constituent abundance data can help identify biomarkers, distinguish disease subtypes, and identify toxicity mechanisms.

[0018] Stem cells are found in all multicellular organisms. Stem cells are characterized by their ability to self-renew by mitotic cell division and to differentiate into diverse specialized cell types. Broadly, two types of mammalian stem cells are: 1) embryonic stem cells isolated from the inner cell mass of the blastocyst, and 2) adult stem cells found in adult tissues. In the developing embryo, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as the body's repair system, replenishing specialized cells and maintaining the normal turnover of regenerative organs such as blood, skin, or stomach tissue.

[0019] Currently, stem cells can be grown and transformed by cell culture into specialized cells with characteristics consistent with cells of various tissues such as muscle or nerve. In medical therapies, highly plastic adult stem cells from various sources including umbilical cord blood and bone marrow are routinely used. Embryonic cell lines and autologous embryonic stem cells created by therapeutic cloning have also been proposed as promising candidates for future therapies.

[0020] The classical definition of a stem cell typically refers to two properties: self-renewal, the ability to undergo numerous cell division cycles while maintaining an undifferentiated state, and developmental potential, the ability to differentiate into specialized cell types. In some embodiments, a stem cell is either totipotent or pluripotent, i.e., the stem cell can give rise to any mature cell type, although multipotent or unipotent progenitor cells may also be referred to as stem cells.

[0021] Developmental potential identifies the potential differentiation capacity of a stem cell (the potential to differentiate into different cell types). Totipotent stem cells (also known as omnipotent stem cells) can differentiate into embryonic and extraembryonic cell types. These cells can construct a viable complete organism. These cells are created by the fusion of an egg cell and a sperm cell. Cells created by the first few divisions of a fertilized egg are also totipotent. Pluripotent stem cells (PSCs) are descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers: endoderm (lining of the stomach, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epidermal tissue and nervous system), i.e., almost any cell. Multipotent stem cells can differentiate into several cells, but only those of a closely related cell family. Oligopotent stem cells can only differentiate into only a few cell types, such as lymphoid or myeloid stem cells. Unipotent cells can only give rise to their own single cell type, but have the property of self-renewal, thereby distinguishing them from non-stem cells (e.g., muscle stem cells).

[0022] Embryonic stem cells and induced pluripotent stem cells have had an unprecedented impact on the ability to study human diseases and the ability to create therapeutically effective alternative tissues in animal models.

[0023] In developmental biology, cell differentiation is the process by which less specialized cells become more specialized cell types. Most efforts to direct the differentiation of human PSCs into therapeutic cell types are based on studies of embryonic organogenesis. Examples include the generation of hepatocytes and pancreatic endocrine cells, which have shown functional potential in animal models of liver disease and diabetes. Similarly, differentiation of PSCs into intestine can provide therapeutic benefits in diseases such as necrotizing enterocolitis, inflammatory bowel disease, and short bowel syndrome.

[0024] As discussed above, pluripotent stem cells have the potential to differentiate into any of the three germ layers: endoderm (lining of the stomach, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epidermal tissue and nervous system). Thus, pluripotent stem cells can give rise to any fetal or adult cell type. However, the fate of a particular pluripotent stem cell is controlled by numerous cell signaling pathways and many factors. Furthermore, pluripotent stem cells do not have the potential to contribute to extraembryonic tissues such as the placenta and therefore cannot develop into a fetus or adult animal on their own.

[0025] To date, gastric tissue has not been generated from human pluripotent stem cells (hPSCs). The success of efforts to differentiate PSCs into lung, liver, pancreas, and intestinal cells depends on a molecular understanding of the embryogenesis of these organs 6~10 . Unfortunately, a problem in the art is that there are many differences in the understanding of gastric development following endoderm formation. Thus, to direct the differentiation of hPSCs into gastric tissue, the Applicant has identified signaling pathways that regulate several important early stages of gastric development, including foregut specification and patterning, stomach specification, and finally gastric epithelial growth and differentiation. In addition, to create more functional and complex three-dimensional tissues, the Applicant aimed to induce several morphogenetic processes that occur during gastric development, including the morphogenesis of the foregut tube and the formation of gastric epithelial structures including glands and pits.

[0026] As described herein, a method and system are constructed to mimic fetal gastric tissue development in culture using temporal growth factor manipulation. Specifically, a method and system are constructed to direct the differentiation of both PSCs, human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs) into gastric tissue in vitro. These factors approximate the stages of fetal intestinal development: activin-induced definitive endoderm (DE) formation; FGF / Wnt / BMP-induced posterior foregut patterning; and finally, the regulation of retinoic acid and EFG signaling, which promotes the growth, morphogenesis, and cell differentiation of gastric tissue into functional gastric cell types and morphologies, including gastric glands and gastric pits, proliferative zones, surface and antral mucous cells, and endocrine cells expressing gastrin, ghrelin, and somatostatin, resulting in a pro-gastric culture system that directs human intestinal development in vitro.

[0027] The applicant has identified a novel fetal signaling pathway that enables efficient stepwise differentiation of human PSCs into gastric cells, gastric tissue, and / or three-dimensional gastric tissue (hGO) with complex structures and cell compositions. The applicant has further found that developing hGO undergoes nearly the same molecular and morphological differentiation stages as the antrum during mouse development, and that the resulting gastric organoids can contain a series of mucous cells, endocrine cells, and progenitor cells with a three-dimensional composition equivalent to that of normal antral epithelium and fetal / postnatal stomach.

[0028] The disclosed human gastric cells, gastric tissue, and / or gastric organoids (hGO) may be used as an in vitro system for identifying novel mechanisms of human gastric development and physiological functions, and may also be used as a model for the pathophysiological response of gastric epithelium to Helicobacter pylori (H. pylori). The disclosed gastric cells, gastric tissue, and / or gastric hGO and methods provide new opportunities for drug discovery and modeling of early gastric cancer. Furthermore, this specification discloses the first three-dimensional generation of the human embryonic foregut, which is a promising starting point for generating other foregut organ tissues, including the lung and pancreas.

[0029] In one aspect, a method of inducing the formation of gastric cells, gastric tissue, and / or gastric hGO from progenitor cells is disclosed. This method may include a) activating one or more signaling pathways within the progenitor cells, wherein the one or more signaling pathways are selected from the WNT signaling pathway, the WNT / FGF signaling pathway, and the FGF signaling pathway, thereby obtaining gastric cells, gastric tissue, and / or gastric hGO that are progeny of the progenitor cells. This method may further include step b) inhibiting one or more signaling pathways within the progenitor cells. The one or more signaling pathways to be inhibited may include the BMP signaling pathway.

[0030] This method may further include contacting the progenitor cells with retinoic acid. The step of contacting the progenitor cells with retinoic acid may be performed after the steps of activating and inhibiting described above.

[0031] This method may further include contacting the gastric organoids with EGF at a concentration and / or length of time sufficient to increase the diameter of the gastric organoids to greater than about 1 mm in diameter, or greater than about 2 mm in diameter, or greater than about 3 mm in diameter, or greater than about 4 m M-mode In one aspect, the one or more signaling pathways may be selected from the Wnt signaling pathway, the Wnt / β-catenin signaling, the Wnt / APC signaling, and the Wnt / PCP pathway signaling.

[0032] In one aspect, the step of activating the Wnt signaling pathway may include contacting the progenitor cells with one or more molecules selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16.

[0033]

[0034] ​In one aspect, the step of activating the FGF signaling pathway may include contacting the progenitor cells with one or more molecules selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

[0035] In one aspect, the step of inhibiting the BMP signaling pathway may include contacting the progenitor cells with a BMP inhibitor. In one aspect, the BMP inhibitor may be selected from dorsomorphin, LDN189, DMH-1, noggin, and combinations thereof. In one aspect, the BMP inhibitor may be noggin.

[0036] In one aspect, the activating step may include contacting the progenitor cells with Wnt3a, FGF4, and a BMP inhibitor over a specific time period referred to as the incubation time. The contacting steps may be performed simultaneously or, in other aspects, the contacting steps may be performed sequentially.

[0037] In one aspect, progenitor cells that may include definitive endoderm may be contacted with a signaling agent that may include a combination of 1) Wnt3a or a GSK inhibitor (e.g., CHIRON) and 2) FGF4 over a first incubation time. The first incubation time may further include a BMP inhibitor. After the first incubation time, the progenitor cells may be subjected to a second incubation time where the progenitor cells are contacted with retinoic acid (RA). In one aspect, the first incubation time and the second incubation time overlap. In some embodiments, the first incubation time and the second incubation time do not overlap.

[0038] In one aspect, the first and / or second incubation time, and / or the sum of the first and second incubation times can be 24 to 120 hours, or about 36 to about 108 hours, or about 48 to about 96 hours, or about 60 to about 84 hours. In one aspect, the first incubation time can be at least about 24 hours.

[0039] In one aspect, the second incubation time (wherein the progenitor cells can be contacted with RA) starts about 72 hours after the first incubation time. In a further aspect, the second incubation time starts after the culture has formed foregut spheroids from the progenitor cells. Next, the foregut spheroids can be transferred to a three-dimensional matrix under growth conditions suitable for the formation of gastric organoids, for example, by applying the foregut spheroids to Matrigel™ (Corning, BD Bioscience). After transfer to Matrigel, the foregut spheroids are contacted with RA for a third incubation time, where continuous 3D growth can occur. Next, the spheroids may be contacted with EGF for a fourth incubation time, which may overlap with the third incubation time. The third incubation time can be about 24 hours.

[0040] In one aspect, the progenitor cells may be contacted with Wnt3a at a concentration of 50 to 1500 ng / ml, or about 100 to about 1200 ng / ml, or about 200 to about 1000 ng / ml, or about 300 to about 900 ng / ml, or about 400 to about 800 ng / ml, or about 500 to about 700 ng / ml.

[0041] In one aspect, the progenitor cells can be selected from embryonic stem cells, embryonic germ cells, induced pluripotent stem cells, mesoderm cells, embryonic endoderm cells, posterior endoderm cells, and hindgut cells.

[0042] In one aspect, the progenitor cells can be embryonic endoderm cells derived from pluripotent stem cells.

[0043] In one aspect, the progenitor cells can be pluripotent stem cells such as embryonic stem cells, embryonic germ cells, or induced pluripotent stem cells.

[0044] In one aspect, the definitive endoderm cells can be obtained by contacting pluripotent stem cells with one or more molecules selected from activin, the BMP subgroup of the TGF-β superfamily of growth factors; nodal, activin A, activin B, BMP4, Wnt3a, and combinations thereof.

[0045] In one aspect, stomach tissue can be produced in vitro from one or more progenitor cells.

[0046] In one aspect, the one or more progenitor cells can be selected from embryonic stem cells, mesoderm cells, definitive endoderm cells, posterior endoderm cells, anterior endoderm cells, foregut cells, and hindgut cells.

[0047] In one aspect, the pluripotent stem cells can be mammalian pluripotent stem cells including, but not limited to, human pluripotent stem cells or mouse pluripotent stem cells.

[0048] In one aspect, the human pluripotent stem cells can be selected from human embryonic stem cells, human embryonic germ cells, and human induced pluripotent stem cells.

[0049] In one aspect, a kit is provided that includes stomach cells, tissue, or organoids produced in vitro from one or more progenitor cells.

[0050] In one aspect, a method is provided for identifying the absorption effect of stomach cells or tissue. The method can include contacting stomach cells, tissue, or organoids derived from progenitor cells with a compound; and detecting the absorption level of the compound by the stomach cells or tissue.

[0051] In one aspect, a method for identifying the toxicity of a compound to gastric cells or tissues is provided. The method may include contacting gastric cells, tissues, or organoids derived from progenitor cells with the compound; and detecting the absorption level of the compound by the gastric cells or tissues.

