Culture medium and culture method for inducing human pluripotent stem cells to differentiate into ameloblast-like organoids

Abstract

This application belongs to the field of culture medium development and application technology, specifically relating to a culture medium and culture method for inducing the directed differentiation of human pluripotent stem cells into ameloblast organoids. This application achieves highly efficient directed differentiation of human pluripotent stem cells into ameloblasts through a four-stage culture medium and phased concentration regulation of Wnt pathway activators. This not only improves differentiation efficiency but also enhances organoid maturation, resulting in induced ameloblast organoids with typical vesicular structures, expression of AMGN and Epfn, and cell polarization. Obvious AMGN secretion can be observed within the vesicle cavities, exhibiting typical characteristics of in vivo ameloblasts.

Description

Technical Field

[0001] This application belongs to the field of culture medium development and application technology, specifically involving culture medium and culture method for inducing human pluripotent stem cells to differentiate into ameloblast organoids. Background Technology

[0002] Tooth enamel, the hardest tissue in the human body, is formed by the secretion and mineralization of ameloblasts. Enamel defects caused by dental caries, trauma, and genetic diseases are very common in clinical practice. However, ameloblasts rapidly undergo apoptosis after tooth eruption, leading to the loss of the enamel's self-repair ability. Current research on genetic diseases of tooth enamel largely relies on mouse models, but due to developmental and physiological differences between species, the results cannot accurately reflect the true pathological process of human diseases.

[0003] In recent years, organoid culture technology has made groundbreaking progress, enabling the formation of three-dimensional tissues in vitro from human pluripotent stem cells through self-organization, which can mimic the structure and function of corresponding organs in vivo. In 2023, a team from Yonsei University in South Korea, and in 2024, Professor Hannele Ruohola-Baker's team in Saudi Arabia, respectively developed methods for inducing ameloblast organoids. However, the proportion of ameloblast-specific markers highly expressed in the obtained organoids was low, and cell polarization was not significant, indicating insufficient organoid maturity.

[0004] The main technical problems of traditional techniques include: the proportion of ameloblast markers expressed by the induced ameloblast organoids is only 30%-50%, and the differentiation efficiency is low; the induced organoids are 3D dense solid structures, lacking the sac-like structure that epithelial organoids should have, and there are few internally polarized ameloblasts.

[0005] Therefore, it is necessary to further explore the process of inducing human pluripotent stem cells to differentiate into ameloblast organoids. Summary of the Invention

[0006] Based on this, one embodiment of this application provides a culture medium and a culture method for inducing human pluripotent stem cells to differentiate into ameloblast organoids.

[0007] This application provides a culture medium for inducing human pluripotent stem cells to differentiate into ameloblast organoids, comprising: culture medium I, culture medium II, culture medium III and / or culture medium IV;

[0008] The culture medium I includes: basal culture medium and EGF, b-FGF, B27 additive, penicillin-streptomycin antibiotic, BMP4 protein and RA, with or without Wnt pathway activator, and if it contains Wnt pathway activator, the concentration of Wnt pathway activator does not exceed 5 μM;

[0009] The culture medium II includes: basal culture medium and EGF, b-FGF, B27 additive, penicillin-streptomycin antibiotic, BMP4 protein, RA and 5μM-20μM Wnt pathway activator;

[0010] The culture medium III includes: basal culture medium and EGF, β-FGF, B27 additive, penicillin-streptomycin antibiotics, BMP4 protein, RA, fetal bovine serum, sodium β-glycerophosphate, vitamin C, calcium chloride, TGF-β1, and 5 μM-15 μM Wnt pathway activator.

[0011] The culture medium IV includes: basal culture medium and fetal bovine serum, sodium β-glycerophosphate, vitamin C, calcium chloride, penicillin-streptomycin antibiotics, EGF, TGF-β1 and 5 μM-15 μM Wnt pathway activator.

[0012] In some embodiments, the culture medium I comprises: basal culture medium and 10 ng / mL-30 ng / mL LEGF, 10 ng / mL-30 ng / mL b-FGF, 1.5 v / v%-2.5 v / v% B27 additive, 0.5 v / v%-1.0 v / v% penicillin-streptomycin antibiotics, 10 ng / mL-30 ng / mL BMP4 protein, 0.1 μM-5 μM RA and 0.01 μM-5 μM Wnt pathway activator or without Wnt pathway activator;

[0013] The culture medium II includes: basal culture medium and 10 ng / mL-30 ng / mL EGF, 10 ng / mL-30 ng / mL b-FGF, 1.0 v / v%-2.0 v / v% B27 additive, 0.5 v / v%-1.0 v / v% penicillin-streptomycin antibiotics, 10 ng / mL-30 ng / mL BMP4 protein, 0.1 μM-5 μM RA and 5 μM-20 μM Wnt pathway activator;

[0014] The culture medium III comprises: basal culture medium and 10 ng / mL-20 ng / mL EGF, 5 ng / mL-15 ng / mL β-FGF, 0.5 v / v%-1 v / v% B27 additive, 0.5 v / v%-1.0 v / v% penicillin-streptomycin antibiotics, 5 ng / mL-15 ng / mL BMP4 protein, 0.05 μM-2 μM RA, 3 v / v%-5 v / v% fetal bovine serum, 5 mM-15 mM sodium β-glycerophosphate, 15 μg / mL-40 μg / mL vitamin C, 0.1 mM-3 mM calcium chloride, and 0.5 ng / mL-3 ng / mL TGF-β1 and 5 μM-15 μM Wnt pathway activator;

[0015] The culture medium IV comprises: basal culture medium and 5 v / v%-10 v / v% fetal bovine serum, 10 mM-30 mM sodium β-glycerophosphate, 30 μg / mL-70 μg / mL vitamin C, 0.1 mM-5 mM calcium chloride, 0.5 v / v%-1.0 v / v% penicillin-streptomycin antibiotics, 5 ng / mL-15 ng / mL EGF, 1 ng / mL-5 ng / mL TGF-β1 and 5 μM-15 μM Wnt pathway activator.

[0016] In some embodiments, the basal culture medium each independently includes one or more of DMEM / F12 medium, DMEM high-glucose medium, and α-MEM medium.

[0017] In some embodiments, the Wnt pathway activator includes CHIR-99021.

[0018] Another aspect of this application provides a method for inducing human pluripotent stem cells to differentiate into ameloblast organoids, comprising: culturing human pluripotent stem cells using the culture medium described above for inducing human pluripotent stem cells to differentiate into ameloblast organoids.

