Method for induced differentiation of human pluripotent stem cells into functional intestinal organs

Abstract

The invention provides a method for induced differentiation of human pluripotent stem cells into functional intestinal organoid, and belongs to the field of stem cell technology and organoid engineering. The core of the invention is to provide a set of culture medium formula system with definite components and clear stages, and the intestinal organs can be efficiently guided to differentiate to various functional cell lineages including epithelial cells, neuroendocrine cells and endothelial cells, so that mature organs highly simulating natural intestinal tracts in cell composition are formed. Through a systematized culture medium formula, a standardized operation process and a multi-stage time sequence induction strategy, the next-generation 3D intestinal organ with structural integrity, cell diversity and functional activity at the same time is successfully constructed. The invention provides an efficient, reliable, repeatable and easy-to-expand advanced in-vitro model for intestinal development, disease mechanism, drug screening, toxicity evaluation and regenerative medicine research, and has important scientific research and transformation application value.

Description

Technical Field

[0001] This invention relates to the field of stem cell technology, and more specifically to a method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids. Background Technology

[0002] The gut is a crucial organ for digestion, nutrient absorption, and maintaining immune homeostasis in the human body. Its complex structure, composed of crypts and villi, contains various functionally diverse cell types, such as intestinal epithelial cells, goblet cells, Paneth cells, and enteroendocrine cells. Traditional gut research models, such as two-dimensional (2D) cell line cultures and animal models, have significant limitations in simulating human intestinal physiology and pathology. 2D cell lines cannot reproduce the three-dimensional structure and cellular diversity of the gut, while animal models face bottlenecks such as species differences, low throughput, high cost, and ethical concerns.

[0003] In recent years, the emergence of three-dimensional (3D) intestinal organoid technology has brought revolutionary breakthroughs to intestinal research. Organoids are three-dimensional micro-organoids that are self-assembled and differentiated in vitro from adult stem cells or pluripotent stem cells, capable of highly mimicking the complex structure, cell type, and specific functions of the source tissue. In 2009, Clevers' team successfully cultured mouse intestinal Lgr5+ adult stem cells into intestinal organoids with crypt-village-like structures in a Matrigel matrix containing specific growth factors (such as EGF, Noggin, and R-spondin1). Since then, human intestinal organoid technology has also developed rapidly, becoming a powerful tool for studying intestinal development, disease mechanisms, host-microbe interactions, and evaluating drug toxicity and efficacy.

[0004] Despite the significant success of existing technologies, current mainstream 3D intestinal organoid culture systems still face numerous unresolved technical challenges, severely limiting their standardization and large-scale application. Existing methods (such as Canadian patent CA3062600A1) disclose a method for preparing intestinal organoids from pluripotent stem cells, including the following steps: differentiating pluripotent stem cells into endoderm-like cells; differentiating the obtained endoderm into intestinal stem cells; culturing the obtained intestinal stem cells to form spheroids; and forming the differentiated spheroids into intestinal organoids. These steps include culturing in the presence of MEK1 / 2 inhibitors, DNA methylation inhibitors, TGF-β receptor inhibitors, and γ-secretase inhibitors, in addition to epidermal growth factor, BMP inhibitors, and Wnt signaling activators. However, the organoids generated by this invention often exhibit significant heterogeneity in size, morphology, and cellular composition, lacking a unified standardized quality control system. Furthermore, conventional organoid models typically lack key microenvironmental components such as immune cells, vascular systems, neural innervation, and microbiota, making it impossible to fully simulate the complex physiological and pathological states in vivo. Due to the factors mentioned above, the translation of existing intestinal organoid technologies from laboratory research to clinical and industrial fields such as precision medicine and new drug development faces enormous challenges. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, a method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids and its induction culture medium are provided to solve the problem that the intestinal organoids produced by existing 3D intestinal organoid culture systems are only at the structural biomimicry level.

[0006] To achieve the above objectives, the first aspect of the present invention provides a method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids, characterized by comprising the following induction stages performed in sequence: (a) Endoderm induction phase: Human pluripotent stem cells were induced to differentiate into endoderm cells in a first induction medium containing activin, BMP4 signaling pathway activator and PI3K signaling pathway inhibitor; (b) Movi-foregut endoderm induction phase: The endoderm cells were induced to differentiate into movi-foregut endoderm cells in a second induction medium containing FGF signaling pathway activator and BMP signaling pathway activator; (c) Intestinal expansion and maturation stage: The hindgut endoderm cells were induced to form and expand into intestinal-like structures in a third induction medium containing multiple growth factors; (d) Intestinal epithelial differentiation and maturation stage: The intestinal tube-like structure was induced to differentiate into 3D intestinal organoids with crypt-village structure in a fourth induction medium containing Wnt signaling pathway agonists and BMP signaling pathway antagonists.

[0007] Furthermore, the activator in the first induction medium is selected from any one or more combinations of recombinant human activator A and animal-free recombinant human activator A.

[0008] Furthermore, the PI3K signaling pathway inhibitor is selected from any one of LY294002, GDC-0941, and Alpelisib.

[0009] Furthermore, the first induction medium also contains the ROCK inhibitor Y27632; furthermore, the FGF signaling pathway activator in the second induction medium is selected as FGF BASIC, and the BMP signaling pathway activator is selected as BMP4.

[0010] Furthermore, the second induction medium also contains non-essential amino acids, GlutaMax, and B27 additives.

[0011] Furthermore, the inducing factors in the third induction medium include HGF, EGF, and VEGF.

[0012] Furthermore, the third induction medium also contains an adenylate cyclase activator.

[0013] Furthermore, the third induction medium also contains non-essential amino acids, GlutaMax, B27 additive, N2 additive, and Oncostatin M, Dexamethasone, and Forskolin.

[0014] Furthermore, the Wnt signaling pathway activator in the fourth induction medium is selected from CHIR99021 or R-spondin-1, and the BMP4 signaling pathway antagonist is selected from Noggin or Chordin.

[0015] Furthermore, the fourth induction medium also includes N2 additive, B27 additive, GlutaMax, and EGF.

[0016] Furthermore, the first induction medium, the second induction medium, the third induction medium, and the fourth induction medium also include a basic synthetic cell culture medium, wherein the basic synthetic cell culture medium is selected from any one or more combinations of RPMI 1640 medium, DMEM / F12 medium, Advanced DMEM / F12 medium, and IMDM medium.

[0017] Furthermore, the first induction culture medium includes basic synthetic cell culture medium, 0.5-1X N2 additive, 0.5-1X B27 additive, 50-500 ng / ml recombinant human activator A, 20-100 ng / mL BMP4, 0.1-10 μM LY294002 and 1-10 μM Y27632.