[0052] In one aspect, a composition comprising de novo-generated three-dimensional human gastric organoids (hGO) and a method for making it by directed differentiation of human pluripotent stem cells (hPSC) are disclosed. Such hGO can be used for modeling gastric development as well as the early events that occur during Helicobacter pylori (H. pylori) infection.

[0053] In one aspect, a method for generating hGO in vitro by directed differentiation of human pluripotent stem cells (hPSC) is disclosed. This human gastric tissue can be used for modeling human gastric development and diseases. A method for inducing definitive endoderm (DE) to form a three-dimensional intestinal structure is also disclosed. In one aspect, this can be accomplished by activating FGF and WNT signaling while simultaneously inhibiting BMP signaling to promote foregut fate. Next, when the foregut spheroids are directed to become posterior foregut and gastric fates by manipulation of retinoic acid and EGF signaling, hGO can be obtained.

[0054] The development of hGO may undergo similar molecular and morphogenetic changes as those in the development of the mouse antrum, which forms gastric glands and pits, a proliferative zone, surface and antral mucous cells, and endocrine cells expressing gastrin, ghrelin, and somatostatin. By using hGO to model human gastric development, it has been determined that EGF signaling suppresses endocrine cell development upstream of the transcription factor NEUROGENIN 3. The applicant has further found that hGO faithfully recapitulates the early stages of gastric diseases induced by Helicobacter pylori (H. pylori), including rapid activation of c-Met signaling and epithelial proliferation. Taken together, these studies depict a new robust in vitro system for elucidating the mechanisms underlying human gastric development and diseases.

[0055] Pluripotent stem cells derived from germ cells In one aspect, the method can include obtaining 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, while the embryonic stem cells are derived from the totipotent cells of the early mammalian embryo and are capable of unlimited undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, which is an early embryo. Methods for obtaining embryonic stem cells from undifferentiated germ cells are well known in the art. For example, although certain cell types are exemplified herein, those skilled in the art will understand that the methods and systems described herein are applicable to any stem cell.

[0056] Additional stem cells that can be used in embodiments of the present invention include, but are not limited to, those provided by or described in the databases managed by the National Stem Cell Bank (NSCB), the Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF); the Wi Cell Research Institute's WISC Cell Bank; the University of Wisconsin Stem Cell and Regenerative Medicine Center (UW - SCRMC); Novocell, Inc. (San Diego, Calif.); Cellartis AB (Goteborg, Sweden); ES Cell International Pte Ltd (Singapore); the Technion at the Israel Institute of Technology (Haifa, Israel); as well as the stem cell databases managed by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that can be used in embodiments of the present invention include, but are not limited to, SA01 (SA001); SA02 (SA002); ES01 (HES - 1); ES02 (HES - 2); ES03 (HES - 3); ES04 (HES - 4); ES05 (HES - 5); ES06 (HES - 6); BG01 (BGN - 01); BG02 (BGN - 02); BG03 (BGN - 03); TE03 (13); TE04 (14); TE06 (16); UC01 (HSF1); UC06 (HSF6); WA01 (H1); WA07 (H7); WA09 (H9); WA13 (H13); WA14 (H14).

[0057] In some embodiments, the stem cells may be further modified to incorporate additional characteristics. Exemplary modified cell lines include, but are not limited to, H1 OCT4-EGFP; H9 Cre-LoxP; H9 hNanog-pGZ; H9 hOct4-pGZ; H9 in GFPhES; and H9 Syn-GFP.

[0058] For further details regarding embryonic stem cells, see, for example, Thomson et al., 1998, “Embryonic Stem Cell Lines Derived from Human Blastocysts”, Science 282(5391):1145-1147; Andrews et al., 2005, “Embryonic stem(ES)cells and embryonal carcinoma(EC)cells:opposite sides of the same coin”, Biochem Soc Trans 33:1526-1530; Martin 1980, “Teratocarcinomas and mammalian embryogenesis”, Science 209(4458):768-776; Evans and Kaufman, 1981, “Establishment in culture of pluripotent cells from mouse embryos”, Nature 292(5819):154-156; Klimanskaya et al., 2005, “Human embryonic stem cells derived without feeder cells”, Lancet 365(9471):1636-1641; (each of which is hereby incorporated by reference in its entirety herein).

[0059] Alternative pluripotent stem cells can be derived from embryonic germ cells (EGCs), which are cells that give rise to the gametes of sexually reproducing organisms. EGCs are derived from primordial germ cells found in the genital ridges of late embryos and have many of the characteristics of embryonic stem cells. When primordial germ cells in the embryo develop, they become stem cells that produce reproductive gametes (sperm or eggs) in the adult. In mice and humans, it is possible to grow embryonic germ cells in tissue culture under appropriate conditions. Both EGCs and ESCs are pluripotent. For the purposes of the present invention, the term "ESC" is used in a broad sense and sometimes includes EGCs.

[0060] Induced pluripotent stem cells (iPSCs) In some embodiments, iPSCs are obtained by transfecting certain stem cell-related genes into non-pluripotent cells, such as adult fibroblasts. Transfection is typically achieved using a viral vector such as a retrovirus. Genes to be transfected include the master transcriptional regulators Oct-3 / 4 (Pouf51) and Sox2, although other genes may also enhance the induction efficiency. After 3-4 weeks, a small number of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells and are typically isolated by morphological selection, by doubling time, or by reporter gene and antibiotic selection. As used herein, iPSCs can include, but are not limited to, first-generation iPSCs, second-generation iPSCs, and human induced pluripotent stem cells in mice. In some embodiments, a retroviral system can be used to transform human fibroblasts into pluripotent stem cells using four central genes: Oct3 / 4, Sox2, Klf4, and c-Myc. In alternative embodiments, a lentiviral system can be used to transform somatic cells with OCT4, SOX2, NANOG, and LIN28. Genes whose expression can be induced in iPSCs can include, but are not limited to, Oct-3 / 4 (e.g., Pou5fl); 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, and LIN28.

[0061] In some embodiments, iPSCs can be created using non-viral-based techniques. In some embodiments, adenoviruses can be used to deliver the four necessary genes into the DNA of mouse skin and liver cells, resulting in cells identical to embryonic stem cells. Since adenoviruses do not integrate any of their own genes into the target host, the risk of creating tumors is eliminated. In some embodiments, reprogramming can be achieved by plasmids without any viral transfection system, but the efficiency is extremely low. In other embodiments, iPSCs are created using direct delivery of proteins, thus eliminating the need for viruses or gene modification. In some embodiments, a similar methodology can be used to create mouse iPSCs: repeated treatment of cells with specific proteins supplied to the cells by 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] For further details regarding embryonic stem cells, see, for example, Kaji et al., 2009, “Virus free induction of pluripotency and subsequent excision of reprogramming factors”, Nature 458:771-775; Woltjen et al., 2009, “piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells”, Nature 458:766-770; Okita et al., 2008, “Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors”, Science 322(5903):949-953; Stadtfeld et al., 2008, “Induced Pluripotent Stem Cells Generated without Viral Integration”, Science 322(5903):945-949; and Zhou et al., 2009, “Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins”, Cell Stem Cell 4(5):381-384; each of which is hereby incorporated by reference in its entirety.

[0063] In some embodiments, exemplary iPS cell lines include, but are not limited to, iPS-DF19-9; iPS-DF19-9; iPS-DF4-3; iPS-DF6-9; iPS (Foreskin); iPS (IMR90); and iPS (IMR90).

[0064] iPSCs have been shown to have the ability to differentiate into fully differentiated tissues in a manner similar to ESCs. For example, iPSCs differentiated into neurons expressing βIII-tubulin, tyrosine hydroxylase, AADC, DAT, ChAT, LMX1B, and MAP2. The presence of catecholamine-related enzymes may suggest that iPSCs can differentiate into dopaminergic neurons similar to hESCs. Stem cell-related genes were shown to be downregulated after differentiation. Also, it has been shown that iPSCs can differentiate into cardiomyocytes that spontaneously began to beat. The cardiomyocytes expressed TnTc, MEF2C, MYL2A, MYHCβ, and NKX2.5. Stem cell-related genes were downregulated after differentiation.

[0065] Stomach organ and development Prior to the invention of the present applicant, there was no system available for converting progenitor cells such as embryonic stem cells and / or iPSCs into stomach tissue.

[0066] In some embodiments, PSCs such as ESCs and iPSCs undergo directed differentiation in a stepwise manner through formation of definitive endoderm (DE), then a three-dimensional gut structure (foregut spheroid), and then formation of posterior foregut / stomach tissue into three-dimensional gastric organoids (hGOs).

[0067] In some embodiments, PSCs such as ESCs and iPSCs undergo directed differentiation in a non-stepwise manner, where molecules (e.g., growth factors, ligands) for promoting DE formation and molecules for subsequent tissue formation are added simultaneously.

[0068] Definitive endoderm The gastric epithelium is derived from a single layer of cells called the definitive endoderm (DE) of the embryo. The anterior DE forms the foregut and its associated organs including the lung, esophagus, stomach, liver, and pancreas, and the posterior DE forms the midgut and hindgut, which form the small and large intestines as well as parts of the urogenital system. The DE gives rise to the epithelium of the digestive tract and respiratory tract in vivo. Studies using mouse, chick, and frog embryos suggest that establishment of the anterior-posterior pattern of the DE during gastrulation is essential for subsequent foregut and hindgut development. In some embodiments, pluripotent stem cells (PSCs) such as ESCs and iPSCs undergo directed differentiation in a stepwise manner first into definitive endoderm (DE), then into anterior / foregut epithelium (e.g., foregut spheroids), and then into gastric tissue. The BMP, Wnt, and FGF signaling pathways are thought to be critically important in this process. Activation of WNT and FGF serves to promote gut morphogenesis, and inhibition of BMP signaling promotes foregut fate. The simple columnar epithelium of the foregut develops first into a pseudostratified columnar epithelium and then into glands and pits containing gastric epithelium as well as a proliferative zone at the base of the villi (which corresponds to the presumptive progenitor domain).

[0069] A robust and efficient process is established for directing differentiation from DE to gastric tissue in vitro. In some embodiments, directed differentiation is achieved by selectively activating specific signaling pathways in iPSCs and / or DE cells. In some embodiments, such signaling pathways include, but are not limited to, those active in the development of gastric tissue, including the Wnt signaling pathway, the Wnt / APC signaling pathway, the FGF signaling pathway, the TGF-β signaling pathway, the BMP signaling pathway; the EGF signaling pathway, and the retinoic acid signaling pathway.

[0070] For further details regarding the generation of DE and / or the function of signaling pathways generally involved in gut development, see, for example, Zorn and Wells, 2009, “Vertebrate endoderm development and organ formation”, Annu Rev Cell Dev Biol 25:221-251; Dessimoz et al., 2006, “FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo”, Mech Dev 123:42-55; McLin et al., 2007, “Repression of Wnt / {beta}-catenin signaling in the anterior endoderm is essential for liver and pancreas development”. Development, 134:2207-2217; Wells and Melton, 2000, Development 127:1563-1572; de Santa Barbara et al., 2003, “Development and differentiation of the intestinal epithelium”, Cell Mol Life Sci 60(7):1322-1332; Sancho et al., 2004, “Signaling Pathways in Intestinal Development and Cancer”, Annual Review of Cell and Developmental Biology 20:695 - 723; Logan and Nusse, 2004, “The Wnt Signaling Pathway in Development and Disease”, Annual Review of Cell and Developmental Biology 20:781 - 810; Taipalel and Beachyl, 2001, “The Hedgehog and Wnt signalling pathways in cancer”, Nature 411:349 - 354; Gregorieff and Clevers, 2005, “Wnt signaling in the intestinal epithelium: from endoderm to cancer”, Genes & Dev. 19:877 - 890; (each of which is hereby incorporated by reference in its entirety herein).