[0019] In some embodiments, the method for inducing human pluripotent stem cells to differentiate into ameloblastic organoids includes: a 2D culture phase and a 3D culture phase.

[0020] The 2D culture stage includes: culturing human pluripotent stem cells in ordinary culture medium for 0.5-1 days to allow them to adhere; culturing them in ectoderm induction medium for 2 days to allow them to differentiate into ectoderm cells; and finally culturing them in medium I for 2-3 days.

[0021] The 3D culture stage includes: suspending and culturing human pluripotent stem cells after 2D culture in culture medium I and a low-adsorption culture container for 1-2 days, then culturing in culture medium II for 1-2 days; then culturing in culture medium III for 1-3 days, and finally culturing in culture medium IV for 2-3 days to obtain ameloblast organoids.

[0022] In some embodiments, the general culture medium includes mTeSR™1 medium and Y27632.

[0023] In some embodiments, the ectodermal culture medium comprises DMEM / F12, 18 v / v%-22 v / v% KSR, 1.5 mM-2.5 mM L-glutamine, and 0.8 × 10⁻⁶ mol / L. -4 M-1.2×10 -4 M non-essential amino acids, 0.4 v / v%–0.6 v / v% penicillin-streptomycin antibiotics, 8 μM–12 μM SB431542, and 8 ng / mL–12 ng / mL BMP. In some embodiments, the human pluripotent stem cells further include pre-culture, digestion, and passage steps before being cultured in the basal medium.

[0024] In some embodiments, the human pluripotent stem cells are cultured in basal culture medium for 2-8 generations, with a cell density of 60%-70% and a cell differentiation rate of <5%.

[0025] In some embodiments, the inoculation ratio used during passage is 1:10 to 1:15.

[0026] In some embodiments, the digestion step includes: adding cell digestion solution, vaccinating human pluripotent stem cells in the basal culture medium, and collecting the cell suspension.

[0027] In some embodiments, the low-adsorption culture vessel includes a U-shaped low-adsorption well plate.

[0028] In some of these embodiments, the inoculation ratio is 1:(14-18).

[0029] In some embodiments, the inoculation process further includes a centrifugation step; the centrifugation parameters include a centrifugal force of 100g-300g and a time of 3min-5min.

[0030] In some of these embodiments, the culture medium is changed every 1-2 days during the culture process.

[0031] In some of these embodiments, the culture conditions include a temperature of 36°C-38°C and a CO2 concentration of 4.5 v / v%-5.5 v / v.

[0032] This application achieves highly efficient directed differentiation of human pluripotent stem cells into ameloblasts through a four-stage culture medium and phased concentration regulation of Wnt pathway activators. The Wnt pathway plays a crucial regulatory role in stem cell differentiation. By maintaining a low concentration or no Wnt activation in the early differentiation stage (medium I), premature activation of the differentiation pathway is avoided. In the mid-differentiation stage (medium II), the Wnt concentration is moderately increased to promote the differentiation of dental epithelial stem cells into ameloblast precursor cells. In the late differentiation stage (medium III and IV), an appropriate Wnt concentration is maintained to ensure the maturation and polarization of ameloblasts. The technical solution of this application not only improves differentiation efficiency but also enhances organoid maturation, resulting in induced ameloblast organoids with typical vesicular structures, expressing AMGN and Epfn, and undergoing cell polarization. Obvious AMGN secretion can be observed within the vesicle cavities, exhibiting typical characteristics of in vivo ameloblasts. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 A schematic diagram illustrating the induction of HPSCs into ameloblast organoids;

[0035] Figure 2 Staining for the organoid cytoskeleton; red represents F-actin in the cytoskeleton, and blue represents DAPI in the nucleus. White arrows indicate polarized cells, which appear as long columnar cells with elongated nuclei; the white scale bar represents 50 μm.

[0036] Figure 3 The cells were stained with ameloblast markers AMBN and EPFN; both groups of cells expressed nearly 100% of EPFN and AMBN, with the white bar indicating 50 μm.

[0037] Figure 4 Staining with ameloblast markers K14 and AMGN; both groups of cells expressed nearly 100% K14 and AMGN. Small white arrows indicate scattered AMGN secretion granules visible inside the cyst cavity; the white scale bar represents 50 μm.

[0038] Figure 5 Results of optimization of culture medium I (organoid formation observed under a light microscope, scale bar, 200 μm).

[0039] Figure 6The effects of different concentrations (μM) of CHIR-99021 in culture media II, III, and IV on ameloblast organoid differentiation were investigated. Results showed that ameloblast markers p63, EPFN, AMELX, CLDN1, AMBN, and KLK4 were most highly expressed in culture media II containing 10 μM CHIR-99021 and in cultures III and IV containing 7.5 μM CHIR-99021. Different letters (e.g., a, b) between groups indicated statistically significant differences (p < 0.05; one-way ANOVA and Tukey's test). Detailed Implementation

[0040] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0041] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0042] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0043] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0044] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0045] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0046] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0047] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0048] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0049] In this application, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions composed of the listed features.

[0050] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0051] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0052] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0053] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0054] The term "HPSCs" stands for human pluripotent stem cells. Human pluripotent stem cells (PSCs) are a type of cell with the potential for self-renewal and multipotent differentiation, capable of differentiating into almost all cell types. While pluripotent stem cells can differentiate into almost all cell types, they cannot form accessory supporting tissues such as the placenta or umbilical cord. They possess the fundamental characteristics of self-renewal and multipotent differentiation.

[0055] The term "KSR" stands for knockout serum replacement. Knockout serum replacement (KSR) is a synthetic protein source widely used in in vitro cell culture, especially in the culture of pluripotent stem cells. KSR is a serum-free product used to replace fetal bovine serum, promoting the growth of undifferentiated pluripotent stem cells. It can be used for routine maintenance and induction of the growth and differentiation of human or non-human primate embryonic stem cells (ESCs) and pluripotent stem cells (iPSCs).

[0056] The term "BMP4" stands for bone morphogenetic protein 4. BMP-4 is a pleiotropic ligand protein belonging to the TGFβ (transforming growth factor β) family. BMP-4 participates in the circulatory system and can activate receptors on vascular cells. It plays a crucial role in vascular and valvular homeostasis, an important process in embryonic development. BMP-4 increases plaque formation through its pro-inflammatory and pro-atherosclerotic effects, promoting oxidative stress, endothelial dysfunction, and osteogenic differentiation.

