Method for differentiating human induced pluripotent stem cells into liver organs as well as product and application thereof

Abstract

The invention provides a method for differentiating human induced pluripotent stem cells into liver organs as well as a product and application thereof. The method comprises the following steps: inoculating the human induced pluripotent stem cells into matrigel, and culturing by adopting a culture medium added with an ROCK inhibitor; replacing the culture medium with an initial culture medium I every other day for initial differentiation culture, and replacing the culture medium with an initial culture medium II every other day for continuous initial differentiation culture; after the initial differentiation culture is finished, replacing the culture medium with a first differentiation culture medium, replacing a fresh culture medium every day for first-stage differentiation culture, and continuously culturing until the stage is finished; digesting the cells subjected to the first-stage differentiation culture, inoculating the cells into matrigel after digesting, and performing second-stage differentiation culture by adopting a second differentiation culture medium; replacing the culture medium with a third differentiation culture medium for third-stage differentiation culture, and continuously culturing until the end of the stage; and continuously culturing by adopting a maintenance and maturation culture medium, and promoting further differentiation and maturation of the cells until the liver organs are obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and particularly relates to a method for differentiating human induced pluripotent stem cells into liver organoids, as well as products and applications thereof. Background Art

[0002] In recent years, with the rapid development of regenerative medicine and stem cell technology, the liver organoid culture system based on pluripotent stem cells (PSCs) has become an important tool for studying liver diseases, drug screening, and regenerative medicine. Human induced pluripotent stem cells (iPSCs) have strong differentiation potential. Liver organoids derived from iPSCs are multicellular aggregates formed by iPSC technology through three-dimensional (3D) culture in vitro, which have a certain organizational structure and maintain some liver physiological functions. Liver organoids have physiological functions and metabolic properties similar to hepatocytes in the human body and can be used as an ideal model for toxicity assessment. Combining liver organoids with gene editing technology can obtain genetic liver disease models for related drug screening. Transplanting liver organoids into patients to replace damaged or lost liver tissue is expected to become an effective treatment for end-stage liver diseases and shows great potential in liver regenerative medicine.

[0003] There are various methods for preparing liver organoids, mainly including three-dimensional culture techniques based on stem cells, hepatic progenitor cells, or primary hepatocytes. Among them, the method for differentiating liver organoids based on pluripotent stem cells (iPSCs or ESCs) includes: differentiating pluripotent stem cells into endodermal cells, further inducing differentiation into hepatic progenitor cells, and under three-dimensional culture conditions, the hepatic progenitor cells self-organize to form liver organoids.

[0004] However, in practical applications, there is still room for optimization in the existing culture system, such as problems like improving cell survival rate and controlling the differentiation of heterologous cells. Therefore, developing a liver organoid culture method with high cell survival rate and effective control of heterologous cell differentiation has important application value. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a method for differentiating human induced pluripotent stem cells into liver organoids, as well as products and applications thereof. The culture method provided by the present invention significantly improves the cell survival rate, promotes the differentiation of target hepatocytes, and simultaneously inhibits the differentiation of non-target cells, thereby obtaining higher-quality liver organoids. While maintaining the simplicity of the original experimental method, it significantly improves the culture efficiency and quality of organoids, providing new possibilities for in vitro research of liver diseases and regenerative medicine applications.

[0006] To achieve the purpose of this invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for differentiating human induced pluripotent stem cells into hepatic organoids, the method comprising:

[0008] (S1) Inoculating human induced pluripotent stem cells into Matrigel and culturing them in a medium supplemented with a ROCK inhibitor;

[0009] (S2) Replacing the medium with initial medium I every other day for initial differentiation culture, and then replacing the medium with initial medium II every other day to continue the initial differentiation culture;

[0010] (S3) After the subculture is completed, replacing the medium with differentiation medium I, replacing the fresh medium every day, and performing the first-stage differentiation culture until the end of this stage;

[0011] (S4) Digesting the cells after the first-stage differentiation culture, inoculating them into Matrigel after digestion, and performing the second-stage differentiation culture with differentiation medium II;

[0012] (S5) Replacing the medium with differentiation medium III and performing the third-stage differentiation culture until the end of this stage;

[0013] (S6) Continuing the culture with a maintenance and maturation medium to promote further differentiation and maturation of the cells until hepatic organoids are obtained.