[0071] Any method for generating definitive endoderm from pluripotent cells (e.g., iPSC or ESC) can be applied 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 that can be used in these methods include, but are not limited to, embryonic stem cells. Embryonic stem cells may be derived from the inner cell mass of the embryo or from the embryonic gonadal ridge. Embryonic stem cells or germ cells can originate from various animal species, including but not limited to various mammalian species including humans. In some embodiments, human embryonic stem cells are used to generate definitive endoderm. In some embodiments, human embryonic germ cells are used to generate definitive endoderm. In some embodiments, iPSCs are used to generate definitive endoderm.

[0072] In some embodiments, one or more growth factors are used in the differentiation process from pluripotent stem cells to DE cells. Examples of one or more growth factors used in the differentiation process include growth factors from the TGF-β superfamily. In such embodiments, the one or more growth factors include nodal / activin and / or the BMP subgroup 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, Wnt3a, or any combination of these growth factors.

[0073] In some embodiments, embryonic stem cells or induced pluripotent cells (iPSCs) and germ cells are treated with one or more growth factors for 6 hours or more; 12 hours or more; 18 hours or more; 24 hours or more; 36 hours or more; 48 hours or more; 60 hours or more; 72 hours or more; 84 hours or more; 96 hours or more; 120 hours or more; 150 hours or more; 180 hours or more; or 240 hours or more.

[0074] In some embodiments, embryonic stem cells or germ cells and iPSCs are treated with one or more growth factors at a concentration of 10 ng / ml or more; 20 ng / ml or more; 50 ng / ml or more; 75 ng / ml or more; 100 ng / ml or more; 120 ng / ml or more; 150 ng / ml or more; 200 ng / ml or more; 500 ng / ml or more; 1,000 ng / ml or more; 1,200 ng / ml or more; 1,500 ng / ml or more; 2,000 ng / ml or more; 5,000 ng / ml or more; 7,000 ng / ml or more; 10,000 ng / ml or more; or 15,000 ng / ml or more. In some embodiments, the concentration of the growth factor is maintained at a constant level throughout the treatment. In other embodiments, the concentration of the growth factor is varied during the treatment. In some embodiments, the growth factor is suspended in a medium containing fetal bovine serum (FBS) at various HyClone concentrations. Those skilled in the art will understand that the regimens described herein are applicable to any known growth factor, alone or in combination. When two or more growth factors are used, the concentration of each growth factor may be varied independently.

[0075] In some embodiments, a cell population enriched in definitive endoderm cells is used. In some embodiments, the definitive endoderm cells are isolated or substantially purified. In some embodiments, the isolated or substantially purified definitive endoderm cells express the SOX17, FOXA2, and / or CXCR4 markers at a higher level compared to the OCT4, AFP, TM, SPARC, and / or SOX7 markers.

[0076] Also contemplated is a method of enriching a cell population comprising definitive endoderm. In some embodiments, the definitive endoderm cells are isolated or substantially purified from a mixed cell population by contacting those cells with a reagent that binds to a molecule that is present on the surface of the definitive endoderm cells but not on the surface of other cells in the mixed cell population, and then isolating the cells that have bound to the reagent. In certain embodiments, the cell constituent present on the surface of the definitive endoderm cells is CXCR4.

[0077] Still other embodiments of the invention relate to CXCR4 antibodies, SDF-1 ligand or other CXCR4 ligands, which can be used to obtain definitive endoderm cells in enriched, isolated or substantially purified form. For example, CXCR4 antibodies, SDF-1 ligand or another CXCR4 ligand can be used as a reagent in methods such as affinity-based separation or magnetic-based separation to enrich, isolate or substantially purify a preparation of definitive endoderm cells that bind to the reagent.

[0078] In some embodiments, the definitive endoderm cells and hESCs are treated with one or more growth factors. Such growth factors can include growth factors of the TGF-β superfamily. In such embodiments, the one or more growth factors include Nodal / Activin and / or the BMP subgroup 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, Wnt3a, or any combination of these growth factors.

[0079] As additional methods for obtaining or generating DE cells that can be used in the present invention, but not limited to, U.S. Patent No. 7,510,876 to D’Amour et al.; U.S. Patent No. 7,326,572 to Fisk et al.; Kubol et al., 2004, “Development of definitive endoderm from embryonic stem cells in culture”, Development 131:1651-1662; D’Amour et al., 2005, “Efficient differentiation of human embryonic stem cells to definitive endoderm”, Nature Biotechnology 23:1534-1541; and Ang et al., 1993, “The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3 / forkhead proteins”, Development 119:1301-1315; (each of which is hereby incorporated by reference in its entirety herein) may be mentioned.

[0080] Directed differentiation of posteriorized DE In some embodiments, activin-induced definitive endoderm (DE) can be further passed through FGF / Wnt / noggin-induced anterior endoderm patterning, foregut specification and morphogenesis, and finally a progastric culture system to promote gastric tissue growth, morphogenesis and cell differentiation into functional gastric cell types including surface mucous cells, mucous gland cells, endocrine, and progenitor cells. In some embodiments, human PSCs are efficiently directed to differentiate in vitro into gastric epithelium containing mucous, endocrine, and progenitor cell types. It will be understood that molecules such as growth factors can be added at any stage of development to promote a particular type of gastric tissue formation.

[0081] In some embodiments, anterior definitive endoderm cells further differentiate into one or more specialized cell types.

[0082] In some embodiments, soluble FGF and Wnt ligands and a BMP antagonist are used to mimic early foregut specification in culture, and direct the differentiation of definitive endoderm (DE) derived from iPSCs or ESCs into foregut epithelium that efficiently gives rise to all of the major gastric cell types. In humans, directed differentiation of DE is achieved by selectively activating specific signaling pathways important for gastric development.

[0083] In vitro generation of the human stomach / gastric occurs in stages that approximate fetal intestinal development; endoderm formation, anterior endoderm patterning, foregut morphogenesis, fetal stomach, antrum and fundus development, epithelial morphogenesis, formation of prospective progenitor domains, and differentiation into functional cell types of the stomach.

[0084] One of ordinary skill in the art will understand that directed differentiation in the present invention can occur by modifying the expression of any Wnt signaling protein in combination with any FGF ligand. In some embodiments, this modification is overexpression of Wnt3, specifically Wnt3a. In some embodiments, this modification is overexpression of Wnt1 or other Wnt ligands.

[0085] One of ordinary skill in the art will understand that directed differentiation in the present invention can occur by modifying the signaling activity of the Wnt signaling pathway in combination with modifying the signaling activity of the FGF signaling pathway. In some embodiments, this modification is by use of small molecule modulators that activate the aforementioned pathways. For example, small molecule modulators of the Wnt pathway include, but are not limited to, lithium chloride; 2-amino-4,6-disubstituted pyrimidines (hetero)aryl pyrimidines; IQ1; QS11; NSC668036; DCA β-catenin; 2-amino-4-[3,4-(methylenedioxy)-benzyl-amino]-6-(3-methoxyphenyl)pyrimidine.

[0086] In alternative embodiments, cell components related to the Wnt and / or FGF signaling pathways, such as natural inhibitors or antagonists of these pathways, can be inhibited to result in the activation of the Wnt and / or FGF signaling pathways.

[0087] In some embodiments, the cell components are inhibited by other cell components or exogenous molecules. Exemplary natural inhibitors of Wnt signaling include, but are not limited to, Dkk1, SFRP proteins, and FrzB. In some embodiments, exogenous molecules can include, but are not limited to, small molecules such as WAY-316606; SB-216763; or BIO (6-bromoindirubin-3'-oxime).

[0088] For further details, see, for example, Liu et al., “A small-molecule agonist of the Wnt signaling pathway”, Angew Chem Int Ed Engl. 44(13):1987-1990(2005); Miyabayashi et al., “Wnt / beta-catenin / CBP signaling maintains long-term murine embryonic stem cell pluripotency”, Proc Natl Acad Sci USA. 104(13):5668-5673(2007); Zhang et al., “Small-molecule synergist of the Wnt / beta-catenin signaling pathway”, Proc Natl Acad Sci U S A. 104(18):7444-7448(2007); Neiiendam et al., “An NCAM-derived FGF-receptor agonist, the FGL-peptide, induces neurite outgrowth and neuronal survival in primary rat neurons”, J. Neurochem. 91(4):920-935(2004); Shan et al., “Identification of a specific inhibitor of the dishevelled PDZ domain”, Biochemistry 44(47):15495-15503(2005); Coghlan et al., “Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription”, Chem. Biol. 7(10):793-803(2000); Coghlan et al.“Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription”, Chemistry&Biology 7(10):793-803; and Pai et al., “Deoxycholic acid activates beta-catenin signaling pathway and increases colon cell cancer growth and invasiveness”, Mol Biol Cell. 15(5):2156-2163(2004); (each of which is hereby incorporated by reference in its entirety) are referred to.

[0089] In some embodiments, these pathways are activated using siRNA and / or shRNA that target cellular components associated with the Wnt and / or FGF signaling pathways. Those skilled in the art will understand that target cellular components can include, but are not limited to, SFRP proteins; GSK3, Dkk1, and FrzB.

[0090] For further details regarding RNAi-based technologies, see, for example, Couzin, 2002, Science 298:2296-2297; McManus et al., 2002, Nat.Rev.Genet. 3,737-747; Hannon, G.J., 2002, Nature 418,244-251; Paddison et al., 2002, Cancer Cell 2,17-23; Elbashir et al., 2001. EMBO J. 20:6877-6888; Tuschl et al., 1999, Genes Dev. 13:3191-3197; Hutvagner et al., Sciencexpress 297:2056-2060; (each of which is hereby incorporated by reference in its entirety).

[0091] Fibroblast growth factor (FGF) is a family of growth factors involved in angiogenesis, wound healing, and embryonic development. FGF is a heparin-binding protein, and its interaction with cell surface-associated heparan sulfate proteoglycans has been shown to be essential for FGF signaling. FGF is a central player 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. Members FGF11, FGF12, FGF13, and FGF14, also known as FGF homolog factors 1 - 4 (FHF1 - FHF4), have been shown to have functionally distinct differences compared to FGF. These factors have significantly similar sequence homology, but they do not bind to FGFRs and are involved in intracellular processes unrelated to FGF. This group is also known as "iFGF". Members FGF16 - FGF23 are relatively new and not well-characterized. FGF15 is the mouse ortholog of human FGF19 (thus human FGF15 does not exist). Human FGF20 was identified based on its homology to Xenopus FGF-20 (XFGF-20). In contrast to the local activities of other FGFs, FGF15 / FGF19, FGF21, and FGF23 have more systemic effects.

[0092] In some embodiments, one of ordinary skill in the art will understand that any FGF can be used in combination with proteins of the Wnt signaling pathway. In some embodiments, soluble FGFs can include, but are not limited to, FGF4, FGF2, and FGF3.

[0093] In some embodiments, the cellular components of the FGF signaling pathway are inhibited by other cellular components or exogenous molecules. Exemplary natural inhibitors of FGF signaling include, but are not limited to, the Sprouty protein family and the Spred protein family. As discussed above, the FGF signaling pathway can be activated using proteins, small molecules, and nucleic acids.

[0094] One of ordinary skill in the art will understand that the methods and compositions described herein are provided by way of example in relation to the Wnt and FGF signaling pathways. Similar methods and compositions are applicable to other signaling pathways disclosed herein.

