CN-121538154-B - 3D intestinal organoid differentiation method based on human pluripotent stem cells, induction medium and application thereof

Abstract

The application discloses a 3D intestinal organoid differentiation method based on human pluripotent stem cells, an induction medium and application thereof, relating to the technical field of stem cells, the culture medium formula combination can lead the intestinal organoid to differentiate various cell types such as epithelial cells, neuroendocrine cells, endothelial cells and the like, and is a key mark of successful differentiation and functional maturation of the intestinal organoid. The 3D intestinal organoid differentiation method provided by the application differentiates the generated intestinal organoid into the histological structures such as the intestinal crypt and the like, and can enable the intestinal organoid to creep in a maintenance stage, so that the intestinal organoid is simulated from the structure bionic to the function, and the significance of the intestinal organoid is far superior to that of a pure morphological simulation. Through a systematic culture medium formula, a definite operation flow and a multi-stage induction strategy, the 3D intestinal organoids with structural integrity, cell diversity and functional activity are successfully constructed, and the system provides an efficient, reliable and expandable in-vitro model platform for intestinal biological research and related application.

Inventors

• QI ZHEN
• LIU ZONGJUN
• LI XIAOWEN
• SHEN MIN
• WANG KANG
• XING LINA
• CHEN YAN

Assignees

• 上海能山生物科技有限公司

Dates

Publication Date

20260512

Application Date

20260121

Claims (2)

1. 1. A combination of media for inducing differentiation of human pluripotent stem cells into intestinal organoids, the combination consisting of: The first induction culture medium consists of an RPMI 1640 culture medium, a 1X B27 additive, 50-200 ng/ml of Activin A, 1-10 ng/ml of FGF4 and 1-10 mu M of Y27632; The second induction culture medium consists of RPMI 1640 culture medium, 1X nonessential amino acid, 1X GlutaMax, 0.5-1X B27 additive, 10-50 ng/ml FGF4, 10-50 ng/ml BMP4 and 1-10 mu M Y27632; The third induction culture medium consists of an RPMI 1640 culture medium, 1X nonessential amino acid, 1X GlutaMax, 0.5-1X B27 additive, 80-200 ng/ml FGF4 and 200-500 ng/ml Wnt signal pathway activator, wherein the Wnt signal pathway activator is Wnt3a; A fourth induction medium consisting of ADVANCED DMEM/F-12 medium, 0.5X N2 additive, 0.5X B27 additive, 1X GlutaMax, 1X nonessential amino acid, 20-100 ng/ml EGF, 50-300 ng/ml R-spondin-1, 10-200 ng/ml Noggin and 1-20 mu M Y-27632; A fifth induction medium, which consists of ADVANCED DMEM/F-12 medium, 0.5X N2 additive, 0.5X B27 additive, 1X GlutaMax, 1X nonessential amino acid, 20-100 ng/ml EGF, 50-300 ng/ml R-spondin-1 and 10-200 ng/ml Noggin; The maintenance medium consists of ADVANCED DMEM/F-12 medium, 1X GlutaMax, 0.1-1 mu M all-trans retinoic acid and 1-10 mu M Forskolin.
2. 2. A method for 3D intestinal organoid differentiation based on human pluripotent stem cells, comprising the steps of: Primary differentiation is carried out on the human pluripotent stem cells in a first induction culture medium to obtain a primary differentiation product, wherein the first induction culture medium consists of RPMI 1640 culture medium, 1X B27 additive, 50-200 ng/ml Activin A, 1-10 ng/ml FGF4 and 1-10 mu M Y27632; Transferring the primary differentiation product into a second induction culture medium for secondary differentiation to obtain a secondary differentiation product, wherein the second induction culture medium consists of RPMI 1640 culture medium, 1X non-essential amino acid, 1X GlutaMax, 0.5-1X B27 additive, 10-50 ng/ml FGF4, 10-50 ng/ml BMP4 and 1-10 mu M Y27632; transferring the secondary differentiation product into a third induction culture medium for tertiary differentiation to obtain a tertiary differentiation product, wherein the third induction culture medium consists of RPMI 1640 culture medium, 1X non-essential amino acid, 1X GlutaMax, 0.5-1X B27 additive, 80-200 ng/ml FGF4 and 200-500 ng/ml Wnt3 a; Transferring the tertiary differentiation product into a fourth induction culture medium for four-stage differentiation to obtain a four-stage differentiation product, wherein the fourth induction culture medium consists of ADVANCED DMEM/F-12 culture medium, 0.5X N2 additive, 0.5X B27 additive, 1X GlutaMax, 1X nonessential amino acid, 20-100 ng/ml EGF, 50-300 ng/ml R-spondin-1, 10-200 ng/ml Noggin and 1-20 mu M Y-27632; Transferring the four-stage differentiation product into a fifth induction culture medium for five-stage differentiation to obtain a five-stage differentiation product, wherein the fifth induction culture medium consists of ADVANCED DMEM/F-12 culture medium, 0.5X N2 additive, 0.5X B27 additive, 1X GlutaMax, 1X nonessential amino acid, 20-100 ng/ml EGF, 50-300 ng/ml R-spondin-1 and 10-200 ng/ml Noggin; and dynamically culturing the five-stage differentiation product in a maintenance culture medium to obtain a mature intestinal organoid, wherein the maintenance culture medium consists of ADVANCED DMEM/F-12 culture medium, 1X GlutaMax, 0.1-1 mu M of all-trans retinoic acid and 1-10 mu M of Forskolin.