[0014] The present invention has made an innovative improvement to the existing method for preparing hepatic organoids. By adding a ROCK inhibitor to the culture system and using it only on the first day of the culture system, the present invention has found that this improvement strategy significantly improves the cell survival rate, promotes the differentiation of target hepatocytes, and at the same time inhibits the differentiation of non-target cells, thereby obtaining higher-quality hepatic organoids. While maintaining the simplicity of the original experimental method, the improvement scheme of the present invention significantly improves the culture efficiency and quality of organoids, providing new possibilities for in vitro research on liver diseases and applications in regenerative medicine.

[0015] Preferably, in (S1), the composition of the medium supplemented with a ROCK inhibitor includes: RPMI1640 medium, activin A, bone morphogenetic protein 4, and a ROCK inhibitor.

[0016] Preferably, the concentration of activin A in the medium supplemented with a ROCK inhibitor is 50-200 ng / mL, such as 50 ng / mL, 120 ng / mL, 150 ng / mL, 180 ng / mL, or 200 ng / mL, etc., and the concentration of bone morphogenetic protein 4 is 25-100 ng / mL, such as 25 ng / mL, 50 ng / mL, 75 ng / mL, or 100 ng / mL, etc.

[0017] Preferably, the concentration of the ROCK inhibitor in the culture medium supplemented with the ROCK inhibitor is 2-20 μM, for example, it can be 2 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, 12 μM, 14 μM, 15 μM, 16 μM, 18 μM or 20 μM, etc., and preferably 5-15 μM.

[0018] In the present invention, a concentration of the ROCK inhibitor higher than 20 μM may affect the differentiation of the organoids, while a concentration lower than 2 μM has no obvious effect.

[0019] Preferably, the ROCK inhibitor is Y-27632.

[0020] The method for culturing liver organoids of the present invention can obtain organoids expressing a variety of hepatocyte markers and can be repeated by other laboratories. In the culture system of the present invention, the small molecule compound Y-27632 is added, and after the addition, the culture and differentiation of the organoids are significantly improved. It is added only on the first day, which increases the cell survival rate while promoting differentiation and reducing the differentiation of miscellaneous cells.

[0021] The method of the present invention overcomes the problems existing in the existing liver organoid culture technology, such as poor reproducibility, low efficiency, slow growth of organoids, more other miscellaneous cells, low proportion of hepatocytes in liver organoids and large proportion of other stromal cells.

[0022] Preferably, in (S1), the seeding amount of the human induced pluripotent stem cells is (0.75-2)×10 5 cells / cm 2 well, where "0.75-2" can be, for example, 0.75, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2, etc.

[0023] During seeding, inoculation is carried out according to the well area of the culture plate. For example, if the well area is 10 cm 2 , then (0.75-2)×10 6 cells / well are inoculated.

[0024] Preferably, in (S2), the composition of the initial culture medium I includes: RPMI1640 medium, activin A and fetal bovine serum.

[0025] Preferably, the concentration of activin A in the initial culture medium I is 50-200 ng / mL, for example, it can be 50 ng / mL, 120 ng / mL, 150 ng / mL, 180 ng / mL or 200 ng / mL, etc., and the concentration of fetal bovine serum is 0.1-0.3%, for example, it can be 0.1%, 0.15%, 0.2%, 0.25% or 0.3%, etc.

[0026] Preferably, in (S2), the composition of the second initial medium includes: RPMI1640 medium, activin A, and fetal bovine serum.

[0027] Preferably, the concentration of activin A in the second initial medium is 50 - 200 ng / mL, such as 50 ng / mL, 120 ng / mL, 150 ng / mL, 180 ng / mL, or 200 ng / mL, etc., and the concentration of fetal bovine serum is 1 - 3%, such as 1%, 1.5%, 2%, 2.5%, or 3%, etc.