[0018] Furthermore, the second induction medium comprises basal synthetic cell culture medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5-1X of B27 additive, 10-100 ng / ml of FGF BASIC and 10-50 ng / ml of BMP4.

[0019] Furthermore, the third induction medium comprises IMDM medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5-1X of B27 additive, 0.5-1X of N2 additive, 10-50 ng / ml of EGF, 10-50 ng / ml of HGF, 50-200 ng / ml of VEGF, 10-50 ng / ml of Oncostatin M, 0.05-0.5 μM of Dexamethasone, and 1-10 μM of Forskolin.

[0020] Furthermore, the fourth induction medium includes a basic synthetic cell culture medium, 0.5-1X N2 additive, 0.5-1X B27 additive, 1X GlutaMax, 50-200 ng / ml EGF, 100-400 ng / ml R-spondin-1, and 50-200 ng / ml Noggin.

[0021] Furthermore, the 3D intestinal organoids are dynamically cultured in a maintenance medium to obtain mature intestinal organoids, wherein the maintenance medium includes Advanced DMEM / F-12 medium and 1X GlutaMax.

[0022] A second aspect of the present invention provides an intestinal organoid, which is generated by differentiating human pluripotent stem cells into functional intestinal organoids using any of the methods described above.

[0023] A third aspect of the present invention provides a method for evaluating the pharmacokinetics or toxicity of a test substance using intestinal organoids as described above.

[0024] The fourth aspect of the present invention provides the use of the intestinal organoids described above in the preparation of medicaments or transplant formulations for treating intestinal injury, intestinal dysfunction or inflammatory bowel disease.

[0025] The beneficial effects of this invention are as follows: the method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids uses a simple culture medium with relatively easy preparation and readily available raw materials. This saves on culture costs while ensuring normal function, and the organoids can be stably preserved for a long period. This invention provides specific components of the induction culture medium for each stage, greatly reducing the uncertainty of formulation and reliance on operator experience. The raw materials for the induction culture medium are highly commercialized; all components are available from common suppliers, ensuring a stable supply chain and facilitating rapid technology promotion and large-scale production. The preparation methods for each stage of the culture medium are clearly defined, which is beneficial for experimental repeatability and batch consistency, making it suitable for high-throughput screening and standardized production. The induction culture medium used in this invention has a simplified composition, avoiding redundancy. Compared to existing complex culture medium systems, this invention, through optimized ratios, reduces the amount of expensive growth factors used while ensuring differentiation efficiency.

[0026] The culture medium formulation provided by the method of inducing human pluripotent stem cells to differentiate into functional intestinal organoids in this invention can enable intestinal organoids to differentiate into various cell types such as epithelial cells, neuroendocrine cells, and endothelial cells, which is a key indicator of successful differentiation and functional maturity of intestinal organoids.

[0027] This invention describes a method for inducing the differentiation of human pluripotent stem cells into functional intestinal organoids. The resulting intestinal organoids exhibit histological structures such as intestinal crypts and demonstrate peristalsis during the maintenance phase, moving beyond structural biomimicry to functional simulation. This significance extends far beyond simple morphological imitation. The intestinal organoids formed by this differentiation method possess the ability to express multiple drug-metabolizing enzymes, enabling in vitro evaluation of drug absorption, metabolism, toxicity, and drug-drug interactions. The intestinal organoids formed by this differentiation method can be used for pathological model construction, mechanistic studies, drug screening, and personalized medicine. Furthermore, the intestinal organoids formed by this differentiation method can serve as transplantation materials for intestinal tissue engineering and regenerative therapy, particularly suitable for research on the repair of refractory intestinal diseases such as inflammatory bowel disease.

[0028] This invention successfully constructed 3D intestinal organoids with structural integrity, cell diversity, and functional activity through a systematic culture medium formulation, a well-defined operational procedure, and a multi-stage induction strategy. Its technical solution combines standardization, economy, stability, and functional simulation, demonstrating strong translational potential not only for basic research but also for drug screening, toxicity evaluation, disease modeling, and regenerative medicine. This system provides an efficient, reliable, and scalable in vitro model platform for intestinal biology research and related applications.

[0029] This invention provides a method for inducing the differentiation of human pluripotent stem cells into functional intestinal organoids. Differentiation begins directly after the human pluripotent stem cells form embryoid bodies, eliminating the need for post-differentiation digestion. This method offers higher differentiation efficiency and success rates, enabling the efficient and stable differentiation of 3D intestinal organoids with structural and functional simulations from pluripotent stem cells. It also attempts to integrate microenvironmental elements such as vascularization and neuralization in the early stages of differentiation, potentially making it more suitable for constructing "next-generation organoids" for regenerative medicine or complex disease models. Furthermore, by simultaneously inhibiting the PI3K signaling pathway (e.g., using LY294002) while inducing endoderm differentiation using traditional activin A, the synchronicity and directionality of cell differentiation are significantly improved, reducing the residual pluripotent cells. This early "dual-signal synergistic regulation" lays a crucial foundation for high-quality differentiation in subsequent stages. Attached Figure Description

[0030] Other features, objects, and advantages of this application will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a photograph of the day100 intestinal organoid generated by the method of inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to an embodiment of the present invention.

[0031] Figure 2 This diagram illustrates the process of intestinal organoid differentiation induced by human iPSCs.

[0032] Figure 3 Optical photographs of different stages of intestinal organoid differentiation induced by human iPSCs.

[0033] Figure 4 This is an immunofluorescence image of the intestinal organoids differentiated in this embodiment.

[0034] Figure 5 HE-stained images of the intestinal organoids differentiated in this embodiment.

[0035] Figure 6 This is a comparison diagram of the morphology of intestinal organoids differentiated from Example 1 and Comparative Example 1. Detailed Implementation

[0036] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0037] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0038] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0039] The human-derived iPSCs (hiPSCs) used were purchased from iXCells Biotechnologies USA, Inc., catalog number: 30HU-002, batch number: 400279-2-hPSC-A26F (https: / / ixcellsbiotech.com / ). The ROCK kinase inhibitor Y-27632 was purchased from MCE, catalog number HY-10071; RPMI 1640 medium was purchased from Thermo-Gibco, catalog number C22400500BT; Non-essential amino acids (NEAA) were purchased from Thermo-Gibco, catalog number 11140050; The glutamine supplement GlutaMax was purchased from Thermo-Gibco, product number 35050-061. B27 supplement (insulin-free) was purchased from Thermo-Gibco, product number A3695201; FGF BASIC (basic fibroblast growth factor) was purchased from nearshore protein, catalog number GMP-C046; BMP4 (bone morphogenetic protein 4) was purchased from Nearshore Protein, catalog number CR93; IMDM medium was purchased from Thermo-Gibco, catalog number 21056023; N2 additive was purchased from Thermo-Gibco, product number A1370701; Epidermal growth factor (EGF) was purchased from Dongkang, product number Y00801; Hepatocyte growth factor (HGF) was purchased from MCE, catalog number HY-P700604; Vascular endothelial growth factor (VEGF) was purchased from Nearshore Protein, catalog number C083. Oncostatin M was purchased from Proteintech, catalog number 17870223; Dexamethasone was purchased from MCE, product number HY-14648; Forskolin was purchased from MCE, product number HY-15371; Advanced DMEM / F-12 medium was purchased from Thermo-Gibco, catalog number 12634-010; R-spondin-1 (R-chondrin 1) was purchased from Dongkang, catalog number Y03501; Noggin was purchased from nearshore protein, catalog number CB89; The PI3K kinase inhibitor LY294002 was purchased from MCE, catalog number HY-10108; Recombinant human activin A was purchased from nearshore protein, catalog number GMP-C687.