Description

3D intestinal organoid differentiation method based on human pluripotent stem cells, induction medium and application thereof Technical Field The invention relates to the technical field of stem cells, in particular to a 3D intestinal organoid differentiation method based on human pluripotent stem cells, an induction medium and application thereof. Background The intestinal tract is a key organ for human digestion, nutrient absorption and immune homeostasis maintenance, and has a complex structure, consists of crypts and villi, and contains various cell types with different functions, such as intestinal epithelial cells, goblet cells, panhnia cells, enteroendocrine cells and the like. Traditional intestinal tract research models, such as two-dimensional (2D) cell line culture and animal models, have significant limitations in simulating human intestinal physiology and pathology. The 2D cell line cannot reproduce the three-dimensional structure and cell diversity of the intestinal tract, and the animal model has the bottlenecks of species difference, low flux, high cost, ethical problems and the like. In recent years, the advent of three-dimensional (3D) intestinal organoids has brought a revolutionary breakthrough in intestinal tract research. Organoids are three-dimensional micro-organs formed by self-assembly and differentiation of adult stem cells or pluripotent stem cells in vitro, capable of highly mimicking the complex structure, cell type and specific function of the tissue of origin. In 2009, clevers team was first successful in culturing intestinal organoids with crypt-villous structures in Matrigel matrix containing specific growth factors (e.g., EGF, noggin, R-spondin 1) using mouse intestinal lgr5+ adult stem cells. Thereafter, human intestinal organoid technology has also rapidly developed as a powerful tool for studying intestinal development, disease mechanisms, host-microorganism interactions, drug toxicity and efficacy assessment. Despite the great success of the prior art, the current mainstream 3D intestinal organoid culture system still has a number of technical pain points to be solved urgently, which severely limits its standardized and scaled application. Existing methods (such as Canadian patent CA3062600A 1) disclose a method for preparing an intestinal organoid from pluripotent stem cells comprising the steps of differentiating pluripotent stem cells into endoderm-like cells, differentiating the endoderm obtained in the step into intestinal stem cells, culturing the intestinal stem cells obtained in the step to form spheroids, and differentiating the spheroids formed in the step to form an intestinal organoid, the step comprising culturing in the presence of MEK1/2 inhibitors, DNA methylation inhibitors, TGF-beta receptor inhibitors and gamma-secretase inhibitors in addition to epidermal growth factors, BMP inhibitors and Wnt signal activators. However, the organoids produced by the invention often have large heterogeneity in size, morphology and cell composition, and lack a unified standardized quality control system. In addition, conventional organoid models often lack critical microenvironment components such as immune cells, vasculature, innervation, and microbiota, which do not completely mimic complex physiological and pathological conditions in the body. Due to the above factors, the conversion of existing intestinal organoids technology from laboratory research to clinical and industrial fields such as precision medicine, new drug development, etc. is facing a great challenge. Disclosure of Invention In order to overcome the defects existing in the prior art, a 3D intestinal organoid differentiation method based on human pluripotent stem cells and an induction medium thereof are provided, so as to solve the problem that the intestinal organoids generated by the culture of the existing 3D intestinal organoid culture system are only on the structure bionic level. To achieve the above object, a first aspect of the present invention provides a combination of media for inducing differentiation of human pluripotent stem cells into intestinal organoids, the combination comprising: A first induction medium comprising RPMI 1640 medium, B27 additive, activin a, FGF4 and Y27632; A second induction medium comprising RPMI 1640 medium, non-essential amino acids, glutaMax, B27 additive, FGF4, BMP4, and Y27632; A third induction medium comprising RPMI 1640 medium, non-essential amino acids, glutaMax, B27 additive, FGF4, and Wnt signaling pathway activator; A fourth induction medium comprising ADVANCED DMEM/F-12 medium, N2 additive, B27 additive, glutaMax, nonessential amino acids, EGF, R-spondin-1, noggin, and Y-27632; a fifth induction medium comprising ADVANCED DMEM/F-12 medium, N2 additive, B27 additive, glutaMax, nonessential amino acids, EGF, R-spondin-1, and Noggin; a maintenance medium comprising ADVANCED DMEM/F-12 medium, glutaMax, all-trans retinoic acid, and Forskolin the second