[0095] In some embodiments, the DE cultures can be treated for 6 hours or more; 12 hours or more; 18 hours or more; 24 hours or more; 36 hours or more; 48 hours or more; 60 hours or more; 72 hours or more; 84 hours or more; 96 hours or more; 120 hours or more; 150 hours or more; 180 hours or more; 200 hours or more; 240 hours or more; 270 hours or more; 300 hours or more; 350 hours or more; 400 hours or more; 500 hours or more; 600 hours or more; 700 hours or more; 800 hours or more; 900 hours or more; 1,000 hours or more; 1,200 hours or more; or 1,500 hours or more by one or more molecules of the signaling pathways described herein.

[0096] In some embodiments, the DE cultures are treated with one or more molecules of the signal transduction pathways described herein at a concentration of 10 ng / ml or more; 20 ng / ml or more; 50 ng / ml or more; 75 ng / ml or more; 100 ng / ml or more; 120 ng / ml or more; 150 ng / ml or more; 200 ng / ml or more; 500 ng / ml or more; 1,000 ng / ml or more; 1,200 ng / ml or more; 1,500 ng / ml or more; 2,000 ng / ml or more; 5,000 ng / ml or more; 7,000 ng / ml or more; 10,000 ng / ml or more; or 15,000 ng / ml or more. In some embodiments, the concentration of the signal transduction molecule is maintained constant throughout the treatment. In other embodiments, the concentration of the molecules of the signal transduction pathway is varied during the treatment. In some embodiments, the signal transduction molecules in the present invention are suspended in a medium containing DMEM and fetal bovine serum (FBS). The FBS may be at a concentration of 2% or more; 5% or more; 10% or more; 15% or more; 20% or more; 30% or more; or 50% or more. Those skilled in the art will understand that the regimens described herein are applicable to any known molecule of the signal transduction pathways described herein, including, but not limited to, any molecule in the Wnt and FGF signal transduction pathways, alone or in combination.

[0097] In embodiments where the DE cultures are treated with two or more signal transduction molecules, those signal transduction molecules can be added simultaneously or separately. When using two or more molecules, the concentration of each can be varied independently.

[0098] Differentiation from PSCs to DE cultures and subsequently to various intermediate mature gastric cell types can be determined by the presence of development stage-specific cell markers. In some embodiments, DE formation is determined using the expression of representative cell constituents. Representative cell constituents include, but are not limited to, CMKOR1, CXCR4, GPR37, RTN4RL1, SLC5A9, SLC40A1, TRPA1, AGPAT3, APOA2, C20orf56, C21orf129, CALCR, CCL2, CER1, CMKOR1, CRIP1, CXCR4, CXorf1, DIO3, DIO30S, EB-1, EHHADH, ELOVL2, EPSTI1, FGF17, FLJ10970, FLJ21195, FLJ22471, FLJ23514, FOXA2, FOXQ1, GATA4, GPR37, GSC, LOC283537, MYL7, NPPB, NTN4, PRSS2, RTN4RL1, SEMA3E, SIAT8D, SLC5A9, SLC40A1, SOX17, SPOCK3, TMOD1, TRPA1, TTN, AW166727, AI821586, BF941609, AI916532, BC034407, N63706, and AW772192.

[0099] For further cell compositions suitable for the detection of DE formation, reference can be made, for example, to U.S. Patent Application No. 11 / 165,305 filed on June 23, 2005; U.S. Patent Application No. 11 / 317,387 filed on December 22, 2005; U.S. Patent Application No. 11 / 021,618 filed on December 23, 2004; U.S. Patent Application No. 11 / 021,618, No. 11 / 115,868 filed on April 26, 2005; U.S. Patent Application No. 11 / 317,387 filed on December 22, 2005; U.S. Patent Application No. 11 / 474,211 filed on June 23, 2006; U.S. Patent Application No. 11 / 165,305 filed on June 23, 2005; U.S. Patent Application No. 11 / 587,735 filed on August 29, 2008; U.S. Patent Application No. 12 / 039,701 filed on February 28, 2008; U.S. Patent Application No. 12 / 414,482 filed on March 30, 2009; U.S. Patent Application No. 12 / 476,570 filed on June 2, 2009; U.S. Patent Application No. 12 / 093,590 filed on July 21, 2008; U.S. Patent Application No. 12 / 582,600 filed on October 20, 2009 (each of which is hereby incorporated by reference in its entirety herein).

[0100] In some embodiments, the expression of SOX2 is used to reveal the tendency of foregut formation after incubating DE with FGF4 and Wnt3a + noggin for a period of time, such as 12 hours or more; 18 hours or more; 24 hours or more; 36 hours or more; 48 hours or more; 60 hours or more; or 90 hours or more. In some embodiments, a longer incubation time is required to achieve a stable anterior endoderm phenotype when measured by the prolongation of CDX2 expression. In such embodiments, the incubation time can be 60 hours or more; 72 hours or more; 84 hours or more; 96 hours or more; 108 hours or more; 120 hours or more; 140 hours or more; 160 hours or more; 180 hours or more; 200 hours or more; 240 hours or more; or 300 hours or more.

[0101] Alternatively, in some embodiments, the absence of cell components, such as the hindgut marker CDX2, can be used to reveal directed foregut formation. In some embodiments, the gastric transcription factors PDX1, KLF5, and SOX9 can be used to represent gastric development. In some embodiments, GATA4 and / or GATA6 protein expression can be used to represent gastric development. In these embodiments, the incubation time can be 12 hours or more; 18 hours or more; 24 hours or more; 36 hours or more; 48 hours or more; 60 hours or more; or 90 hours or more. Alternatively, the incubation time can be 60 hours or more; 72 hours or more; 84 hours or more; 96 hours or more; 108 hours or more; 120 hours or more; 140 hours or more; 160 hours or more; 180 hours or more; 200 hours or more; 240 hours or more; or 300 hours or more.

[0102] In some embodiments, immunohistochemistry using primary and / or secondary antibodies that target molecules of the relevant signaling pathway determines abundance data of cell components, such as protein and / or gene expression levels. In other embodiments, microarray analysis determines abundance data of cell components, such as protein and / or gene expression levels.

[0103] Alternatively, morphological changes can be used to represent the progression of directed differentiation. In some embodiments, foregut spheroids can be further subjected to three-dimensional culture conditions for further maturation. In addition, gastric organoids can be observed for 6 days or more; 7 days or more; 9 days or more; 10 days or more; 12 days or more; 15 days or more; 20 days or more; 25 days or more; 28 days or more; 32 days or more; 36 days or more; 40 days or more; 45 days or more; 50 days or more; or 60 days or more.

[0104] Directed Differentiation of Pluripotent Stem Cells In some embodiments, the pluripotent stem cells are converted into gastric cell types by a "one-step" process. For example, one or more molecules (such as activin A) that can differentiate pluripotent stem cells into DE cultures are combined with additional molecules (such as Wnt3a / FGF4 activators and BMP inhibitors) that can promote the directed differentiation of DE cultures, such that the pluripotent stem cells are directly treated.

[0105] Utility and Kit Embodiments In some embodiments, the gastric tissue or related cell types described herein can be used to screen drugs with respect to the gastric uptake and / or transport and / or processing mechanisms of Helicobacter pylori (H. Pylori). For example, this can be done in a high-throughput manner to screen for the most readily absorbed or effective drugs, and can enhance phase I clinical trials conducted to test drug gastric uptake and gastric toxicity. This can include the cellular and intracellular transport mechanisms of small molecules, peptides, metabolites, salts. The gastric tissue disclosed herein can further be used to evaluate the compatibility of any agent and / or device intended to come into contact with the gastric tissue for the purpose of evaluating biocompatibility.

[0106] In some embodiments, the gastric cells, gastric tissue and / or gastric hGO described herein can be used to identify the molecular basis of normal human gastric development.

[0107] In some embodiments, the gastric cells, gastric tissue and / or gastric hGO described herein can be used to identify the molecular basis of congenital defects that affect human gastric development.

[0108] In some embodiments, the gastric cells, gastric tissues, and / or gastric hGO described herein can be used to correct congenital gastric defects caused by gene mutations. Specifically, the iPSC technology and the genetically normal gastric tissues or related cell types described herein can be used to correct mutations that affect human gastric development. In some embodiments, alternative tissues can be created using the gastric tissues or related cell types described herein. Examples of genetic diseases include, but are not limited to, Neurog3 mutations and enteric anendocrinosis, PTF1a mutations and neonatal diabetes, and PDX1 mutations that affect gastric enteroendocrine cells.

[0109] In some embodiments, the gastric cells, gastric tissues, and / or gastric hGO described herein can be used to create alternative gastric tissues for diseases or conditions such as peptic ulcer disease, Menetrier's disease, or for gastric cancer patients.

[0110] In some embodiments, the gastric cells, gastric tissues, and / or gastric hGO described herein can be used to study the microbiotic interactions with the human host epithelium and host immunity.

[0111] In some embodiments, the gastric tissues or related cell types described herein, specifically enteroendocrine cells, can be used to study the feeding behavior mediated by gastric endocrine and the hormonal regulation of metabolism.

[0112] In some embodiments, the gastric cells, gastric tissues, and / or gastric hGO described herein, specifically enteroendocrine cells that produce the hormones gastrin or ghrelin, can be used to study and improve metabolic regulation in patients with, for example, obesity, metabolic syndrome, or type 2 diabetes.

[0113] In some embodiments, the gastric cells, gastric tissue, and / or gastric hGO described herein can be used to replace any damaged or excised gastric tissue in a subject in need thereof.

[0114] In some embodiments, the gastric cells, gastric tissue, and / or gastric hGO described herein can be used to screen the toxicity and efficacy of any drug acting on the gastric tissue.

[0115] In some embodiments of determining the absorption level of a compound using the gastric cells, gastric tissue, and / or gastric hGO described herein, the compound is contacted with the gastric cells, gastric tissue, and / or gastric hGO; and the absorption level of the compound by the gastric cells, gastric tissue, and / or gastric hGO can be quantified. In some embodiments, the compound may be labeled with a radioisotope, a fluorescent label, and / or a primary or secondary visible marker.

[0116] In some embodiments, a diagnostic kit or package is developed that includes the gastric cells, gastric tissue, and / or gastric hGO described herein and is based on one or more of the foregoing utilities.

[0117] Although the invention has been described in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the invention as defined in the appended claims. Furthermore, it must be understood that all examples in this disclosure are provided as non-limiting examples.

Examples

[0118] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. Those skilled in the art should understand that the techniques disclosed in the following examples correspond to methods that have been found to function well in the practice of the invention, and thus may be considered examples of embodiments for their implementation. However, those skilled in the art should understand that, in view of the present disclosure, many changes can be made in the specific embodiments disclosed, and still achieve similar or analogous results without departing from the spirit and scope of the invention.

[0119] Pluripotent stem cell culture Human embryonic stem cell lines WA01 (H1) and WA09 (H9) were obtained from WiCell. ESC lines and iPSC lines were maintained as colonies under feeder-free conditions on HESC-qualified Matrigel (BD Biosciences) in mTesR1 medium (Stem Cell Technologies). Cells were passaged every 4 days using dispase (Invitrogen) in the conventional manner.

[0120] DE induction (Summarized in Figure 14) Human ES and iPS cells were plated as single cells at 150,000 cells per well in mTesR1 medium + ROCK inhibitor Y27632 (10 μM; Stemgent) in 24-well plates coated with Matrigel (BD Biosciences). The ROCK inhibitor enhances the survival of stem cells after plating for differentiation. Starting the next day, cells were treated with activin A (100 ng ml-1; Cell Guidance Systems) for 3 days in RPMI 1640 (Invitrogen) containing gradually increasing concentrations of 0%, 0.2%, and 2.0% defined fetal bovine serum (dFBS; Invitrogen).