[0040] The first aspect of the present invention provides a method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids, comprising four sequential induction phases: (a) an endoderm induction phase, (b) a hindbrain-foregut endoderm induction phase, (c) an intestinal expansion and maturation phase, and (d) an intestinal epithelial differentiation and maturation phase. Specifically: Endoderm induction stage: Human pluripotent stem cells are induced to differentiate into endoderm cells in a first induction medium containing activin, a BMP4 signaling pathway activator, and a PI3K signaling pathway inhibitor. The activin in this first induction medium can be selected from any one or more combinations of recombinant human activin A and animal-free recombinant human activin A. The PI3K signaling pathway inhibitor can be selected from any one of LY294002, GDC-0941, and Alpelisib. In addition to the above core components, the first induction medium may also contain necessary basic synthetic cell culture media (such as RPMI 1640 medium), ROCK inhibitors (such as Y27632), N2 additives, and B27 additives.

[0041] The endoderm induction phase involves enhancing endoderm differentiation of stem cells under dynamic conditions in the primary induction medium. This phase can last 2–5 days, ideally 4 days. Stem cells are cultured at 5–10% CO2 and 30–37°C, with medium changes every two days. It is important to emphasize that this phase, by simultaneously inducing endoderm differentiation with traditional activin A and inhibiting the PI3K signaling pathway (e.g., using LY294002), significantly improves the synchronicity and directionality of cell differentiation, reducing the residual pluripotent cells. This early "dual-signal synergistic regulation" lays a crucial foundation for high-quality differentiation in subsequent stages.

[0042] The foregut endoderm induction phase: Endoderm cells are induced to differentiate into foregut endoderm cells in a second induction medium containing FGF signaling pathway activators and BMP signaling pathway activators. The FGF signaling pathway activator in the second induction medium can be FGF BASIC, and the BMP signaling pathway activator can be BMP4. In addition, the second induction medium may also contain essential components such as a basic synthetic cell culture medium (e.g., RPMI 1640 medium), non-essential amino acids, GlutaMax, and B27 supplements.

[0043] The hindgut endoderm induction phase involves transferring the primary differentiation products to a secondary induction medium for secondary differentiation under dynamic conditions, with the medium being changed periodically. This hindgut endoderm induction phase can last 4–6 days, ideally 5 days. The primary differentiation products are cultured at 5–10% CO2 and 30–37°C, with the medium changed every two days.

[0044] It is important to emphasize that FGF BASIC, or fibroblast growth factor 2, promotes cell proliferation and survival. It works synergistically with BMP4 to drive cell fate transformation towards hindgut mesoderm or intestinal progenitor cells. BMP4, or bone morphogenetic protein 4, participates in mesoderm and hindgut development, inducing mesoderm differentiation and laying the foundation for subsequent intestinal epithelial-mesenchymal interactions. At the signaling pathway regulation level, FGF BASIC, as a key mitogen and morphogenetic protein, strongly promotes the proliferation of endoderm-derived cells and maintains their stemness and differentiation potential by continuously activating downstream pathways such as MAPK. Precisely antagonistic to FGF BASIC is BMP4, a ligand in the bone morphogenetic protein signaling pathway, which, at specific spatiotemporal concentrations, can induce cell differentiation into specific lineages. The precise balance between the two in terms of time and concentration together constructs a "bidirectional regulatory switch" that drives cell fate determination: FGF BASIC signaling promotes cell population expansion and maintains pluripotency, while BMP4 signaling initiates and guides specific differentiation programs in this population, thereby mimicking the precise regulation of growth factor gradients and antagonistic effects during in vivo intestinal development.

[0045] Furthermore, RPMI 1640 medium, as a basic synthetic cell culture medium, provides the essential nutrients and pH buffer system required for cell proliferation and differentiation, laying a stable physicochemical environment for the development of intestinal organoids. The supplementation of non-essential amino acids and GlutaMax effectively reduces the metabolic burden on cells and stabilizes the supply of glutamine, ensuring efficient protein synthesis and energy metabolism during long-term culture. The B27 additive provides cells with key hormones, antioxidants, and lipids, comprehensively supporting cell survival, maintenance, and directed differentiation.

[0046] Therefore, FGF BASIC in the second induction medium promotes proliferation, while BMP4 induces mesodermal differentiation, synergistically laying the foundation for enteric development. The second induction medium provides optimized and sufficient nutrients, hormones, and antioxidants, minimizing apoptosis. Its B27 component is rich in antioxidants (such as vitamin E, vitamin C, glutathione, and superoxide dismutase). At a 1X concentration, these components neutralize excess reactive oxygen species produced by cellular metabolism, preventing oxidative damage.

[0047] Intestinal expansion and maturation stage: In a third induction medium containing multiple growth factors, the hindgut endoderm cells are induced to form and expand into intestinal-like structures; wherein, the third induction medium may also contain a basic synthetic cell culture medium (such as IMDM medium), adenylate cyclase activator (such as Forskolin), non-essential amino acids, GlutaMax, B27 additive, N2 additive, Oncostatin M, and Dexamethasone.

[0048] The intestinal expansion and maturation stage involves transferring secondary differentiation products to a third induction medium for tertiary differentiation, with the medium being changed periodically. This stage lasts from day 2 to 5, ideally day 2, and is used to construct a complex microenvironment to promote multi-lineage differentiation (epithelial, vascular, neural, etc.). In the third induction medium, HGF promotes morphogenesis; VEGF induces vascular endothelial differentiation; Dexamethasone stabilizes cell state; and Forskolin promotes neuroendocrine function. Secondary differentiation products are cultured at 5–10% CO2 and 30–37°C, with the medium changed every two days.