[0057] The term "b-FGF" stands for basic fibroblast growth factor. Basic fibroblast growth factor (bFGF) is a multifunctional cell growth factor with various biological functions and important roles. bFGF is a key growth factor in stem cell culture, crucial for maintaining the undifferentiated state of cells, especially in human stem cell culture where it is needed to inhibit cell differentiation.

[0058] The term "EGF" stands for epidermal growth factor. Epidermal growth factor (EGF) is a multifunctional growth factor with a wide range of biological effects. EGF can stimulate the proliferation of various cell types and the metabolism of normal epidermal cells.

[0059] The term "RA" stands for retinoic acid. Retinoic acid (RA), also known as vitamin A acid, is a metabolite of vitamin A with important biological activities, playing a key role in cell growth, differentiation, and organogenesis. Retinoic acid is a natural activator of the nuclear receptor RAR (Retinoic Acid Receptor), regulating the activation and expression of messenger RNA in the cell nucleus. It regulates the expression of target genes through interactions with RAR and RXR (Retinoid X Receptor).

[0060] The term "TGF-β1" stands for transforming growth factor-β1. Transforming growth factor β1 (TGF-β1) is a pleiotropic cytokine in the transforming growth factor β superfamily, possessing a wide range of biological functions. TGF-β1 plays an important role in cell proliferation, differentiation, immune regulation, and maintaining homeostasis. It regulates cellular behavior by binding to receptors on the cell surface, activating downstream signaling pathways.

[0061] The term "penicillin-streptomycin bispecific antibiotic," often simply referred to as "bispecific antibiotic," is a commercially available antibiotic mixture widely used in cell culture to prevent microbial contamination. This mixture contains two antibiotics, penicillin and streptomycin. Penicillin primarily targets Gram-positive bacteria, acting by interfering with bacterial cell wall synthesis; while streptomycin is effective against both Gram-negative and Gram-positive bacteria, inhibiting bacterial protein synthesis by binding to the 30S subunit of the bacterial ribosome. The combined use of these two antibiotics provides a broader antibacterial spectrum, effectively preventing contamination by most bacteria.

[0062] To address the core technical problem of low differentiation efficiency of ameloblast organoids, this application provides a culture medium and culture method for inducing the directed differentiation of human pluripotent stem cells into ameloblast organoids.

[0063] The first aspect of this application provides a culture medium for inducing the directed differentiation of human pluripotent stem cells into ameloblast organoids, comprising: culture medium I, culture medium II, culture medium III, and culture medium IV; wherein culture medium I comprises: basal culture medium and 0.01 μM-5 M Wnt pathway activator or without Wnt pathway activator; culture medium II comprises: basal culture medium and 5 μM-20 μM Wnt pathway activator; culture medium III comprises: basal culture medium and 5 μM-15 μM Wnt pathway activator; and culture medium IV comprises: basal culture medium and 5 μM-15 μM Wnt pathway activator.

[0064] This application achieves highly efficient directed differentiation of human pluripotent stem cells into ameloblasts through a four-stage culture medium system and phased concentration regulation of Wnt pathway activators. The Wnt pathway plays a crucial regulatory role in stem cell differentiation. By maintaining a low concentration or no Wnt activation in the early differentiation stage (culture medium I), premature activation of the differentiation pathway is avoided; in the mid-differentiation stage (culture medium II), the Wnt concentration is moderately increased to promote the differentiation of dental epithelial stem cells into ameloblast precursor cells; and in the late differentiation stage (culture mediums III and IV), an appropriate Wnt concentration is maintained to ensure the maturation and polarization of ameloblasts. This phased regulatory mechanism significantly improves the differentiation efficiency of ameloblast organoids, increasing the expression rate of ameloblast markers from 30%-50% in traditional techniques to nearly 100%.

[0065] In some embodiments, the culture medium I further includes 10 ng / mL-30 ng / mL EGF, 10 ng / mL-30 ng / mL b-FGF, 1.5 v / v%-2.5 v / v% B27 additive, 0.5 v / v%-1.0 v / v% penicillin-streptomycin antibiotics, 10 ng / mL-30 ng / mL BMP4 protein, and 0.1 μM-5 μM RA. Specifically, EGF can be selected from any value among 20 ng / mL, 25 ng / mL, and 30 ng / mL; b-FGF can be selected from any value among 10 ng / mL, 15 ng / mL, 20 ng / mL, and 25 ng / mL; B27 additive can be selected from any value among 1.0 v / v%, 1.5 v / v%, and 2.0 v / v%; penicillin-streptomycin bispecific antibody can be selected from any value among 0.5 v / v%, 0.8 v / v%, and 1.0 v / v%; BMP4 protein can be selected from any value among 10 ng / mL, 15 ng / mL, 20 ng / mL, and 25 ng / mL; and RA can be selected from any value among 0.1 μM, 0.5 μM, 1.0 μM, and 2.0 μM.

[0066] In some embodiments, the culture medium II further includes 10 ng / mL-30 ng / mL EGF, 10 ng / mL-30 ng / mL b-FGF, 1.5 v / v%-2.5 v / v% B27 additive, 0.5 v / v%-1.0 v / v% penicillin-streptomycin antibiotics, 10 ng / mL-30 ng / mL BMP4 protein, and 0.1 μM-5 μM RA. Specifically, EGF can be selected from any value among 10 ng / mL, 15 ng / mL, 20 ng / mL, and 25 ng / mL; b-FGF can be selected from any value among 10 ng / mL, 15 ng / mL, 20 ng / mL, and 25 ng / mL; B27 additive can be selected from any value among 1.0 v / v%, 1.5 v / v%, and 2.0 v / v%; penicillin-streptomycin bispecific antibody can be selected from any value among 0.5 v / v%, 0.8 v / v%, and 1.0 v / v%; BMP4 protein can be selected from any value among 10 ng / mL, 15 ng / mL, 20 ng / mL, and 25 ng / mL; and RA can be selected from any value among 0.1 μM, 0.5 μM, 1.0 μM, and 2.0 μM.