[0121] Differentiation of definitive endoderm (DE) To differentiate PSCs, in 24-well dishes coated with Matrigel, single cells were plated at a density of 150,000 cells per well in mTesR1 containing the ROCK inhibitor Y-27632 (10 μM; Stemgent) using accutase (Stem Cell Technologies). The next day, the PSCs differentiated into DEs as previously described. 11、35 . Cells were exposed to activin A (100 ng / ml; Cell Guidance Systems) for 3 days in RPMI 1640 medium (Invitrogen) containing increasing concentrations of 0%, 0.2%, and 2.0% known composition fetal bovine serum (dFBS; Invitrogen). In addition, BMP4 (50 ng / ml; R&D Systems) was added on the first day of DE induction.

[0122] Endoderm patterning and gut tube morphogenesis Following DE induction, cells were treated with growth factors / antagonists for 3 days in RPMI 1640 containing 2.0% dFBS. To generate posterior foregut spheroids, DEs were treated for 3 days with noggin (200 ng / ml; R&D Systems), FGF4 (500 ng / ml; R&D Systems), and either WNT3A (500 ng / ml; R&D Systems) or CHIR99021 (2 μM; Stemgent). CHIR99021 is a small molecule that stimulates the Wnt signaling pathway. RA (2 μM; Sigma Aldrich) was added on the final day. Three-dimensional growth and specification of the foregut. Posterior foregut spheroids were embedded in Matrigel (BD Biosciences) as previously described 10、12 , and then grown in advanced DMEM / F12 (Invitrogen) supplemented with N2 (Invitrogen), B27 (Invitrogen), L-glutamine, 10 μM HEPES, penicillin / streptomycin, and EGF (100 ng / ml; R&D Systems). For foregut specification, RA and noggin were added during the first 3 days of three-dimensional growth. For endocrine cell specification, the EGF concentration was reduced to 10 ng / ml on day 30.-1 Lower it to

[0123] Endoderm patterning and foregut spheroid formation Following DE induction, cells were cultured in RPMI 1640 medium containing 2.0% dFBS and growth factors: WNT3A (500 ng / ml; R&D Systems), CHIR99021 (2 μM; Stemgent); FGF4 (500 ng / ml; R&D Systems), and noggin (200 ng / ml; R&D Systems). The medium was changed daily. After 3 days, a combination of WNT3A (or CHIR99021), FGF4, and noggin resulted in floating foregut spheroids in the culture wells. For posteriorization of the foregut endoderm, RA (2 μM; Sigma Aldrich) was added on day 3 of WNT / FGF / noggin treatment.

[0124] Tertiary culture of gastric organoids The spheroids were transferred to a three-dimensional in vitro culture system as previously described 5、10、12 . Briefly, the spheroids were collected, resuspended in 50 μl Matrigel (BD Biosciences), and plated in three-dimensional droplets. After solidifying Matrigel in a tissue culture incubator for 10 - 15 minutes, the spheroids were overlaid with advanced DMEM / F12 containing intestinal medium: N2 (Invitrogen), B27 (Invitrogen), L-glutamine, 10 μM HEPES, penicillin / streptomycin, and EGF (100 ng / ml; R&D Systems). RA and noggin were added to this intestinal medium for the first 3 days. The medium was changed every 3 - 4 days as needed. On day 20, the organoids were collected and replated in fresh Matrigel at approximately 1:12 dilution.

[0125] Generation of dox-inducible hNEUROG3 hESC line To create the overexpression construct, hNEUROG3 cDNA (Dana-Farber / Harvard Cancer Center DNA Resource Core; clone HsCD00345898) was cloned into the pInducer20 lentiviral vector (kindly provided by T. Westbrook) using Gateway Cloning (Invitrogen). High-titer lentiviral particles were generated by the CCHMC Viral Vector Core. H1 hESCs were dissociated with Accutase and plated as single-cell suspensions in mTesR1 containing 10 μM Y-27632 and exposed to lentivirus for 4 hours. mTesR1 was replaced daily, and after 2 days, G418 (200 μg / ml) was added to the medium to select for integrated clones. G418-resistant cells were maintained indefinitely in the antibiotic, but otherwise cultured and passaged as normal. 36 ) were generated by the CCHMC Viral Vector Core. H1 hESCs were dissociated with Accutase and plated as single-cell suspensions in mTesR1 containing 10 μM Y-27632 and exposed to lentivirus for 4 hours. mTesR1 was replaced daily, and after 2 days, G418 (200 μg / ml) was added to the medium to select for integrated clones. G418-resistant cells were maintained indefinitely in the antibiotic, but otherwise cultured and passaged as normal.

[0126] Generation and Characterization of iPSC Lines Primary human foreskin fibroblasts (HFFs) were cultured from neonatal human foreskin tissue obtained from two donors through the Department of Dermatology, University of Cincinnati, and kindly provided by Susanne Wells, PhD. HFFs were cultured in fibroblast medium consisting of DMEM (Invitrogen) supplemented with 10% FCS (Hyclone) and used for reprogramming between passages 5 and 8. The EBNA1 / OriP-based episomal plasmids pCLXE-hOct3 / 4-shp53, pCLXE-hSox2-Klf4, pCLXE-hLmyc-Lin28, and pCLXE-GFP used in this study have been described previously 37, obtained from Addgene (ID numbers: 27077, 27078, 27080, and 27082 respectively). The optimized Human Fibroblast Nucleofector Kit (VPD-1001; Lonza) was used for the transfection of HFFs with episomal plasmids. Briefly, for each transfection, 1 × 106 HFFs were pelleted by centrifugation at 200 × g for 10 minutes at room temperature, resuspended in 100 μl of room temperature Nucleofector solution, and nucleofected with 1.25 μg of each episomal plasmid (program U20). Cells from two transfections (a total of 2 × 106 cells) were replated into fibroblast medium in a 10 cm tissue culture plate and cultured at 37 °C / 5% CO2. Six days after transfection, 4.5 × 105 HFFs were replated into fibroblast medium in a gelatin-coated 10 cm dish containing 1.07 × 106 irradiated mouse embryonic fibroblasts (MEFs). Starting on day 7 after transfection, the cells were fed daily with DMEM / F12 medium supplemented with 20% knockout serum replacement, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 0.1 mM non-essential amino acids, and 4 ng ml-1 basic FGF (all from Invitrogen). After approximately two weeks, isolated colonies with an hESC-like morphology were manually excised and replated into mTesR1 medium (Stem Cell Technologies) in a tissue culture dish coated with hESC-qualified matrigel (Becton Dickinson). After adaptation to mTeSR1 / matrigel culture, iPSCs that maintained robust growth and an hESC-like morphology with minimal spontaneous differentiation were expanded for cryopreservation and characterization.

[0127] Standard intermediate spread and G-banded karyotype were determined by the CCHMC Cytogenetics Laboratory. For teratoma formation, iPSCs from three wells of a 6-well dish were combined and gently resuspended in ice-cold DMEM / F12. Immediately prior to injection, matrigel was added to a final concentration of approximately 33%, and the cells were injected subcutaneously into immunodeficient NOD / SCID γ C- / - mice. Tumors formed within 6 - 12 weeks. Excised teratomas were fixed, embedded in paraffin, and sections were stained with hematoxylin and eosin for histological examination.

[0128] Exemplary protocol for gastric organoids The following table shows an exemplary processing protocol for generating gastric organoids from progenitor cells.

[0129]

Table 1-1

Table 1-2

[0130] Protocol for gastric fundus specification The applicant first sought to identify genes that are specifically expressed in the gastric fundus during embryogenesis but not in the antrum. The digestive tracts of E14.5 mouse embryos were microdissected and divided into four regions: the forestomach (including the esophagus), the gastric fundus, the antrum, and the duodenum. See Figure 15. Next, the regional markers of these regions were analyzed by qPCR. Figure 15 shows the expression of control genes known to be expressed in various regions. The gastric fundus and antrum can be distinguished from the forestomach and duodenum by their high expression of Sox2 and Gata4, and the absence of P63 and Cdx2. Importantly, Pdx1 (a marker of the antrum) is expressed at a much higher level in antrum tissue compared to the gastric fundus, indicating accurate dissection.

[0131] Using bioinformatics analysis of publicly available microarray datasets of embryonic mouse endoderm and adult human stomach tissue, a list of candidate genes that may be preferentially expressed in the fundus but not in the antrum was generated. Expression of these putative markers in the E14.5 mouse segment was examined by qPCR. Irx1, Irx2, Irx3, Irx5, and Pitx1 are indeed expressed at higher levels in the fundus compared to the antrum. Thus, these markers may be used as indicators of fundus specification in hPSC-derived foregut cultures. See Figure 16.

[0132] Next, the function of Wnt signaling in the regulation of fundus-antrum patterning on days 6 - 9 of the gastric organoid differentiation protocol was tested. Addition of Wnt3a (at 100 ng / mL and 500 ng / mL) had no effect on spheroid gene expression, but it also induced expression of the Wnt target gene Axin2. Thus, the effect of the small molecule CHIR99021 (CHIR; 2 μM) was tested. CHIR99021 stimulates Wnt signaling in a receptor-independent manner. Exposure to CHIR resulted in robust suppression of Pdx1 expression levels, which was consistent with fundus specification. CHIR did not induce expression of the intestinal marker Cdx2. See Figure 17. Figure 18 shows that exposure to CHIR induced high levels of expression of the fundus-specific markers IRX3 and IRX5, consistent with the suppression of Pdx1.

[0133] Helicobacter pylori infection Helicobacter pylori strain G27 and the mutant strain G27 (ΔCagA) lacking CagA 39 as previously described 40, grown on blood agar plates consisting of Columbia agar base (Fisher Scientific), 5% horse blood (Colorado Serum Company), 5 μg / ml vancomycin, and 10 μg / ml trimethoprim. For organoid injection, Helicobacter pylori was resuspended in Brucella broth at a concentration of 1×10⁹ bacteria / ml and loaded into a Nanoject II (Drummond) microinjector device. Approximately 200 nl (containing 2×10⁵ bacteria) was injected directly into the lumen of each organoid, and the injected organoids were cultured for 24 hours. Brucella broth was injected as a negative control.

[0134] Materials and Methods Immunofluorescence Staining All tissues were fixed in 4% paraformaldehyde either at room temperature for 1 hour for cryopreservation or at 4°C overnight for paraffin processing. For cryosections, tissues were protected in 30% sucrose at 4°C overnight, then embedded in OCT (Tissue-Tek) and cut into 10 μm sections. For paraffin sections, tissues were processed through a series of ethanol steps followed by xylene, then paraffin-embedded and cut into 7 μm sections. Tissue culture cells were fixed at room temperature for 15 minutes and stained directly. For staining, cryosections were thawed at room temperature and rehydrated in PBS, while paraffin sections were deparaffinized and subjected to antigen retrieval. The slides were blocked in 5% normal donkey serum (Jackson Immuno Research) in PBS + 0.5% Triton-X at room temperature for 30 minutes. Primary antibodies (listed in Table 1 of "Methods") were diluted in blocking buffer and incubated overnight at 4°C. The slides were washed with PBS, incubated with secondary antibodies for 1 hour at room temperature, and coverslips were mounted using Fluoromount-G (Southern Biotech). Confocal images were acquired with a Nikon A1Rsi inverted confocal microscope.

[0135] RNA Isolation and qPCR Total RNA was isolated from tissues using the Nucleospin RNA II kit (Machery-Nagel). Reverse transcription was performed from 100 ng of RNA using the Superscript VILO cDNA Synthesis kit (Invitrogen) according to the manufacturer's protocol. qPCR was performed using the Quantitect SybrGreen Master Mix (Qiagen) with a CFX-96 real-time PCR detection system (BioRad). Analyses were performed using the ΔΔCT method. PCR primers were designed using sequences from qPrimerDepot (http: / / primerdepot.nci.nih.gov) and are listed in Table 2.