[0049] In the third induction medium, IMDM medium is a nutrient-rich basal medium suitable for high-density cell culture, supporting the growth and development of complex cell communities (such as the epithelial-mesenchymal complex). Non-essential amino acids are a mixture to supplement amino acids insufficiently synthesized by the cells themselves, supporting rapid metabolism and protein synthesis, and promoting differentiation. GlutaMax is a stable source of glutamine, providing energy and nitrogen, maintaining cell health, and preventing ammonia toxicity. B27 additive is a neural culture supplement containing antioxidants, hormones, and vitamins, providing key nutrients, reducing oxidative damage, and supporting cell survival and early intestinal progenitor cell development. N2 additive is a supplement for neural crest and epithelial cell culture, promoting intestinal epithelial cell polarization and early neuroendocrine / neuronal differentiation. EGF (epidermal growth factor) promotes epithelial cell proliferation and drives the expansion of intestinal epithelial progenitor cells. HGF (hepatocyte growth factor) promotes cell migration and morphogenesis, and may participate in the early construction of intestinal crypt-village structures and cell migration. VEGF (vascular endothelial growth factor) promotes angiogenesis and induces endothelial cell differentiation, providing initial vascular network support for organoids. Oncostatin M (tumor suppressor M) can induce organoid cells to transition from a proliferating, undifferentiated state to a state closer to mature, functional adult cells in vivo. Dexamethasone (glucocorticoid) has anti-inflammatory and differentiation-regulating effects, potentially stabilizing cell state, promoting functional maturation, and inhibiting nonspecific differentiation. Forskolin (adenylate cyclase activator) increases intracellular cAMP levels, promoting the differentiation of intestinal endocrine cells and the functional maturation of intestinal neurons, preparing for peristaltic function.

[0050] In summary, in this step, the IMDM medium provides the essential amino acids, vitamins, and inorganic salts necessary for cell growth, thus constructing a nutritional framework for the development of intestinal organoids. Non-essential amino acids and GlutaMax optimize cell metabolism by supplementing key nitrogen sources and stabilizing glutamine supply, supporting protein synthesis and energy balance during long-term culture. The B27 and N2 additives synergistically provide multiple supports, including hormones, trace elements, and neurotrophic factors, to maintain cell viability and promote directed differentiation.

[0051] At the level of growth factors and signal regulation, EGF continuously activates the MAPK and PI3K / AKT pathways, driving the proliferation and differentiation of intestinal epithelial cells; HGF enhances cell migration and morphogenesis by binding to c-MET receptors, promoting the formation and expansion of organoid structures; VEGF indirectly supports nutrient permeation and the establishment of the microenvironment within organoids by regulating vascular endothelial growth signals. Meanwhile, Oncostatin M, as an IL-6 family cytokine, regulates the expression of inflammation and regeneration-related genes, synergistically promoting epithelial barrier maturation; Dexamethasone, as a glucocorticoid, helps maintain the stability of the culture system by inhibiting inflammatory responses and regulating gene transcription; and Forskolin, as an adenylate cyclase activator, further strengthens the intracellular signaling network by increasing cAMP levels, thus better promoting organoid morphogenesis and functional differentiation.

[0052] Intestinal epithelial differentiation and maturation stages: In a fourth induction medium containing a Wnt signaling pathway agonist and a BMP signaling pathway antagonist, the intestinal tube-like structures are induced to differentiate into 3D intestinal organoids with crypt-village structures. The Wnt signaling pathway activator in the fourth induction medium can be selected from CHIR99021 or R-spondin-1, and the BMP4 signaling pathway antagonist can be selected from Noggin or Chordin. Furthermore, the fourth induction medium may also include essential components such as a basic synthetic cell culture medium (e.g., Advanced DMEM / F-12 medium), N2 supplement, B27 supplement, GlutaMax, and EGF.

[0053] During the intestinal epithelial differentiation and maturation stages, the three-dimensional structure of the intestinal epithelium matures, forming crypt-villi axes. In the fourth induction medium, R-spondin-1 enhances Wnt signaling, promoting crypt formation; Noggin inhibits the BMP signaling pathway, promoting villi differentiation; and EGF drives epithelial expansion. The intestinal epithelial differentiation and maturation stages involve transferring tertiary differentiation products to the fourth induction medium for quaternary differentiation, with periodic medium changes. The intestinal epithelial differentiation and maturation stages last for 20–50 days, ideally 30 days. Tertiary differentiation products are cultured at 5–10% CO2 and 30–37°C, with medium changes every two days.

[0054] In this step, R-spondin-1, as an activator of the Wnt signaling pathway, maintains cell stemness by strongly stabilizing Wnt signaling and continuously activating downstream stemness-related genes. EGF, by activating pathways such as MAPK and PI3K-AKT, jointly promotes intestinal epithelial cell differentiation. Noggin, a bone morphogenetic protein (BMP) antagonist, maintains intestinal stem cell stemness by inhibiting the BMP signaling pathway. The three work synergistically to promote the proliferation and differentiation of intestinal epithelial cells and intestinal stromal cells, causing the hindgut bulb to gradually expand into a hollow, spherical intestinal organoid.

[0055] Furthermore, as a preferred embodiment, each of the basic synthetic cell culture media in the first induction medium, second induction medium, third induction medium, and fourth induction medium can be selected from any one or more combinations of RPMI 1640 medium, DMEM / F12 medium, Advanced DMEM / F12 medium, and IMDM medium, or any culture media that achieves similar or the same effect should fall within the protection scope of the present invention, and is not limited to the culture media provided in the examples.

[0056] As a preferred embodiment of the culture medium combination, the first induction medium comprises RPMI 1640 medium, 0.5-1X N2 additive, 0.5-1X B27 additive, 50-500 ng / ml recombinant human activator A, 20-100 ng / ml BMP4, 0.1-10 μM LY294002, and 1-10 μM Y27632. The second induction medium comprises RPMI 1640 medium, 1X of non-essential amino acids, 1X GlutaMax, 0.5-1X B27 additive, 10-100 ng / ml FGF BASIC, and 10-50 ng / ml BMP4. The third induction medium includes IMDM medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5-1X of B27 additive, 0.5-1X of N2 additive, 10-50 ng / ml of EGF, 10-50 ng / ml of HGF, 50-200 ng / ml of VEGF, 10-50 ng / ml of Oncostatin M, 0.05-0.5 μM of Dexamethasone, and 1-10 μM of Forskolin. The fourth induction medium includes Advanced DMEM / F 12 medium, 0.5-1X of N2 additive, 0.5-1X of B27 additive, 1X of GlutaMax, 50-200 ng / ml of EGF, 100-400 ng / ml of R-spondin-1, and 50-200 ng / ml of Noggin.