[0067] In some embodiments, the culture medium III further includes 10 ng / mL-20 ng / mL EGF, 5 ng / mL-15 ng / mL b-FGF, 1.5 v / v%-2.5 v / v% B27 additive, 0.5 v / v%-1.0 v / v% penicillin-streptomycin antibiotics, 5 ng / mL-15 ng / mL BMP4 protein, 0.05 μM-2 μM RA, 3 v / v%-5 v / v% fetal bovine serum, 5 mM-15 mM sodium β-glycerophosphate, 15 μg / mL-40 μg / mL vitamin C, 0.1 mM-3 mM calcium chloride, and 0.5 ng / mL-3 ng / mL TGF-β1. Specifically, EGF can be selected from any value among 10 ng / mL, 15 ng / mL, and 20 ng / mL; β-FGF can be selected from any value among 5 ng / mL, 10 ng / mL, and 15 ng / mL; B27 additive can be selected from any value among 1.5 v / v%, 2 v / v%, and 2.5 v / v%; penicillin-streptomycin bispecific antibody can be selected from any value among 0.5 v / v%, 0.8 v / v%, and 1.0 v / v%; BMP4 protein can be selected from any value among 5 ng / mL, 10 ng / mL, and 15 ng / mL; RA can be selected from any value among 0.05 μM, 0.1 μM, 0.5 μM, 1.0 μM, and 2.0 μM; fetal bovine serum can be selected from any value among 3 v / v%, 4 v / v%, and 5 v / v%; β-glycerophosphate sodium can be selected from any value among 5 mM, 10 mM, and 15 mM; vitamin C can be selected from 15... Any value among μg / mL, 25 μg / mL, and 40 μg / mL; calcium chloride can be selected from any value among 0.1 mM, 1 mM, 2 mM, and 3 mM; TGF-β1 can be selected from any value among 0.5 ng / mL, 1.5 ng / mL, and 3 ng / mL.

[0068] In some embodiments, the culture medium IV further includes 5 v / v%-10 v / v% fetal bovine serum, 10 mM-30 mM sodium β-glycerophosphate, 30 μg / mL-70 μg / mL vitamin C, 0.1 mM-5 mM calcium chloride, 0.5 v / v%-1.0 v / v% penicillin-streptomycin antibiotics, 5 ng / mL-15 ng / mL EGF, and 1 ng / mL-5 ng / mL TGF-β1. Specifically, fetal bovine serum can be selected from any value among 5 v / v%, 8 v / v%, and 10 v / v%; sodium β-glycerophosphate can be selected from any value among 10 mM, 20 mM, and 30 mM; vitamin C can be selected from any value among 30 μg / mL, 50 μg / mL, and 70 μg / mL; calcium chloride can be selected from any value among 0.1 mM, 2 mM, and 5 mM; penicillin-streptomycin antibiotics can be selected from any value among 0.5 v / v%, 0.8 v / v%, and 1.0 v / v%; EGF can be selected from any value among 5 ng / mL, 10 ng / mL, and 15 ng / mL; and TGF-β1 can be selected from any value among 1 ng / mL, 3 ng / mL, and 5 ng / mL.

[0069] In some embodiments, the basal culture medium includes one or more of DMEM / F12 medium, DMEM high-glucose medium, and α-MEM medium. Specifically, the basal culture medium may be selected from DMEM / F12 medium, DMEM high-glucose medium, α-MEM medium, or any combination thereof.

[0070] In some embodiments, the Wnt pathway activator includes CHIR-99021. Specifically, CHIR-99021 is a GSK-3β inhibitor that can effectively activate the Wnt signaling pathway and plays an important role in the regulation of stem cell differentiation.

[0071] This application also provides a method for inducing human pluripotent stem cells to differentiate into ameloblastic organoids, comprising the following steps: culturing human pluripotent stem cells in the aforementioned culture medium, including a 2D culture stage and a 3D culture stage; the 2D culture stage includes: culturing human pluripotent stem cells in ordinary culture medium for 0.5-1 days to allow them to adhere; culturing them in ectoderm induction medium for 2 days to allow them to differentiate into ectoderm cells; and finally culturing them in culture medium I for 2-3 days; the 3D culture stage includes: culturing the 2D-cultured human pluripotent stem cells in culture medium I and a low-adsorption culture vessel under suspension conditions for 1-2 days; culturing them in culture medium II for 1-2 days; then culturing them in culture medium III for 1-3 days; and finally culturing them in culture medium IV for 2-3 days to obtain ameloblastic organoids. This method achieves highly efficient induction of ameloblastic organoids through the organic combination of 2D and 3D culture and the systematic application of four-stage culture media.

[0072] In some embodiments, the general culture medium includes mTeSR™1 medium and Y27632. Specifically, mTeSR™1 medium is a serum-free medium specifically designed for the culture of human pluripotent stem cells, and Y27632 is a ROCK kinase inhibitor that can improve cell viability.

[0073] In some embodiments, the ectodermal culture medium comprises DMEM / F12, 18 v / v%-22 v / v% KSR, 1.5 mM-2.5 mM L-glutamine, and 0.8 × 10⁻⁶ mol / L. -4 M-1.2×10 -4 M non-essential amino acids, 0.4 v / v%-0.6 v / v% penicillin-streptomycin bispecific antibody, 8 μM-12 μM SB431542 and 8 ng / mL-12 ng / mL BMP4.

[0074] In some embodiments, the human pluripotent stem cells further include pre-culture, digestion, and passage steps before being cultured in the conventional culture medium.

[0075] In some embodiments, the human pluripotent stem cells are cultured in a normal culture medium for 2-8 generations, with a cell density of 60%-70% and a cell differentiation rate of <5%.

[0076] In some embodiments, the inoculation ratio used during passage is 1:10 to 1:15.

[0077] In some embodiments, the digestion step includes: adding cell digestion solution, vaccinating human pluripotent stem cells in the basal culture medium, and collecting the cell suspension.

[0078] In some embodiments, the low-adsorption culture vessel includes a U-shaped low-adsorption well plate.

[0079] In some of these embodiments, the inoculation ratio is 1:(14-18).

[0080] In some embodiments, the inoculation process further includes a centrifugation step; the centrifugation parameters include a centrifugal force of 100g-300g and a time of 3min-5min.

[0081] In some embodiments, the culture medium is changed every 1-2 days during the culture process, and the culture conditions include a temperature of 36°C-38°C and a CO2 concentration of 4.5 v / v%-5.5 v / v%. Specifically, the temperature can be selected from any value among 36°C, 37°C, and 38°C; the CO2 concentration can be selected from any value among 4.5 v / v%, 5.0 v / v%, and 5.5 v / v%.