[0136] Immunoprecipitation and Western blot analysis Organoids infected with Helicobacter pylori (H. pylori) were recovered from Matrigel in ice-cold PBS and centrifuged at 150 g for 5 minutes. The tissue was lysed in M-PER mammalian protein extraction reagent (Thermo Scientific) supplemented with protease inhibitor (Roche). 10 μg of total protein from the cell lysate was immunoprecipitated with anti-c-Met antibody (2 μg; Cell Signaling 4560) at 4 °C for 16 hours. Next, protein A / G agarose beads (20 μl; Santa Cruz Biotechnology) were added and the samples were incubated at 4 °C for 16 hours. The immunoprecipitates were washed three times with PBS and then resuspended in Laemmli loading buffer containing β-mercaptoethanol (40 μl; BioRad). The samples were run on a 4–20% Tris-glycine gradient gel (Invitrogen) at 80 V for 3.5 hours. The gel was transferred to a nitrocellulose membrane (Whatman Protran, 0.45 μm) at 105 V for 1.5 hours. The membrane was blocked in KPL Detector blocking solution (Kirkeaard & Perry Laboratories) for 1 hour at room temperature and then incubated with the primary antibody overnight at 4 °C. Primary antibodies used: anti-phosphotyrosine (Santa Cruz, sc-7020; 1:100), anti-c-Met (Abcam, ab59884; 1:100), and anti-Helicobacter pylori (H. pylori) CagA (Abcam, ab90490; 1:100). The membrane was washed and incubated with Alexa Fluor anti-mouse 680 (Invitrogen; 1:1000) secondary antibody. The blot was imaged using an Odyssey infrared imaging software system (Licor).

[0137] Discussion hPSCs differentiated into definitive endoderm (DE). 11DE gives rise to the gastrointestinal and respiratory epithelia in vivo. The next two important events in the development of all endodermal organs are the patterning of DE along the anterior-posterior (A-P) axis and gut morphogenesis, which result in the formation of Sox2+ foregut anteriorly and Cdx2+ mid- and hindgut posteriorly (E8.5, 14-somite stage mouse embryo, as highlighted in Figure 1A). This morphogenesis and tissue interactions between endoderm and mesoderm seem to be critically important for proper organogenesis both in vivo and in vitro. WNT3A and FGF4 have previously been demonstrated to act synergistically to do the following: posteriorize hPSC-derived DE, promote mesenchymal expansion, and induce the formation of gut-like structures expressing the mid- and hindgut marker CDX2. 10、12 Figure 1A shows that the Sox2 protein characterizes the foregut endoderm and the Cdx2 protein characterizes the mid / hindgut endoderm in an e8.5 (14-somite stage) mouse embryo. Figure 1B shows that inhibition of BMP suppressed the mid / hindgut fate and promoted the expression of the foregut marker SOX2. PCR analysis of patterning markers in hPSC-DE cultures exposed for 3 days in medium alone (control) or medium containing the indicated growth factor / antagonist. The combined activity of WNT and FGF induced Cdx2 expression as previously reported 10 whereas the BMP antagonist noggin was sufficient to suppress Cdx2 expression and induce high levels of the foregut marker SOX2. * p < 0.05 compared to the control. ** p < 0.005 compared to WNT / FGF. Figure 1C shows that foregut spheroids generated using Wnt / FGF / noggin have high levels of SOX2 protein by whole-mount immunofluorescence staining and mRNA when compared to spheroids generated with Wnt and FGF alone, which have high levels of CDX2. *, p < 1.0×10-6. Figure 1D shows that the posterior foregut in e8.5, 14-somite stage mouse embryos gives rise to the stomach and pancreas and has high levels of Hnf1β protein. Figure 1E shows that exposure of the culture to RA on the final day of the spheroid formation step induces the expression of HNF1β in the SOX2-expressing epithelium, resulting in the formation of posterior foregut spheroids. * , p < 0.005. Figure 1F shows a schematic diagram summarizing the patterning effects of noggin and RA in the formation of both anterior and posterior foregut endoderm. Scale bar, 100 μm. Error bars represent standard deviation.

[0138]

Table 2

[0139]

Table 3

[0140] To promote the formation of foregut structures in hPSC-derived DE, the applicant sought to separate the ability of WNT / FGF to stimulate gut tube morphogenesis from their role in promoting posterior endoderm fate. Based on in vivo studies in model organisms of development 13、14 , the applicant tested the function of BMP signaling in the regulation of A-P patterning and found that WNT / FGF requires BMP activity to induce the hindgut program. Specifically, inhibition of BMP signaling with the antagonist noggin suppressed CDX2 and induced the foregut marker SOX2 in DE cultures after 3 days in the presence of WNT / FGF (Figures 1B - 1C and 5). Importantly, inhibition of BMP signaling had no effect on the ability of WNT / FGF to promote mesenchymal expansion and the construction of gut tube structures, and thus the ability to + result in the formation of foregut spheroids.

[0141] Figure 5 shows that BMP signaling is required in parallel with the activation of WNT and FGF to promote posterior fate. Figure 5A shows that the GSK3β inhibitor CHIR99021 (CHIR; 2 μM) induced the same posteriorizing effect as recombinant WNT3A, and this effect could be blocked by BMP inhibition. Figure 5B shows that CHIR induced intestinal morphogenesis and spheroid formation occurred as in the case of WNT3A. Figure 5C shows the immunofluorescent staining of monolayer cultures, from which a high CDX2 induction efficiency in the CHIR / FGF-treated endoderm and SOX2 induction in the noggin-treated and CHIR / FGF / noggin-treated endoderm were confirmed. Figure 5D shows the qPCR analysis of the BMP target genes MSX1 / 2, which indicates that there is no increase in BMP activity in response to Wnt / FGF, but the target genes are suppressed in response to noggin, demonstrating the presence of endogenous BMP signaling. Figure 5E shows that the addition of BMP2 (100 ng mL-1) did not substitute for or enhance the ability of Wnt / FGF to posteriorize the endoderm. These data indicate that the posteriorizing effect of Wnt / FGF is not mediated by upregulation of BMP signaling, but endogenous BMP activity is indeed required. Scale bar, 1 mm in Figure 5B; 100 μm in Figure 5C. Error bars represent standard deviation.

[0142] Spheroid morphogenesis is a robust process in both hESC lines and hiPSC lines (Figure 6A), with over 90% of spheroid cells expressing SOX2 (Figure 1C), indicating efficient specification to the foregut lineage. Thus, a new epistatic relationship among WNT, FGF, and BMP has been identified by the applicant, where all three pathways cooperate to promote mid- and hindgut fate, while WNT and FGF, separately from BMP, function to drive the construction of the gut tube structure from the endoderm and mesoderm.

[0143] Figures 2A - 2G show that gastric organoid differentiation is an efficient and cell line - independent process. Figure 2A, a table comparing spheroid formation and characteristics among two hESC lines (H1 and H9) and one iPSC line (72.3). Figure 2B, immunofluorescent staining of day 34 hGO derived from H1 and iPSC 72.3 cell lines. Organoids derived from iPSC exhibit the same morphological and molecular characteristics as those from hESC. Figure 2C, quantification of organ epithelial cell types in day 34 hGO. More than 90% of the epithelium is the antrum (indicated by PDX1 expression and lack of PTF1A expression), while the expression of markers related to other organs derived from the endoderm, including CDX2 (intestine), albumin (liver), and p63 (squamous epithelium), is less than 5%. Figures 2D - G, characterization of the induced pluripotent stem cell line iPSC 72.3. Figure 2D, iPSC 72.3 exhibits normal morphological characteristics of pluripotent stem cell colonies when compared to the H1 hESC line, and Figure 2E, has a normal 46;XY karyotype. Figure 2F, iPSC 72.3 expresses the pluripotency markers OCT3 / 4 and NANOG, and Figure 2G, demonstrates pluripotency by differentiation into endoderm, mesoderm, and ectoderm lineages in an in vivo teratoma assay. Scale bar, 100 μm. Error bars represent standard deviation.

[0144] In vivo, both the fundus and antrum domains of the stomach, in addition to the pancreas, liver, and duodenum, express Sox2 + arise from the posterior segment of the foregut endoderm. SOX2 + To direct foregut spheroids towards a gastric fate, the Applicant sought to identify signaling pathways that promote posterior foregut fate. Considering its role in the development of organs derived from the posterior foregut 15~17 , the Applicant focused on retinoic acid (RA) signaling. In vivo, the posterior foregut is characterized by the expression of Hnf1β (Figure 1D). The Applicant found that exposure to RA for 24 hours on the last day (days 5 - 6) of the patterning / spheroid - forming stage (FGF4 / WNT3A / noggin) results in robust activation of posterior foregut markers and SOX2 / HNF1β +It was determined that the formation of posterior foregut spheroids was brought about (Figs. 1E and 7). Thus, precise temporal and combinatorial manipulation of the RA, WNT, FGF, and BMP signaling pathways enables the creation of three-dimensional posterior foregut spheroids.

[0145] Figs. 7A-7D show that retinoic acid posteriorizes the foregut endoderm. Fig. 7A shows a schematic illustration of the foregut patterning experiment. It shows that DE cultures were treated with Wnt (CHIR) / FGF / noggin for 3 days to create Sox2-positive foregut spheroids, and RA was added for 24 hours on the third day of patterning. Fig. 7B shows a bright-field image showing that the number of spheroids generated from foregut monolayer cultures is increased by RA. Fig. 7C shows an enlarged weak image of Fig. 1D, an immunofluorescence image of a 14-somite stage embryo in which the Hnf1β protein is localized to the posterior part of the foregut. The region of the embryo enclosed by the frame is shown in Fig. 1D. Fig. 7D shows a qPCR analysis of gene expression in foregut spheroids treated with RA. The posterior foregut markers HNF1β and HNF6 are robustly induced by exposure to RA for 24 hours. * , p < 0.05. Scale bar, 1 mm in Fig. 7B; 100 μm in Fig. 7C. Error bars represent standard deviation.

[0146] The molecular mechanisms that direct the posterior foregut into individual organ systems are mostly unelucidated. In the early stages of development, prospective organ domains are characterized by distinct gene expression patterns: Sox2 + for the gastric fundus, Sox2 / Pdx1 + for the antrum, Pdx1 / Ptf1α + for the pancreas, and Pdx1 / Cdx2 + for the duodenum (Fig. 2B). The applicant used these molecular markers to identify the signaling pathways that direct posterior foregut spheroid cultures into the gastric lineage. After transferring the spheroids to three-dimensional culture conditions and further treating them with RA for 72 hours (days 6-9), a more than 100-fold increase in PDX1 mRNA levels occurred while maintaining high SOX2 expression (Fig. 2C). Importantly, the expression of the pancreatic-specific marker PTF1α was not induced, so as observed in other studies9 , RA treatment did not promote pancreatic fate. These data demonstrate that the combination of RA signaling and three-dimensional growth efficiently directs posterior foregut spheroids to the SOX2 / PDX1 + epithelium, which is an indicator of the early vestibular fate.