[0057] Furthermore, the method of inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to the present invention also includes dynamically culturing the induced 3D intestinal organoids in a maintenance medium to obtain mature intestinal organoids. In a specific embodiment, the maintenance medium may include Advanced DMEM / F-12 medium and 1X GlutaMax. During the maintenance culture phase, the intestinal organoids are kept viable for a long time and maintain basic function, providing stable nutritional support and maintaining metabolic and structural integrity, but no signaling molecules specifically promoting higher functions are added. The quaternary differentiation products are dynamically cultured at 5-10% CO2, 70 rpm, and 30-37°C, with the medium changed every two days.

[0058] It is worth mentioning that, compared with the prior art, the induction culture media of this embodiment have the following beneficial effects, including but not limited to: In the first induction medium, RPMI 1640 medium serves as the basic synthetic cell culture medium, providing a nutrient and buffering system to maintain a stable cell growth environment. N2 supplementation acts as a supplement for neural crest and epithelial cell culture, promoting intestinal epithelial cell polarization and early neuroendocrine / neuronal differentiation. B27 supplementation, containing antioxidants, hormones, and vitamins, is a neural culture supplement providing key nutritional factors, reducing oxidative damage, and supporting cell survival and early intestinal progenitor cell development. Recombinant Human Activin A belongs to the TGF-β superfamily; it activates the intracellular SMAD2 / 3 signaling pathway, initiating the expression of a series of downstream genes. BMP4 plays a crucial and dual role in intestinal organoid differentiation, with its effects highly dependent on the specific stage and environment. In short, BMP4 primarily inhibits intestinal differentiation in the early differentiation stage, while playing a vital morphogenetic role in the maintenance and regional specialization of mature intestinal organoids. LY294002 is a widely used PI3K inhibitor that plays a significant role in regulating intestinal organoid differentiation by inhibiting the PI3K / AKT signaling pathway. The ROCK inhibitor Y27632 can effectively reduce apoptosis and promote cell adhesion and survival in the primary induction phase, and protect cells in the differentiation initiation phase, improving differentiation efficiency and cell cluster integrity.

[0059] In the second induction medium, RPMI 1640 basal medium, a basal synthetic cell culture medium, provides the essential nutrients and pH buffer system required for cell proliferation and differentiation, establishing a stable physicochemical environment for the development of intestinal organoids. The supplementation of non-essential amino acids and GlutaMax effectively reduces the metabolic burden on cells and stabilizes glutamine supply, ensuring efficient protein synthesis and energy metabolism during long-term culture. The B27 additive provides cells with key hormones, antioxidants, and lipids, comprehensively supporting cell survival, maintenance, and directed differentiation.

[0060] At the level of signaling pathway regulation, FGF BASIC, as a key mitogen and morphogenetic protein, strongly promotes the proliferation of endoderm-derived cells and maintains their stemness and differentiation potential by continuously activating downstream pathways such as MAPK. Precisely antagonistic to this is BMP4, a ligand for the bone morphogenetic protein signaling pathway, which, at specific spatiotemporal concentrations, can induce cell differentiation into specific lineages. The precise balance between the two in terms of time and concentration constructs a "bidirectional regulatory switch" driving cell fate determination. FGF BASIC signaling promotes cell population expansion and maintains pluripotency, while BMP4 signaling initiates and guides specific differentiation programs within this population, thus mimicking the precise regulation of growth factor gradients and antagonistic effects during in vivo intestinal development, laying the foundation for subsequent intestinal epithelial-mesenchymal interactions.

[0061] In the third induction medium, IMDM medium is a basic synthetic cell culture medium that provides the essential amino acids, vitamins and inorganic salts necessary for cell growth, thus constructing a nutritional framework for the development of intestinal organoids. Non-essential amino acids and GlutaMax optimize cell metabolic state by supplementing key nitrogen sources and stabilizing glutamine supply, supporting protein synthesis and energy balance in long-term culture. B27 and N2 additives synergistically provide multiple supports such as hormones, trace elements and neurotrophic factors, jointly maintaining cell activity and promoting directed differentiation.

[0062] At the level of growth factors and signal regulation, EGF continuously activates the MAPK and PI3K / AKT pathways, driving the proliferation and differentiation of intestinal epithelial cells; HGF enhances cell migration and morphogenesis by binding to c-MET receptors, promoting the formation and expansion of organoid structures; VEGF indirectly supports nutrient permeation and the establishment of the microenvironment within organoids by regulating vascular endothelial growth signals. Meanwhile, Oncostatin M, as an IL-6 family cytokine, regulates the expression of inflammation and regeneration-related genes, synergistically promoting epithelial barrier maturation; Dexamethasone, as a glucocorticoid, helps maintain the stability of the culture system by inhibiting inflammatory responses and regulating gene transcription; and Forskolin, as an adenylate cyclase activator, further strengthens the intracellular signaling network by increasing cAMP levels, thus better promoting organoid morphogenesis and functional differentiation.

[0063] In the fourth induction medium, R-spondin-1, as an activator of the Wnt signaling pathway, maintains cell stemness by strongly stabilizing Wnt signaling and continuously activating downstream stemness-related genes. EGF, through activation of pathways such as MAPK and PI3K-AKT, jointly promotes intestinal epithelial cell differentiation. Noggin, a bone morphogenetic protein (BMP) antagonist, maintains intestinal stem cell stemness by inhibiting the BMP signaling pathway. The three work synergistically to promote the proliferation and differentiation of intestinal epithelial cells and intestinal stromal cells, causing the hindgut bulb to gradually expand into a hollow, spherical intestinal organoid.

[0064] In maintenance media, Advanced DMEM / F-12 medium is an optimized basal medium, providing comprehensive nutrition and stable nutritional support for the long-term survival and functional maintenance of mature intestinal organoids. GlutaMax is a stable source of glutamine, continuously supporting energy metabolism and protein synthesis in various cells within the organoids, maintaining their structural and functional stability.

[0065] It should be further noted that, in this embodiment, before implementing the multi-stage induction differentiation stage, the method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to the present invention further includes S1, the embryoid body formation stage. Specifically, S1, the embryoid body formation stage, includes the self-organization of pluripotent stem cells into embryoid bodies. The specific operation steps are as follows: Pluripotent stem cell suspensions were seeded into 96-well low-absorption U-shaped plates, allowing them to self-assemble into embryoid bodies. The pluripotent stem cells were then seeded into stem cell maintenance culture medium for expansion culture. Theoretically, these pluripotent stem cells could be derived from iPSCs (Induced Pluripotent Stem Cells) or ESCs (Embryonic Stem Cells), both of which can differentiate; however, this embodiment uses iPSCs. The pluripotent stem cells used in this embodiment are human induced pluripotent stem cells (hiPSCs). The intestinal organoid model used in this embodiment was generated from the differentiation of human induced pluripotent stem cells (hiPSCs), which has the following core advantages compared to intestinal organoids derived from traditional primary cells: The intestinal organoid model used in this invention is generated from the differentiation of human induced pluripotent stem cells (hiPSCs), and compared with traditional primary cell-derived intestinal organoids, it has the following core advantages: 1. Stable source, can be expanded indefinitely.