[0082] This protocol significantly improves the differentiation efficiency of ameloblast organoids through a four-stage culture medium system and phased concentration regulation of the Wnt pathway activator, increasing the expression rate of ameloblast markers from 30%-50% in traditional techniques to nearly 100%. By organically combining 2D and 3D culture with the application of low-adsorption culture containers, this protocol promotes the formation of vesicular structures in organoids, solving the problems of dense organoid structures and lack of polarization in traditional techniques. Through precise formulation and phased regulation of culture medium components, this protocol not only improves differentiation efficiency but also enhances organoid maturity, resulting in induced ameloblast organoids with typical vesicular structures, expressing AMGN and Epfn, and undergoing cell polarization. Obvious AMGN secretion can be observed within the vesicle cavities.

[0083] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0084] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0085] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0086] Example 1

[0087] This embodiment provides a method for inducing the directed differentiation of human pluripotent stem cells into ameloblast organoids, comprising the following steps:

[0088] (1) Cultivation of HPSCs

[0089] HPSCs were cultured in Matrigel-coated 6-well plates with 2 mL of mTeSR™ Plus medium added to each well. The plates were then incubated at 37°C with 5% CO2. The medium was changed daily, and the cells were passaged when the cell density reached 65%.

[0090] (2) Digestion and passage of HPSCs

[0091] A. Cell digestion: Add 1 mL of calcium- and magnesium-free PBS to each well of a 6-well plate and discard it; add 1 mL of ACCUTASE™ cell digestion solution, incubate at 37°C for 5 min, discard 700 μL of digestion solution, and continue incubating at 37°C for 3 min. At this point, most cells will float and form small cell clusters. Immediately add 2 mL of mTeSR™1 culture medium (containing 10 μM Y27632) and collect the cell suspension into a 15 mL centrifuge tube.

[0092] B. Cell passage: Seed the cell suspension into new Matrigel-coated 6-well plates at a passage ratio of 1:10. The culture medium used for passage is mTeSR™1 medium (containing 10 μM Y27632). Change the medium the next day and use mTeSR™1 medium without Y27632 for cell culture. Change the medium daily until the cell density reaches 60%-70%, at which point cell passage can be performed.

[0093] (3) Induction protocol for HPSCs to gradually differentiate into ameloblasts

[0094] For ameloblast induction experiments, the 2nd to 8th generation HPSCs require a cell density of 65% and a cell differentiation rate of less than 5%.

[0095] A schematic diagram illustrating the induction of HPSCs into ameloblast organoids is shown below. Figure 1 As shown, the specific process is as follows:

[0096] Day 0: Based on 50,000 / cm 2 HPSCs were seeded into Matrigel-coated 6-well plates at the specified cell density using mTeSR™1 medium supplemented with 10 μM Y27632.

[0097] Day 1: Begin the ectoderm cell induction phase. Discard the old culture medium and add ectoderm cell induction medium containing DMEM / F12, 20% KSR, 2 mM L-glutamine, and 1×10⁻⁶ dextrin. -4 M non-essential amino acids, 0.5 v / v% penicillin-streptomycin bispecific antibody, 10 μM SB431542 and 10 ng / mL BMP4.

[0098] Day 2: Discard the old culture medium and replace it with a new ectoderm cell induction solution.

[0099] Day 3: Initiate the dental epithelial stem cell induction phase. Discard the old culture medium and add dental epithelial stem cell induction medium (medium I), which consists of DMEM / F12, 20 ng / mL EGF, 25 ng / mL b-FGF, 2 v / v% B27 additive, 1 v / v% penicillin-streptomycin antibiotics, 12.5 ng / mL BMP4, and 1 μM RA.

[0100] Day 4: Discard the old culture medium and replace it with new culture medium I.

[0101] Day 5: Discard the old culture medium and replace it with new culture medium I.

[0102] Day 6: Start 3D culture. Discard the old culture medium and add fresh, pre-cooled culture medium I. Use a cell scraper to peel off the adherent cells. At a seeding ratio of 1:16, seed one well of a 6-well plate into 16 wells of a U-shaped low-adsorption 96-well plate with 100 μL of culture medium per well. Centrifuge at 300 g for 3 minutes to start 3D culture.

[0103] Day 8: Initiate dental epithelial organoid induction. Discard the old culture medium and add medium II, which consists of DMEM / F12, 20 ng / mL EGF, 25 ng / mL b-FGF, 2 v / v% B27 additive, 1 v / v penicillin-streptomycin antibiotic, 12.5 ng / mL BMP4, 1 μM RA and 10 μM CHIR-99021.

[0104] Day 10: Ameloblast organoid induction begins. Culture medium III is added, consisting of α-MEM medium, 10 ng / mL EGF, 10 ng / mL β-FGF, 1 v / v% B27, 1 v / v% penicillin-streptomycin antibiotics, 6 ng / mL BMP4, 0.5 μM RA, 5 v / v% fetal bovine serum, 10 mM sodium β-glycerophosphate, 25 μg / mL vitamin C, 1 mM calcium chloride, 1.5 ng / mL LTGF-β1, and 7.5 μM CHIR-99021.

[0105] Day 12: Discard the old culture medium and add culture medium IV, which consists of α-MEM medium, 10 v / v% fetal bovine serum, 20 mM sodium β-glycerophosphate, 50 μg / mL vitamin C, 2 mM calcium chloride, 1 v / v% penicillin-streptomycin antibiotics, 10 ng / m LEGF, 3 ng / mL TGF-β1 and 7.5 μM CHIR-99021.

[0106] Day 14: Ameloblast organoid induction completed. Organoids were collected, frozen sections were prepared, and immunofluorescence staining was performed.

[0107] (4) Identification of ameloblasts derived from HPSCs

[0108] A. Real-time quantitative PCR: After washing once with PBS, intracellular RNA was extracted using the TRIzol method, purified with DNase I, and cDNA was synthesized using Roche reverse transcription reagent. A total PCR reaction volume of 20 μL was prepared using Roche quantitative real-time PCR reagent. The Bio-Rad real-time PCR system was run to initiate PCR. –ΔΔCT Legal analysis of data.