[0147] Figure 2 generally shows the specification and growth of human vestibular stomach organoids. Error bars represent standard deviation. Figure 2A shows a schematic of the in vitro culture system used to direct differentiation from hPSCs to three-dimensional stomach organoids, and Figure 2B shows the definitive markers of developing posterior foregut organs by whole-mount immunofluorescence staining of mouse E10.5 embryos using Sox2, Pdx1, and Cdx2. Co-expression of Sox2 and Pdx1 is unique to the distal part of the gastric epithelium, the prospective vestibulum (a), Sox2 expression characterizes the gastric fundus (f), expression of Pdx1 (and Ptf1a) characterizes the dorsal pancreas (dp) and ventral pancreas (vp), and co-expression of Pdx1 / Cdx2 characterizes the duodenum (d). Figure 2C shows that posterior foregut spheroids cultured for 3 days in the presence of RA (2 μM) in a three-dimensional matrix co-expressed high levels of PDX1 and SOX2, similar to the developing vestibulum, and did not express the pancreatic marker PTF1α ( * , p < 0.05). Figure 2D shows stereomicrographs revealing the morphological changes during the growth from posterior foregut spheroids to gastric organoids. By 4 weeks, the epithelium of hGO presented a complex glandular structure (scale bar, 500 μm). Figure 2E shows a comparison of the developing mouse vestibulum at E14.5 and E18.5 and equivalent stages of hGO development. Sox2 and Pdx1 are co-expressed in the initial pseudostratified epithelium in both mouse vestibulum and hGO. At later stages, Sox2 is downregulated as the epithelium changes to a more mature glandular structure. Pdx1 is maintained in the vestibulum throughout adulthood in vivo and at all stages examined in hGO (scale bar in Figure 2E is 100 μm).

[0148] The applicant herein is SOX2 / PDX1 +Using spheroids, we identified pathways that promote the growth and morphogenesis of the initial gastric epithelium, and found that high concentrations of EGF (100 ng mL -1 ) were sufficient to promote robust growth of human gastric antral organoids (hGO). Over 3 - 4 weeks, spheroids less than 100 μm in diameter grew into organoids 2 - 4 mm in diameter. At a later stage of culture (around day 27), the hGO epithelium underwent a series of morphogenetic changes reminiscent of the late stages of fetal gastric development, during which a simple flat multi - layer epithelium transitioned into a sophisticated and complex glandular epithelium (Figure 2D). The initial growth of foregut spheroids is EGF - dependent (data not shown); furthermore, epithelial expansion and glandular morphogenesis do not occur when EGF is removed from the medium on day 27 (Figure 8). These results support the previously published findings indicating the important role of EGF in promoting proper growth of the gastric mucosa 19、20 .

[0149] Figure 8 shows that EGF is required for glandular morphogenesis in gastric organoids. Bright - field images and immunostaining demonstrate that EGF is required for epithelial morphogenesis and gland formation in the late stage of hGO differentiation. On day 27, when EGF is removed from the growth medium before glandular morphogenesis, the hGO epithelium maintains a simple cuboid - like structure and is unable to form glands. Scale bar, 100 μm

[0150] By comparing hGO growth to the development of the fetal mouse stomach, it became clear that hGO development is surprisingly similar to in vivo gastric organogenesis. Initially (E12 - 14 in mice and day 13 hGO), both have a multi - layer epithelium containing mitotic cells concentrated towards the luminal surface (Figures 9 and 10), and the interphase nuclear migration process is shown 21 . The initial hGO is properly polarized and contains secondary lumens that are roughly explained by the expression of the apical marker aPKC 22 (Figure 10).

[0151] Between E16.5 and early postnatal stages, the vestibulum transforms into a simple columnar epithelium presenting a highly structured mechanism consisting of glands and crypts (Figs. 2E and 9). Between 13 and 34 days in vitro, hGO epithelium undergoes a similar transition to form a tall columnar epithelium with glandular structures similar to those of the late fetal vestibulum (Fig. 2E). Analysis of the expression of the transcription factors Sox2, Pdx1, Gata4, and Klf5 revealed stereotypical spatiotemporal expression patterns associated with these morphogenetic processes both in vivo and in vitro (Fig. 9). Initially, all of these factors are co-expressed in the immature stratified epithelium. However, at later stages, Sox2 expression is downregulated as the epithelium forms the initial glands and crypts, while the expression of the other factors is maintained indefinitely. Based on these data, it is inferred that hGO at day 13 corresponds to a similar developmental stage as the E12–14 mouse vestibulum, while hGO at day 34 is closer to the late fetal to early postnatal vestibulum. Furthermore, it is concluded that hGO recapitulates normal embryonic development and that the molecular and morphogenetic processes occurring during vestibular development are conserved between rodents and humans.

[0152] Figure 9 shows a comparison of transcription factor expression during the development of the mouse vestibule and human gastric organoids. Four embryonic stages (E12.5, E14.5, E16.5, and E18.5) and one postnatal stage (P12) of in vivo vestibular development were analyzed for transcription factor expression: Sox2, Pdx1, Gata4, Klf5, and FoxF1. The same markers were analyzed at two stages of in vitro hGO development (day 13 and day 34), and it was revealed that organoid development is similar to that which occurs in vivo. At the early stage of vestibular development, the epithelial marker Sox2 is ubiquitously expressed, but at later stages, while other epithelial transcription factors, Pdx1, Gata4, and Klf5, exhibit persistent expression throughout development, Sox2 is downregulated. Both early and late hGOs contain FoxF1-positive mesenchymal cells surrounding the epithelium. Scale bar, 100 μm. Figure 10 shows that early human gastric organoids exhibit stereotypical structure and nuclear behavior. At day 13, hGOs contain a pseudostratified epithelium characterized by apical marker aPKC and basolateral marker E-cadherin, showing apical-basal polarity, similar to the E12.5 mouse vestibule. Furthermore, within the organoid epithelium, secondary lumens (white arrows) covered by the apical membrane are seen. Both the E12.5 mouse vestibule and day 7 hGOs are seen to undergo interkinetic nuclear migration, as indicated by the presence of mitotic nuclei pHH3 only in the apical portion of the cells. Scale bar, 50 μm.

[0153] The foregut spheroids contained mesenchymal components similar to those of the previously described mid- and hindgut spheroids 10 During differentiation into gastric organoids, the mesenchyme expands and expresses important transcription factors associated with vestibular mesenchymal development, including FOXF1 and BAPX1 (Figures 10 and 11). At later stages, the hGO mesenchyme generally expresses vimentin, a marker of immature gastric mesenchyme + submucosal fibroblasts and fewer ACTA2 +It consists of subepithelial myofibroblasts (Figure 11). hGO does not form a differentiated smooth muscle layer as occurs in vivo. Considering such robust epithelial morphogenesis in the absence of any exogenous factors except EGF, it seems likely that mesenchyme plays a role in epithelial development. Therefore, it is surprising that the epithelium does not appear to promote robust differentiation of mesenchyme. This suggests that other stimuli, probably mechanical stimuli, play a role in gastric mesenchymal differentiation.

[0154] Figure 11 shows mesenchymal differentiation in gastric organoids. Figure 11A shows a temporal expression analysis of the vestibular mesenchymal transcription factor BAPX1. Similar to its known embryonic expression pattern, BAPX1 is upregulated in the early stage of hGO differentiation and then downregulated in line with the expression of functional cell type markers. Figure 11B shows, by staining for mesenchymal cell type markers, that hGO at day 34 contains FOXF1 / vimentin-positive submucosal fibroblasts and a smaller number of vimentin / alpha-SM-actin (SMA)-expressing subepithelial fibroblasts. hGO lacks the robust smooth muscle layer shown by SMA / desmin-positive cells in the in vivo vestibule. Scale bar, 100 μm. Error bars represent standard deviation.

[0155] The main functional cell types found in the vestibule are mucous cells that secrete a protective mucus layer covering the gastric epithelium, and endocrine cells that secrete hormones to regulate gastrointestinal physiological functions and metabolic homeostasis. 24 . By day 34, hGO contains superficial mucous cells (MUC5AC / UEAI + ) that secrete mucus into the lumen and have the same tall columnar morphology as their in vivo counterparts. hGO also contains TFF2 / GSII + vestibular gland cells, indicating appropriate differentiation in the vestibular mucus system (Figure 3A). In addition, in hGO, a progenitor cell niche indicated by a basal-restricted proliferation zone and SOX9 expression develops (Figure 4A), but the epithelial proliferation index is variable and ranges from 1 to 10%. Therefore, in vitro hGO contains a physiological gastric epithelium containing both progenitor cell types and differentiated cell types.

[0156] Figure 4 shows that human gastric organoids exhibit an acute response to Helicobacter pylori (H. pylori) infection. Figure 4A shows that on day 28, hGO contained proliferating cells (characterized by Ki67) and SOX9+ progenitor cells limited to the base of the initial gland, similar to the late fetal and postnatal mouse vestibule. Figure 4B shows that the human-specific disease process of H. pylori infection was modeled using hGO. Bacteria were microinjected into the lumen of hGO, and the luminal bacteria were visualized by bright-field microscopy (black arrows) and immunofluorescence staining 24 hours after injection. Figure 4C shows the immunoprecipitation of the oncogene c-Met, demonstrating that H. pylori induced robust activation (tyrosine phosphorylation) of c-Met and that this was a CagA-dependent process. Furthermore, CagA directly interacts with c-Met in human gastric epithelial cells. Figure 4D shows that H. pylori infection caused a two-fold increase in the number of proliferating cells in the hGO epithelium, as measured by EdU incorporation, within 24 hours. * , p < 0.05. Scale bar, 100 μm in a; 25 μm in b. Error bars represent s.e.m.

[0157] Figure 3 demonstrates that human gastric organoids contain normal differentiated antral cell types and can be used for modeling human gastric development. Figure 3A demonstrates that hGOs contain all the major antral cell lineages. hGOs at day 34 have surface mucous cells (Muc5AC) and mucous gland cells (TFF2), and have lectin staining that distinguishes surface mucous UEAI from mucous gland cell GSII. hGOs also contain endocrine cells as characterized by chromogranin A (CHGA). Figure 3B is a schematic diagram of the various roles of EGF in the growth, morphogenesis, and cell type specification during the development of hGOs. High levels of EGF were required at the early stage of gland formation, however, it suppressed endocrine differentiation at the late stage of development; thus, the EGF concentration was decreased at day 30 to generate endocrine cells. Figure 3C shows that all major endocrine hormones are expressed in hGOs when the use of EGF is discontinued, including gastrin, ghrelin, and serotonin (5-HT). Figure 3D shows that high levels of EGF suppress NEUROG3 expression. A significant increase in NEUROG3 expression measured by qPCR at day 34 was brought about by decreasing the EGF concentration at day 30, indicating that EGF acts upstream of NEUROG3 in endocrine specification. * , p < 0.05. Figure 3E shows that NEUROG3 acts downstream of EGF to induce endocrine cell fate. Forced expression of NEUROG3 using a dox-inducible system was sufficient to counteract the endocrine-suppressive effect of high EGF (100 ng mL-1). hGOs were exposed to dox (1 μg mL-1) for 24 hours at day 30 and analyzed at day 34. Dox-treated organoids exhibited robust induction of ChrA-expressing endocrine cells. Scale bar, 100 μm. Error bars represent standard deviation.