[0066] hiPSCs can be stably cultured and passaged in vitro for a long time, providing a continuous and uniform cell source, overcoming technical bottlenecks such as limited primary intestinal tissue acquisition, large individual differences, and limited expansion capacity.

[0067] 2. Avoid ethical restrictions and operate more flexibly.

[0068] It does not rely on human intestinal biopsy tissue or surgical specimens, avoiding related ethical reviews and patient sample acquisition difficulties, and is more convenient for standardized operation and large-scale construction.

[0069] 3. Gene editing is convenient and the model is highly customizable.

[0070] hiPSCs are easy to use for gene editing (such as CRISPR / Cas9 technology), and can be used to introduce specific pathogenic mutations, reporter genes, or perform gene repair. They are convenient for building disease models with clear genetic backgrounds, gene function studies, or drug screening platforms.

[0071] 4. The developmental process can be reproduced, resulting in a more complete study.

[0072] It can simulate the entire process of intestinal differentiation from early endoderm to mature intestinal epithelium, which is beneficial for studying intestinal development, cell fate determination and early disease mechanisms, while primary organoids mainly reflect the state of the adult intestine at the time of sampling.

[0073] 5. Good batch-to-batch consistency, making the data more reliable.

[0074] By standardizing the induction process, intestinal organoids with the same genetic background and consistent differentiation status can be obtained, significantly reducing batch-to-batch variation and improving experimental reproducibility and data comparability.

[0075] 6. Expand personalized medical applications.

[0076] hiPSCs can be obtained by reprogramming patient somatic cells (such as skin fibroblasts or blood cells), and then intestinal organoids carrying patient-specific genetic information can be constructed for personalized disease modeling, drug sensitivity testing and precision medicine research.

[0077] Specifically, in this step, the embryoid formation stage lasts for 1-3 days. The embryoid formation medium can include mTesR Plus and Y-27632. Furthermore, the embryoids are statically cultured at 5-10% CO2 and 30-37°C, with the medium changed daily.

[0078] A second aspect of the present invention also provides an intestinal organoid, which is generated by the aforementioned method of inducing human pluripotent stem cells to differentiate into functional intestinal organoids.

[0079] The method of inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to this invention uses a simple culture medium that is relatively easy to prepare and the required raw materials are readily available. Furthermore, the culture medium formulation provided by this method allows the intestinal organoids to differentiate into various cell types, including epithelial cells, enteroendocrine cells, and endothelial cells, which are key indicators of successful differentiation and functional maturity of the intestinal organoids. In addition, the intestinal organoids differentiated by this method exhibit histological structures such as intestinal crypts and can show peristalsis during the maintenance phase, moving from structural biomimicry to functional simulation, with significance far exceeding simple morphological imitation.

[0080] To further illustrate the method of inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to the present invention, the following examples and comparative examples are used. Example

[0081] This embodiment provides a method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids, including the following steps: Step 1: Pluripotent stem cells self-organize to form embryoid bodies.

[0082] Frozen healthy human blood-derived pluripotent stem cells were removed from the -80℃ freezer and rapidly thawed in a 37℃ water bath. The cells were then gently mixed with 5 ml of DMEM / F12 medium, centrifuged at 4℃, 300 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 1 ml of mTesR Plus and 10 μM Y-27632 solution and counted. Cells were then counted at 3 × 10⁶ cells per well in a 6-well plate. 5 Cells were seeded into plates. After 2 hours, the medium containing Y-27632 was removed, and 3 ml of fresh mTeSR Plus was added. The medium was changed daily. When the cells reached 80-90% confluence in the 6-well plates, the original medium was discarded, 2 mL of TryPLE digestion solution was added, and the cells were incubated at 37°C for 5 minutes to dissociate them. The cell suspension was then collected and centrifuged at 300 rpm for 5 minutes at 4°C. After centrifugation, the supernatant was discarded, and the cells were resuspended in 10 mL of mTeSR Plus medium containing 10 μM Y-27632. The cell suspension was seeded at 200 μL per well into 96-well low-absorption U-shaped plates and incubated statically in a cell culture incubator to promote embryoid formation.

[0083] Step 2: Multi-stage induction and differentiation of intestinal organoids.

[0084] 1. First differentiation induction stage (i.e., endoderm induction stage): Transfer all embryoids to a low-adhesion six-well plate, discard mTeSR Plus medium, add the first induction medium, and carry out the first differentiation induction culture. Culture for 4 days under the conditions of 5~10% CO2, 30~37℃, and 70~100rpm, and change the medium every two days.

[0085] The specific components of the first induction medium include RPMI 1640 basal medium, 1X of N2 additive, 1X of B27 additive, 300 ng / mL of Recombinant Human Activin A, 30 ng / mL of BMP4, 1 μM of LY294002 and 10 μM of Y27632.

[0086] 2. Secondary Differentiation Induction Stage (i.e., Post-Foregut Endoderm Induction Stage): Remove all culture medium from the six-well plate, add secondary induction medium, and perform secondary differentiation induction culture. Culture for 5 days at 5-10% CO2, 30-37℃, and 70-100 rpm. The secondary induction medium needs to be changed every two days to obtain secondary differentiation products.

[0087] The specific components of the second induction medium include RPMI 1640 medium, 1X of non-essential amino acids, 1X of GlutaMax, 1X of B27 additive, 60 ng / ml of FGF BASIC, and 30 ng / ml of BMP4.

[0088] 3. Third induction differentiation stage (i.e., intestinal expansion and maturation stage): Remove all the culture medium from the six-well plate, add the third induction medium for third differentiation induction culture, and incubate statically for 2 days at 5-10% CO2, 30-37℃, and 70-100rpm. Replace the third induction medium every two days to obtain the tertiary differentiation product.

[0089] The specific components of the third induction medium include IMDM medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5X of B27 additive, 0.5X of N2 additive, 30 ng / ml of EGF, 30 ng / ml of HGF, 150 ng / ml of VEGF, 30 ng / ml of Oncostatin M, 0.2 μM of Dexamethasone, and 5 μM of Forskolin.

[0090] 4. Fourth differentiation stage (i.e., intestinal epithelial differentiation and maturation stage): Remove all the culture medium from the six-well plate, add the fourth induction medium for fourth differentiation induction culture, and dynamically culture for 40 days under the conditions of 5-10% CO2, 30-37℃, and 70-100rpm. Replace the third induction medium every two days to obtain the fourth differentiation product.