[0109] B. Immunofluorescence staining: Organoids were fixed in 4% paraformaldehyde for 24 h, dehydrated in 30% sucrose solution for 48 h, treated with OTC embedding solution, and 8 μm frozen sections were obtained from a frozen sectioning machine and stored at -80 ℃. The sections were thawed, incubated at 65 ℃ for 1 h, washed 3 times with PBS, permeabilized for 30 min, blocked for 30 min, incubated with primary antibody at 4 ℃ overnight, and incubated with the corresponding fluorescent secondary antibody at room temperature for 60 min the next day. The cytoskeleton was stained with phalloidin, the nucleus was stained with DAPI, the slides were mounted, and photographed under a fluorescence microscope.

[0110] Example 2

[0111] This embodiment provides a method for inducing the directed differentiation of human pluripotent stem cells into ameloblast organoids, which is basically the same as that in Embodiment 1, except that:

[0112] Culture medium I consisted of: DMEM high-glucose medium and 30 ng / mL EGF, 30 ng / mL b-FGF, 2.0 v / v% B27 additive, 1.0 v / v% penicillin-streptomycin antibiotic, 30 ng / mL BMP4 protein, 5 μM RA and 5 μM CHIR-99021;

[0113] Culture medium II consisted of: DMEM high-glucose medium and 30 ng / mL EGF, 30 ng / mL b-FGF, 2.0 v / v% B27 additive, 1.0 v / v% penicillin-streptomycin antibiotic, 30 ng / mL BMP4 protein, 5 μM RA and 20 μM CHIR-99021;

[0114] Culture medium III consisted of: DMEM high-glucose medium and 20 ng / mL EGF, 15 ng / mL β-FGF, 1 v / v% B27 additive, 1.0 v / v% penicillin-streptomycin antibiotics, 15 ng / mL BMP4 protein, 2 μM RA and 15 μM Wnt pathway activator, 5 v / v% fetal bovine serum, 15 mM sodium β-glycerophosphate, 40 μg / mL vitamin C, 3 mM calcium chloride and 3 ng / mL LTGF-β1;

[0115] Culture medium IV consisted of DMEM high-glucose medium and 10 v / v% fetal bovine serum, 30 mM sodium β-glycerophosphate, 370 μg / mL vitamin C, 5 mM calcium chloride, 1.0 v / v% penicillin-streptomycin antibiotics, 15 ng / mL EGF, 5 ng / mL TGF-β1 and 15 μM CHIR-99021.

[0116] Example 3

[0117] This embodiment provides a method for inducing the directed differentiation of human pluripotent stem cells into ameloblast organoids, which is basically the same as that in Embodiment 1, except that:

[0118] Culture medium I consisted of α-MEM medium and 0.1 ng / mL EGF, 10 ng / mL b-FGF, 1.5 v / v% B27 additive, 0.5 v / v% penicillin-streptomycin antibiotics, 10 ng / mL BMP4 protein, 0.1 μM RA and 0 μM CHIR-99021;

[0119] Culture medium II included: α-MEM medium and 10 ng / mL EGF, 10 ng / mL b-FGF, 1.0 v / v% B27 additive, 0.5 v / v% penicillin-streptomycin antibiotics, 10 ng / mL BMP4 protein, 5 μM RA and 5 μM CHIR-99021;

[0120] Culture medium III consisted of α-MEM medium and 10 ng / mL EGF, 5 ng / mL β-FGF, 0.5 v / v% B27 additive, 0.5 v / v% penicillin-streptomycin antibiotics, 5 ng / mL BMP4 protein, 0.05 μM RA and 5 μM CHIR-99021, 3 v / v% fetal bovine serum, 5 mM β-glycerophosphate sodium, 15 μg / mL vitamin C, 0.1 mM calcium chloride and 0.5 ng / mL TGF-β1;

[0121] Culture medium IV consisted of α-MEM medium and 5 v / v% fetal bovine serum, 10 mM sodium β-glycerophosphate, 30 μg / mL vitamin C, 0.1 mM calcium chloride, 0.5 v / v% penicillin-streptomycin antibiotics, 5 ng / mL EGF, 1 ng / mL TGF-β1 and 5 μM CHIR-99021.

[0122] The results of Example 1 are verified as follows: Figures 2-4 As shown, the results indicate that the induced ameloblast organoids have typical vesicular structures, express AMGN and Epfn, and undergo cell polarization. Obvious AMGN secretion can be observed in the vesicle cavity, exhibiting typical characteristics of ameloblasts in vivo, and the induction efficiency is high.

[0123] Comparative Example 1

[0124] This comparative example provides a method for inducing the directed differentiation of human pluripotent stem cells into ameloblast organoids, which is basically the same as that in Example 1, except that the concentration of CHIR-99021 in culture medium I used during the dental epithelial stem cell induction phase from Day 3 to Day 5 is different, and the specific settings are as follows:

[0125] The 2nd to 8th generation HPSCs are used for ameloblast induction experiments, requiring a cell density of 60%-70% and a cell differentiation rate of less than 5%.

[0126] Day 0: Based on 50,000 / cm 2 HPSCs were seeded into Matrigel-coated 6-well plates at the specified cell density using mTeSR™1 medium supplemented with 10 μM Y27632.

[0127] Day 1: Begin the ectoderm cell induction phase. Discard the old culture medium and add ectoderm cell induction medium containing DMEM / F12, 20% KSR, 2 mM L-glutamine, and 1×10⁻⁶ dextrin. -4 M non-essential amino acids, 0.5 v / v% penicillin-streptomycin bispecific antibody, 10 μM SB431542 and 10 ng / mL BMP4.

[0128] Day 2: Discard the old culture medium and replace it with a new ectoderm cell induction solution.

[0129] Day 3: Begin the dental epithelial stem cell induction phase. Discard the old culture medium and add culture medium I with different concentrations of CHIR-99021.

[0130] ①0CHIR group: DMEM / F12, 20 ng / mL EGF, 25 ng / mL b-FGF, 1 v / v% B27 additive, 1 v / v% penicillin-streptomycin antibiotic, 12.5 ng / mL BMP4, 1 μM RA;

[0131] ②15CHIR group: DMEM / F12, 20 ng / mL EGF, 25 ng / mL b-FGF, 1 v / v% B27 additive, 1 v / v% penicillin-streptomycin antibiotic, 12.5 ng / mL BMP4, 1 μM RA, 15 μM CHIR-99021;

[0132] ②20CHIR group: DMEM / F12, 20 ng / mL EGF, 25 ng / mL b-FGF, 1 v / v% B27 additive, 1 v / v% penicillin-streptomycin antibiotic, 12.5 ng / mL BMP4, 1 μM RA, 20 μM CHIR-99021.