[0158] Also, at day 34 hGOs contain chromogranin-A (CHGA), including four major endocrine cell types in the antrum that express gastrin, ghrelin, somatostatin, and serotonin + Endocrine cells are also abundant (Figure 3C and Figure 12). Interestingly, the inventors found that high levels of EGF suppress endocrine cell formation, and 100 ng ml-1 It was observed that there were less than one endocrine cell per organoid. In contrast, abundant endocrine cells were generated in hGOs cultured with low levels of EGF (10 ng / ml) until day 30 - 34 (Figure 13). Furthermore, high EGF also inhibited the expression of the endocrine - producing transcription factor NEUROG3 (Figure 3D). NEUROG3 has been widely studied in the pancreas and intestine -1 and is required for the formation of most of the gastric endocrine system 25~28 . These data suggest a novel inhibitory effect of EGFR signaling in gastric endocrine cell specification upstream of NEUROG3. To test this model, the applicant used a doxycycline - inducible hNEUROG3 - overexpressing hESC line and found that NEUROG3 expression was sufficient to overcome the endocrine - inhibitory effect of high EGF (100 ng / ml) 29、30 and robust formation of CHGA - positive endocrine cells was achieved (Figure 3E and Figure 13). From these findings, the inventors concluded that EGF inhibits the formation of endocrine progenitor cells by suppressing NEUROG3 and that NEUROG3 is sufficient for the specification of human gastric endocrine cells. -1 +

[0159] ​​Figure 12 shows the in vivo development of endocrine cells in the gastric vestibule. Endocrine cell differentiation in the vestibule becomes apparent initially at E18.5 but is more definitive in the postnatal period (shown as P12). Initially, all expected gastric endocrine subtypes are apparent, including gastrin, ghrelin, somatostatin, and serotonin (5-HT). Scale bar, 100 μm. Figure 13 shows that EGF signaling suppresses the NEUROG3-dependent gastric endocrine specification program. Figure 13A shows that hGO maintained at high EGF (100 ng mL-1) had few endocrine cells on day 34, as shown by staining for the pan-endocrine marker CHGA. When the EGF concentration was decreased on day 24 (10 ng mL-1), a more physiological number of endocrine cells was brought about in the gastric epithelium. Figure 13B shows the generation of hGO from an hESC line stably transfected with a dox-inducible NEUROG3 overexpression transgene to test whether EGF-mediated suppression of endocrine differentiation occurs upstream of NEUROG3. hGO was maintained at high EGF (100 ng mL-1), then treated with doxycycline (1 μg mL-1) for 24 hours on day 30 and then analyzed on day 34. Dox-treated hGO showed robust activation of the endocrine markers CHGA, gastrin, ghrelin, and somatostatin and contained CHGA-positive (Figure 3A), ghrelin-positive, and somatostatin-positive cells with an endocrine morphology. * , p < 0.05. Scale bar, 100 μm. Error bars represent standard deviation.

[0160] According to what clinical evidence shows, in Helicobacter pylori (H. pylori)-mediated diseases, dominant colonization of the vestibule plays an important role 31、32. Therefore, the applicant of the present application tested whether hGO can be used to model the pathophysiological response of the human stomach to the pathogen Helicobacter pylori (H. pylori). To mimic a normal host-pathogen interface, the inventors directly introduced H. pylori into the luminal surface of the epithelium by microinjecting it into the lumen of the organoids and measured epithelial signaling and proliferation (Figure 4). By immunofluorescence, bacteria tightly bound to the hGO epithelium were observed (Figure 4B). Within 24 hours, the applicant observed a significant epithelial response to H. pylori, including robust activation of the gastric cancer gene c-Met 33 and a two-fold increase in epithelial cell proliferation. The H. pylori virulence factor CagA plays a central role in the pathogenesis of the disease. Consistent with published studies 34 , the applicant demonstrated that CagA translocates into organoid epithelial cells and forms a complex with c-Met (Figure 4C). Furthermore, injection of a non-pathogenic H. pylori strain lacking CagA into hGO abrogated the epithelial response, confirming the importance of this factor in H. pylori-mediated human pathogenesis. Therefore, hGO, due to its pathophysiological response to H. pylori, represents an unprecedented model for elucidating events that cause H. pylori-mediated human gastric diseases.

[0161] Additional references 1. Wen, S. & Moss, S. F. Helicobacter pylori virulence factors in gastric carcinogenesis. Cancer Lett. 282, 1-8 (2009). 2. Yuan, Y., Padol, I. T. & Hunt, R. H. Peptic ulcer disease today. Nat Clin Pract Gastroenterol Hepatol 3, 80-89 (2006). 3. Parkin, D. M. The global health burden of infection-associated cancers in the year 2002. Int. J. Cancer 118, 3030 - 3044 (2006). 4. Peek, R. M. Helicobacter pylori infection and disease: from humans to animal models. Dis Model Mech 1, 50 - 55 (2008). 5. Barker, N. et al. Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6, 25 - 36 (2010). 6. Longmire, T. A. et al. Efficient Derivation of Purified Lungand Thyroid Progenitors from Embryonic Stem Cells. Stem Cell 10, 398 - 411 (2012). 7. Mou, H. et al. Generation of Multipotent Lung and Airway Progenitors from Mouse ESCs and Patient-Specific Cystic Fibrosis iPSCs. Stem Cell 10, 385 - 397 (2012). 8. Si-Tayeb, K. et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 51, 297 - 305 (2010). 9. D’Amour, K. A. et al. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 24, 1392-1401 (2006). 10. Spence, J. R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105-109 (2011). 11. D’Amour, K. A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol 23, 1534-1541 (2005). 12. McCracken, K. W., Howell, J. C., Spence, J. R. & Wells, J. M. Generating human intestinal tissue from pluripotent stem cells in vitro. Nature Protocols 6, 1920-1928 (2011). 13. Kumar, M., Jordan, N., Melton, D. & Grapin-Botton, A. Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate. Dev Biol 259, 109-122 (2003). 14. Tiso, N., Filippi, A., Pauls, S., Bortolussi, M. & Argenton, F. BMP signalling regulates anteroposterior endoderm patterning in zebrafish. Mech Dev 118, 29-37 (2002). 15. Wang, Z., Dolle, P., Cardoso, W. V. & Niederreither, K. Retinoic acid regulates morphogenesis and patterning of posterior foregut derivatives. Dev Biol 297, 433 - 445 (2006). 16. Martin, M. et al. Dorsal pancreas agenesis in retinoic acid - deficient Raldh2 mutant mice. Dev Biol 284, 399 - 411 (2005). 17. Molotkov, A., Molotkova, N. & Duester, G. Retinoic acid generated by Raldh2 in mesoderm is required for mouse dorsal endodermal pancreas development. Dev Dyn 232, 950 - 957 (2005). 18. Kawaguchi, Y. et al. The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors. Nat Genet 32, 128 - 134 (2002). 19. Johnson, L. R. & Guthrie, P. D. Stimulation of rat oxyntic gland mucosal growth by epidermal growth factor. Am. J. Physiol. 238, G45 - 9 (1980). 20. Majumdar, A. P. Postnatal undernutrition: effect of epidermal growth factor on growth and function of the gastrointestinal tract in rats. J. Pediatr. Gastroenterol. Nutr. 3, 618 - 625 (1984). 21. Spear, P. C. & Erickson, C. A. Interkinetic nuclear migration: A mysterious process in search of a function. Develop. Growth Differ. 54, 306 - 316 (2012). 22. Grosse, A. S. et al. Cell dynamics in fetal intestinal epithelium: implications for intestinal growth and morphogenesis. Development 138, 4423 - 4432 (2011). 23. Verzi, M. P. et al. Role of the homeodomain transcription factor Bapx1 in mouse distal stomach development. Gastroenterology 136, 1701 - 1710 (2009). 24. Mills, J. C. & Shivdasani, R. A. Gastric Epithelial Stem Cells. Gastroenterology 140, 412 - 424 (2011). 25. Gradwohl, G., Dierich, A., LeMeur, M. & Guillemot, F. neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc Natl Acad Sci USA 97, 1607 - 1611 (2000). 26. Jenny, M. et al. Neurogenin3 is differentially required for endocrine cell fate specification in the intestinal and gastric epithelium. EMBO J 21, 6338 - 6347 (2002). 27. Johansson, K. A. et al. Temporal control of neurogenin3 activity in pancreas progenitors reveals competence windows for the generation of different endocrine cell types. Dev Cell 12, 457 - 465 (2007). 28. Lopez-Diaz, L. et al. Intestinal Neurogenin 3 directs differentiation of a bipotential secretory progenitor to endocrine cell rather than goblet cell fate. Dev Biol 309, 298 - 305 (2007). 29. Schonhoff, S. E., Giel-Moloney, M. & Leiter, A. B. Neurogenin 3-expressing progenitor cells in the gastrointestinal tract differentiate into both endocrine and non-endocrine cell types. Dev Biol 270, 443 - 454 (2004). 30. Lee, C. S., Perreault, N., Brestelli, J. E. & Kaestner, K. H. Neurogenin 3 is essential for the proper specification of gastric enteroendocrine cells and the maintenance of gastric epithelial cell identity. Genes Dev 16, 1488 - 1497 (2002). 31. Olbe, L., Hamlet, A., Dalenback, J. & Fandriks, L. A mechanism by which Helicobacter pylori infection of the antrum contributes to the development of duodenal ulcer. Gastroenterology 110, 1386 - 1394 (2001). 32. Xia, H. H. et al. Antral - type mucosa in the gastric incisura, body, and fundus (antralization): a link between Helicobacter pylori infection and intestinal metaplasia? Am. J. Gastroenterol. 95, 114 - 121 (2000). 33. Churin, Y. et al. Helicobacter pylori CagA protein targets the c - Met receptor and enhances the motogenic response. J. Cell Biol. 161, 249 - 255 (2003). 34. Peek, R. M. et al. Helicobacter pylori cagA+ strains and dissociation of gastric epithelial cell proliferation from apoptosis. J. Natl. Cancer Inst. 89, 863 - 868 (1997). 35. Teo, A. K. K. et al. Activin and BMP4 Synergistically Promote Formation of Definitive Endoderm in Human Embryonic Stem Cells. Stem Cells 30, 631 - 642 (2012). 36. Meerbrey, K. L. et al. 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Claims

1. A human gastric organoid (hGO), a) Luminal and glandular epithelium; b) MUC5AC, which secretes mucus into the lumen ＋ Surface mucous cells, and TFF2 ＋ mucous gland cells; c) chromogranin A (CHGA) positive endocrine cells; A human gastric organoid comprising:

2. The human gastric organoid of claim 1, wherein the human gastric organoid comprises gastric antrum tissue and is further characterized by the expression of antrum-specific marker PDX1.

3. The human gastric organoid of claim 1, wherein the human gastric organoid comprises gastric fundus tissue and is further characterized by expression of gastric fundus-specific markers IRX3 and IRX5, and suppression of PDX1 on gastric antrum tissue.

4. The human gastric organoid of claim 1, wherein the human gastric organoid is derived from definitive endoderm.

5. The human gastric organoid of claim 4, wherein the definitive endoderm is derived from pluripotent stem cells.

6. The human gastric organoids are d) Mesenchyme, including FOXF1 / vimentin positive submucosal fibroblasts and vimentin / ALPHA-SM-actin (SMA) expressing subepithelial fibroblasts; and e) lack of a differentiated smooth muscle layer; The human gastric organoid of claim 1, further characterized by:

7. The human gastric organoid of claim 1, wherein the human gastric organoid has a diameter of 1 mm to 4 mm.

8. The human gastric organoid of claim 1, wherein the human gastric organoid comprises gastric glands and gastric pits.

9. The human gastric organoid of claim 1, wherein the human gastric organoid comprises a progenitor cell niche, as indicated by a proliferation zone confined to the fundus and SOX9 expression.

10. 2. The human gastric organoid of claim 1, further characterized by expression of gastrin, ghrelin, somatostatin, and serotonin (5-HT).

11. The human gastric organoid of claim 1, wherein the human gastric organoid comprises Helicobacter pylori in the lumen.

12. The human gastric organoid of claim 4, wherein the definitive endoderm is derived from an induced pluripotent stem cell.

13. The human gastric organoid of claim 11, wherein the human gastric organoid comprises c-Met phosphorylation.

14. The human gastric organoids are ＋ The human gastric organoid of claim 11, comprising epithelial cell proliferation.

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