[0091] The specific components of the fourth induction medium include Advanced DMEM / F-12 medium, 0.5X N2 additive, 0.5X B27 additive, 1X GlutaMax, 150 ng / ml EGF, 300 ng / ml R-spondin-1, and 150 ng / ml Noggin.

[0092] 5. Maintenance phase: The products of the fourth differentiation stage are dynamically cultured in maintenance medium to obtain intestinal organoids.

[0093] The maintenance medium consisted of Advanced DMEM / F-12 medium and 1X GlutaMax. (Combined) Figure 1 , Figure 1 This is a photograph of the intestinal organoids that were finally differentiated in this embodiment. Figure 1 The spherical object indicated by the white arrow is an intestinal organoid cultured for 100 days.

[0094] Figure 2 This diagram illustrates the process of intestinal organoid differentiation induced by human iPSCs.

[0095] Figure 3 Optical photographs of different stages of intestinal organoid differentiation induced by human iPSCs.

[0096] Figure 4 This is an immunofluorescence image of the intestinal organoids differentiated in this embodiment. MAP2 is a marker of mature neurons; α-SMA is a marker of smooth muscle cells; DAPI is a fluorescent dye for labeling nuclear DNA; EpCAM is a marker of epithelial tissue; and CHGA (Chromogranin A) is a marker of neuroendocrine cells. Figure 4 This indicates that intestinal organoids contain multiple cell types, including epithelial cells and endocrine cells.

[0097] Figure 5 HE-stained images of the intestinal organoids differentiated in this embodiment. Figure 5 This indicates that the intestinal organoids have differentiated into distinct muscle, epithelial, and interstitial layers.

[0098] The present invention provides a method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids through direct 3D differentiation. Cells are deposited, aggregated, and self-organized into embryoid bodies (EBs) in a 96-well low-adhesion U-shaped plate, and then transferred to a 6-well low-adhesion plate containing a first induction medium for culture. No subsequent digestion step is required. The differentiation process does not require multiple transfers (direct 6-well plate differentiation).

[0099] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that LY294002 is not added to the first induction differentiation culture medium in the first induction differentiation stage of the multi-stage induction differentiation of intestinal organoids in step two.

[0100] Combination Figure 6 As shown, the comparative example did not have LY294002 added during the first induction differentiation stage. The resulting intestinal organoids were smaller in size, irregular in shape, and unevenly distributed with epithelial-like structures. No large cavitary structures appeared during the maintenance stage.

[0101] A third aspect of the invention also relates to the use of an intestinal organoid obtained by the differentiation method of the invention.

[0102] As a primary application, various analyses can be provided. The intestinal organoids of the present invention can be used in intestinal model systems and are useful for evaluating pharmacokinetics (absorption, metabolism, etc.) and toxicity in the intestine; that is, the intestinal organoids of the present invention can be utilized in the evaluation of the pharmacokinetics and toxicity of compounds.

[0103] Specifically, the intestinal organoids of the present invention are used to test the metabolism, absorption, membrane permeability, drug interactions, induction of drug-metabolizing enzymes, induction of drug transport proteins, and toxicity of test substances. That is, as one application of intestinal organoids, the present invention provides a method for evaluating the metabolism, absorption, membrane permeability, drug interactions, induction of drug-metabolizing enzymes, induction of drug transport proteins, and toxicity of test substances. This method includes the following steps: Step I: The process of contacting the test substance with the intestinal organoids obtained by the differentiation method of the present invention; Step II: Determine / evaluate the metabolism, absorption, membrane permeability, drug interactions, induction of drug-metabolizing enzymes or drug transporters, or toxicity of the test substance.

[0104] The "contact" in step I is typically performed by adding the test substance to the culture medium. There is no particular limitation on the timing of the addition of the test substance. Therefore, it can be added at some point after culturing has begun in a medium that does not contain the test substance, or it can be started in a medium that already contains the test substance.

[0105] The analyte can be an organic or inorganic compound of various molecular sizes. Examples of organic compounds include nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (glycerophosphates, sphingolipids, glycosylglycerols, cerebrosides, etc.), prostaglandins, isoprene morphologies, terpenes, steroids, polyphenols, catechins, and vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.). Existing or candidate ingredients from pharmaceuticals, nutritional foods, food additives, pesticides, and cosmetics are also preferred analytes. Plant extracts, cell extracts, and culture supernatants can also be used as analytes. By simultaneously adding two or more analytes, interactions and synergistic effects between them can be investigated. The analyte can be of natural origin or synthesized. In the latter case, for example, combinatorial synthesis methods can be used to construct an effective analytical system.

[0106] The contact time of the substance being tested can be set arbitrarily. The contact time can be, for example, from 10 minutes to 3 days, preferably from 1 hour to 1 day. Contact can also be performed in multiple sessions.

[0107] Following step I, the metabolism, absorption, membrane permeability, drug interactions, induction by drug-metabolizing enzymes, induction by drug transporters, or toxicity of the analyte are measured / evaluated (step II). This measurement / evaluation can be performed immediately after step I, i.e., immediately after exposure to the analyte, without a substantial time interval, or it can be performed after a certain period (e.g., 10 minutes to 5 hours). Metabolism can be measured, for example, by detecting metabolites. In this case, the culture medium following step I is typically used as a sample for qualitative or quantitative determination of the desired metabolites. The appropriate measurement method can be selected based on the metabolites; for example, mass spectrometry, liquid chromatography, or immunological methods (e.g., fluorescence immunoassay (FIA) or enzyme immunoassay (EIA)) can be used.

[0108] When metabolites of the analyte are detected, it is determined or evaluated that "the analyte has been metabolized." Furthermore, the amount of metabolites can be used to evaluate the metabolic rate of the analyte. The metabolic efficiency of the analyte can be calculated based on the detection results of the metabolites and the amount of analyte used (typically, the amount added to the culture medium).

[0109] The expression of drug-metabolizing enzymes (cytochrome P450 (especially human CYP3A4, cynomolgus monkey CYP3A8), uridine diphosphate-glucuronide transferases (especially UGT1A8, UGT1A10), sulfatases (especially SULT1A3, etc.)) in intestinal organoids can also be used as indicators to determine the metabolism of the test substance. The expression of drug-metabolizing enzymes can be evaluated at the mRNA or protein level. For example, an increase in the mRNA level of a drug-metabolizing enzyme can be considered an increase in gene expression. Similarly, an increase in the activity of a drug-metabolizing enzyme can be considered a metabolism of the test substance. Similar to using metabolites as indicators, quantitative determination / evaluation can be based on the expression level of drug-metabolizing enzymes.