[0133] Day 4: Discard the old culture medium and replace it with new culture medium I.

[0134] Day 5: Discard the old culture medium and replace it with new culture medium I.

[0135] Day 6: Start 3D culture. Discard the old culture medium and add fresh, pre-cooled culture medium I. Use a cell scraper to peel off the adherent cells. At a seeding ratio of 1:16, seed one well of a 6-well plate into 16 wells of a U-shaped low-adsorption 96-well plate with 150 μL of culture medium per well. Centrifuge at 300 g for 3 minutes to start 3D culture.

[0136] Day 8: Observation of organoid formation under a light microscope. Results are as follows: Figure 5 As shown: 0CHIR group: Cells were able to successfully self-assemble to form 3D organoids with typical vesicle-like structures (e.g., Figure 5 (As shown in the left figure).

[0137] 15CHIR group: Most cells were in a suspended single-cell state, with no obvious 3D self-assembly structure observed, and could not form organoids (such as...). Figure 5 (As shown in the middle image).

[0138] 20CHIR group: Cells were also in a suspended single-cell state, unable to form 3D structures, and some cells showed signs of death (e.g. Figure 5 (As shown in the right figure).

[0139] This comparative example shows that during the dental epithelial stem cell induction phase (Day 3-Day 5), the addition of high concentrations (15-20 μM) of CHIR-99021 to culture medium I significantly inhibited the cell self-assembly ability, resulting in the inability to form 3D organoids.

[0140] Comparative Example 2: Optimization of Culture Media II, III, and IV

[0141] Day 0: Based on 50,000 / cm 2 HPSCs were seeded into Matrigel-coated 6-well plates at the specified cell density using mTeSR™1 medium supplemented with 10 μM Y27632.

[0142] Day 1: Begin the ectoderm cell induction phase. Discard the old culture medium and add ectoderm cell induction medium containing DMEM / F12, 20% KSR, 2 mM L-glutamine, and 1×10⁻⁶ dextrin. -4 M non-essential amino acids, 0.5% penicillin / streptomycin, 10 μM SB431542 and 10 ng / mL BMP4.

[0143] Day 2: Discard the old culture medium and replace it with a new ectoderm cell induction solution.

[0144] Day 3: Initiate the dental epithelial stem cell induction phase. Discard the old culture medium and add dental epithelial stem cell induction medium (medium I), which consists of DMEM / F12, 20 ng / mL EGF, 25 ng / mL b-FGF, 1 v / v% B27 additive, 1 v / v% penicillin-streptomycin antibiotics, 12.5 ng / mL BMP4, and 1 μM RA.

[0145] Day 4: Discard the old culture medium and replace it with new culture medium I.

[0146] Day 5: Discard the old culture medium and replace it with new culture medium I.

[0147] Day 6: Start 3D culture. Discard the old culture medium and add fresh, pre-cooled culture medium I. Use a cell scraper to peel off the adherent cells. At a seeding ratio of 1:16, seed one well of a 6-well plate into 16 wells of a U-shaped low-adsorption 96-well plate with 100 μL of culture medium per well. Centrifuge at 300 g for 3 minutes to start 3D culture.

[0148] Day 8: Start dental epithelial organoid induction. Discard the old culture medium and add culture medium II, which consists of DMEM / F12, 20 ng / ml EGF, 25 ng / ml b-FGF, 1 v / v% B27 additive, 1 v / v% penicillin-streptomycin antibiotic, 12.5 ng / mL BMP4, 1 μM RA, and different concentrations of CHIR-99021 (0 μM, 5 μM, 10 μM, 20 μM).

[0149] Day 10: Ameloblast organoid induction begins. Culture medium III is added, consisting of α-MEM, 10 ng / mLEGF, 10 ng / mL β-FGF, 0.5 v / v% B27, 1 v / v% penicillin-streptomycin antibiotics, 6 ng / mL BMP4, 0.5 μM RA, 5% fetal bovine serum, 10 mM sodium β-glycerophosphate, 25 μg / ml vitamin C, 1 mM calcium chloride, 1.5 ng / mL TGF-β1, and different concentrations of CHIR-99021 (0 μM, 5 μM, 7.5 μM, 15 μM).

[0150] Day 12: Discard the old culture medium and add culture medium IV, which consists of α-MEM medium, 10% fetal bovine serum, 20 mM sodium β-glycerophosphate, 50 μg / mL vitamin C, 2 mM calcium chloride, 1 v / v% penicillin-streptomycin antibiotics, 10 ng / mL EGF, 3 ng / mL TGF-β1 and different concentrations of CHIR-99021 (0 μM, 5 μM, 7.5 μM, 15 μM).

[0151] Day 14: Ameloblast organoid induction was completed. Organoids were collected and subjected to PCR detection. The concentration relationship of CHIR-99021 in media II, III, and IV is shown in Table 1. The results are as follows: Figure 6 As shown:

[0152] Table 1

[0153]

[0154] The results showed that different CHIR treatment conditions had significant effects on the mRNA expression of six genes, exhibiting two main response patterns: EPFN expression was continuously upregulated with increasing CHIR treatment intensity, reaching its highest level in the high-concentration group (20-15 CHIR); while P63, AMELX, CLDN1, AMBN, and KLK4 showed an initial increase followed by a decrease, reaching peak expression in the medium-concentration group (10-7.5 CHIR), and then declining to near the control group level after high-concentration treatment. Overall, the 10-7.5 CHIR treatment had the best upregulation effect on most odontogenic and epithelial differentiation-related genes, suggesting that this condition is the optimal treatment for inducing target gene expression, while excessively high CHIR concentrations may have an inhibitory effect on the expression of some genes.

[0155] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A culture medium for inducing the directed differentiation of human pluripotent stem cells into ameloblast organoids, characterized in that, include: Culture medium I, culture medium II, culture medium III and / or culture medium IV; The culture medium I includes: basal culture medium and EGF, b-FGF, B27 additive, penicillin-streptomycin antibiotic, BMP4 protein and RA, with or without Wnt pathway activator, and if it contains Wnt pathway activator, the concentration of Wnt pathway activator does not exceed 5 μM; The culture medium II includes: basal culture medium and EGF, b-FGF, B27 additive, penicillin-streptomycin antibiotic, BMP4 protein, RA and 5μM-20μM Wnt pathway activator; The culture medium III includes: basal culture medium and EGF, β-FGF, B27 additive, penicillin-streptomycin antibiotics, BMP4 protein, RA, fetal bovine serum, sodium β-glycerophosphate, vitamin C, calcium chloride, TGF-β1, and 5 μM-15 μM Wnt pathway activator. The culture medium IV includes: basal culture medium and fetal bovine serum, sodium β-glycerophosphate, vitamin C, calcium chloride, penicillin-streptomycin antibiotics, EGF, TGF-β1 and 5 μM-15 μM Wnt pathway activator.