[0110] To evaluate the absorption of a analyte, for example, by determining the residual amount of the analyte in the culture medium, the culture medium after step I is typically sampled to quantify the analyte. The assay method can be chosen appropriately based on the analyte. For example, mass spectrometry, liquid chromatography, and immunological methods (such as fluorescence immunoassay (FIA) and enzyme immunoassay (EIA)) can be used. Typically, when a decrease in the concentration of the analyte in the culture medium is confirmed, it is considered that "the analyte has been absorbed." Furthermore, the degree of reduction can be used to determine / evaluate the amount or efficiency of absorption of the analyte. It should be noted that absorption can also be evaluated by measuring the amount of the analyte ingested into the cells.

[0111] It should be noted that metabolic assays / evaluations and absorption assays / evaluations can be performed simultaneously or in parallel.

[0112] As a second use of the intestinal organoids obtained by the differentiation method of the present invention, transplant materials comprising intestinal organoids can be provided. The transplant materials of the present invention can be applied to the treatment of various intestinal diseases (e.g., refractory inflammatory bowel disease). In particular, they are envisioned for use as materials for the regeneration / reconstruction of damaged (including dysfunctional) intestinal tissue, i.e., they are expected to contribute to regenerative medicine. The transplant materials of the present invention can be used directly or after processing such as embedding in matrix gel or collagen gel as transplant materials. Furthermore, their use is also envisioned as screening candidate compounds for therapeutic drugs in various intestinal disease pathological models and elucidating pathological mechanisms.

[0113] The transplant material of this invention can also be used to construct in vivo experimental systems. For example, transplant material containing intestinal organoids made from human pluripotent stem cells can be transplanted into laboratory animals such as mice, rats, guinea pigs, hamsters, pigs, cynomolgus monkeys, macaques, and chimpanzees to create humanized animals (human intestinal models). Such humanized animals are particularly useful for pharmacokinetic and toxicity tests, and are expected to contribute to research on the effects of first-pass effects on oral medications, drug-induced enteritis, and other related topics.

[0114] Intestinal organoids made from iPSC cells from patients with intestinal diseases can be used not only as intestinal pathology models for drug evaluation systems, but also for various experiments in studies aimed at elucidating the mechanisms of pathogenesis, pathogenesis, and / or progression of intestinal diseases.

[0115] The above description is merely a preferred embodiment of this application and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the inventive concept. For example, technical solutions formed by substituting the above features with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. A method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids, characterized in that, The following induction phases are performed in sequence: (a) Endoderm induction phase: Human pluripotent stem cells were induced to differentiate into endoderm cells in a first induction medium containing activin, BMP4 signaling pathway activator and PI3K signaling pathway inhibitor; (b) Movi-foregut endoderm induction phase: The endoderm cells were induced to differentiate into movi-foregut endoderm cells in a second induction medium containing FGF signaling pathway activator and BMP signaling pathway activator; (c) Intestinal expansion and maturation stage: The hindgut endoderm cells were induced to form and expand into intestinal-like structures in a third induction medium containing multiple growth factors; (d) Intestinal epithelial differentiation and maturation stage: The intestinal tube-like structure was induced to differentiate into 3D intestinal organoids with crypt-village structure in a fourth induction medium containing Wnt signaling pathway agonists and BMP signaling pathway antagonists.

2. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The activator in the first induction medium is selected from any one or more combinations of recombinant human activator A and animal-free recombinant human activator A.

3. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The PI3K signaling pathway inhibitor is selected from any one of LY294002, GDC-0941, and Alpelisib.

4. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The first induction medium also contains the ROCK inhibitor Y27632, N2 additive, and B27 additive.

5. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The FGF signaling pathway activator in the second induction medium is FGF BASIC, and the BMP signaling pathway activator is BMP4.

6. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The second induction medium also contains non-essential amino acids, GlutaMax, and B27 additives.

7. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The inducing factors in the third induction medium include HGF, EGF, and VEGF.

8. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The third induction medium also contains an adenylate cyclase activator.

9. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The third induction medium also contains non-essential amino acids, GlutaMax, B27 additive, N2 additive, Oncostatin M, Dexamethasone, and Forskolin.

10. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The Wnt signaling pathway activator in the fourth induction medium is selected from CHIR99021 or R-spondin-1, and the BMP4 signaling pathway antagonist is selected from Noggin or Chordin.

11. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The fourth induction medium also includes N2 additive, B27 additive, GlutaMax and EGF.

12. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The first induction medium, the second induction medium, the third induction medium, and the fourth induction medium further comprise a basic synthetic cell culture medium, wherein the basic synthetic cell culture medium is selected from any one or more combinations of RPMI 1640 medium, DMEM / F12 medium, Advanced DMEM / F12 medium, and IMDM medium.

13. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The first induction culture medium includes basic synthetic cell culture medium, 0.5-1X N2 additive, 0.5-1X B27 additive, 50-500 ng / ml recombinant human activator A, 20-100 ng / mL BMP4, 0.1-10 μM LY294002 and 1-10 μM Y27632.

14. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The second induction medium includes basal synthetic cell culture medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5-1X of B27 additive, 10-100 ng / ml of FGF BASIC and 10-50 ng / ml of BMP4.

15. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The third induction medium includes IMDM medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5-1X of B27 additive, 0.5-1X of N2 additive, 10-50 ng / ml of EGF, 10-50 ng / ml of HGF, 50-200 ng / ml of VEGF, 10-50 ng / ml of Oncostatin M, 0.05-0.5 μM of Dexamethasone, and 1-10 μM of Forskolin.

16. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The fourth induction medium includes basic synthetic cell culture medium, 0.5-1X N2 additive, 0.5-1X B27 additive, 1X GlutaMax, 50-200 ng / ml EGF, 100-400 ng / ml R-spondin-1 and 50-200 ng / ml Noggin.

17. The method for inducing human pluripotent stem cells to differentiate into functional intestinal organoids according to claim 1, characterized in that, The 3D intestinal organoids were dynamically cultured in a maintenance medium to obtain mature intestinal organoids, the maintenance medium comprising Advanced DMEM / F-12 medium and 1X GlutaMax.

18. An intestinal organoid, characterized in that, The intestinal organoids are generated by the method described in any one of claims 1 to 17, which involves inducing human pluripotent stem cells to differentiate into functional intestinal organoids.

19. A method for evaluating the pharmacokinetics or toxicity of a test substance using the intestinal organoids as described in claim 18.

20. The use of the intestinal organoid of claim 18 in the preparation of a medicament or transplant formulation for treating intestinal injury, intestinal dysfunction or inflammatory bowel disease.

Images