2. The culture medium for inducing the directed differentiation of human pluripotent stem cells into ameloblast organoids according to claim 1, characterized in that, The culture medium I includes: basal culture medium and 10 ng / mL-30 ng / mL EGF, 10 ng / mL-30 ng / mL b-FGF, 1.5 v / v%-2.5 v / v% B27 additive, 0.5 v / v%-1.0 v / v% penicillin-streptomycin antibiotics, 10 ng / mL-30 ng / mL BMP4 protein, 0.1 μM-5 μM RA and 0.01 μM-5 μM Wnt pathway activator or without Wnt pathway activator; The culture medium II comprises: basal culture medium and 10 ng / mL-30 ng / mL EGF, 10 ng / mL-30 ng / mL b-FGF, 1.0 v / v%-2.0 v / v% B27 additive, 0.5 v / v%-1.0 v / v% penicillin-streptomycin antibiotics, 10 ng / mL-30 ng / mL BMP4 protein, 0.1 μM-5 μM RA and 5 μM-20 μM Wnt pathway activator; The culture medium III comprises: basal culture medium and 10 ng / mL-20 ng / mL EGF, 5 ng / mL-15 ng / mL β-FGF, 0.5 v / v%-1 v / v% B27 additive, 0.5 v / v%-1.0 v / v% penicillin-streptomycin antibiotics, 5 ng / mL-15 ng / mL BMP4 protein, 0.05 μM-2 μM RA, 3 v / v%-5 v / v% fetal bovine serum, 5 mM-15 mM sodium β-glycerophosphate, 15 μg / mL-40 μg / mL vitamin C, 0.1 mM-3 mM calcium chloride, and 0.5 ng / mL-3 ng / mL TGF-β1 and 5 μM-15 μM Wnt pathway activator; The culture medium IV comprises: basal culture medium and 5 v / v%-10 v / v% fetal bovine serum, 10 mM-30 mM sodium β-glycerophosphate, 30 μg / mL-70 μg / mL vitamin C, 0.1 mM-5 mM calcium chloride, 0.5 v / v%-1.0 v / v% penicillin-streptomycin antibiotics, 5 ng / mL-15 ng / mL EGF, 1 ng / mL-5 ng / mL TGF-β1 and 5 μM-15 μM Wnt pathway activator.

3. The culture medium for inducing the directed differentiation of human pluripotent stem cells into ameloblast organoids according to claim 2, characterized in that, The basal culture media each independently include one or more of DMEM / F12 medium, DMEM high-glucose medium, and α-MEM medium.

4. The culture medium for inducing the directed differentiation of human pluripotent stem cells into ameloblast organoids according to any one of claims 1 to 3, characterized in that, The Wnt pathway activator includes CHIR-99021.

5. A method for inducing human pluripotent stem cells to differentiate into ameloblast organoids, characterized in that, include: Human pluripotent stem cells are cultured using the culture medium for inducing the directed differentiation of human pluripotent stem cells into ameloblast organoids as described in any one of claims 1-4.

6. The method for inducing human pluripotent stem cells to differentiate into ameloblast organoids according to claim 5, characterized in that, include: 2D culture stage and 3D culture stage; The 2D culture stage includes: culturing human pluripotent stem cells in ordinary culture medium for 0.5-1 days to allow them to adhere; culturing them in ectoderm induction medium for 2 days to allow them to differentiate into ectoderm cells; and finally culturing them in medium I for 2-3 days. The 3D culture stage includes: suspending and culturing human pluripotent stem cells after 2D culture in culture medium I and a low-adsorption culture container for 1-2 days, then culturing in culture medium II for 1-2 days; then culturing in culture medium III for 1-3 days, and finally culturing in culture medium IV for 2-3 days to obtain ameloblast organoids.

7. The method for inducing human pluripotent stem cells to differentiate into ameloblast organoids according to claim 6, characterized in that, The 2D culture stage meets one or more of the following conditions: (1) The general culture medium includes mTeSR™1 medium and Y27632; (2) The ectodermal induction medium includes DMEM / F12, 18 v / v%-22 v / v% KSR, 1.5 mM-2.5 mM L-glutamine, and 0.8×10 -4 M-1.2×10 -4 M non-essential amino acids, 0.4 v / v%-0.6 v / v% penicillin-streptomycin bispecific antibody, 8 μM-12 μM SB431542 and 8 ng / mL-12 ng / mL BMP; (3) The human pluripotent stem cells further include pre-culture, digestion and passage steps before being cultured in the ordinary culture medium; (4) The human pluripotent stem cells were cultured in ordinary culture medium for 2-8 generations, with a cell density of 60%-70% and a cell differentiation rate of <5% before culture; and (5) The inoculation ratio used in the passage process is 1:10-1:

15.

8. The method for inducing human pluripotent stem cells to differentiate into ameloblast organoids according to claim 7, characterized in that, The digestion steps include: adding cell digestion solution, vaccinating human pluripotent stem cells in the ordinary culture medium, and collecting the cell suspension.

9. The method for inducing human pluripotent stem cells to differentiate into ameloblast organoids according to claim 6, characterized in that, Human pluripotent stem cells cultured in 2D culture medium I and low-adsorption culture containers are cultured to meet one or more of the following conditions: (1) The low-adsorption culture container includes a U-shaped low-adsorption well plate; (2) The vaccination ratio is 1:(14-18); as well as (3) After inoculation, there is also a centrifugation process; the centrifugation parameters include: centrifugal force of 100g-300g and time of 3min-5min.

10. The method for inducing human pluripotent stem cells to differentiate into ameloblast organoids according to any one of claims 5 to 9, characterized in that, Change the culture medium every 1-2 days during the cultivation process; and / or The cultivation conditions included a temperature of 36℃-38℃ and a CO2 concentration of 4.5 v / v%-5.5 v / v.

Images