[0028] Using the above first initial medium and second initial medium for differentiation culture can efficiently differentiate pluripotent stem cells into endoderm cells to prepare for differentiation into hepatic progenitor cells.

[0029] Preferably, in (S3), the composition of the first differentiation medium includes: Advanced DMEM / F12 medium, B27 supplement, N2 supplement, fibroblast growth factor 4, and GSK3β inhibitor.

[0030] In the present invention, the stock solution concentration of the B27 supplement is 50×, and it is diluted to 0.5× - 2× according to a ratio of 1:100 to 1:25. B27 is a serum-free additive commonly used in nerve cell culture, generally containing antioxidants, proteins, vitamins, and fatty acids. The product formulas provided by each company are similar, and the references are Brewer et al., J Neuroscience Res 35:567 - 576, 1993 and Brewer and Cotman, Brain Res 494:65 - 74, 1989 [1, 2].

[0031] In the present invention, the stock solution of the N2 supplement is 100×. The N2 supplement is a commonly used supplement for nerve cell culture. Based on Bottenstein’s N-1 formula, it is a chemically defined serum-free additive containing transferrin, recombinant insulin, progesterone, putrescine, and selenite, etc. Products provided by each company can be used.

[0032] Preferably, the concentration of the B27 supplement in the first differentiation medium is 0.5× - 2×, the concentration of the N2 supplement is 0.5× - 2×, the concentration of fibroblast growth factor is 250 - 1000 ng / mL, such as 250 ng / mL, 500 ng / mL, 750 ng / mL, or 1000 ng / mL, etc., and the concentration of the GSK3β inhibitor is 1 - 5 μM, such as 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM, etc.

[0033] Preferably, the GSK3β inhibitor is CHIR99021.

[0034] In the present invention, the first differentiation medium is a serum-free medium, and the components therein cooperate with each other to promote the differentiation of cells into hepatic progenitor cells.

[0035] Preferably, the time for the first-stage differentiation culture is 2 to 3 days, for example, it can be 2 or 3, etc.

[0036] Preferably, in (S4), the digestion solution used for digestion is 0.5× - 2× Accutase solution.

[0037] Preferably, in (S4), the seeding amount of the digested cells is (0.75 - 1.5)×10 5 cells / cm 2 well, where "0.75 - 1.5" can be, for example, 0.75, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5, etc.

[0038] Preferably, in (S4), the composition of the second differentiation medium includes: Advanced DMEM / F12 medium, B27 supplement, N2 supplement, GlutaMAX supplement, basic fibroblast growth factor (FGF2), vascular endothelial growth factor, epidermal growth factor, GSK3β inhibitor, TGF-β type I receptor inhibitor, ascorbic acid and antibiotics.

[0039] The second differentiation medium is a serum-free medium, which is used to expand hepatic progenitor cells and promote the future cell differentiation.

[0040] In the present invention, the stock solution of the GlutaMAX supplement is 100×, containing 200 mM L-alanyl-L-glutamine dipeptide, which is a substitute for L-glutamine and has better stability.

[0041] Preferably, in the second differentiation medium, the concentration of B27 supplement is 0.5× - 2×, the concentration of N2 supplement is 0.5× - 2×, the concentration of GlutaMAX supplement is 0.5× - 2×, the concentration of basic fibroblast growth factor is 2.5 - 10 ng / mL, the concentration of vascular endothelial growth factor is 5 - 20 ng / mL, the concentration of epidermal growth factor is 10 - 40 ng / mL, the concentration of GSK3β inhibitor is 1 - 5 μM, the concentration of TGF-β type I receptor inhibitor is 0.1 - 4 μM, the concentration of ascorbic acid is 20 - 200 μg / mL, and the concentration of antibiotics is 0.5 - 2%.

[0042] Among them, 0.5× to 2× can be, for example, 0.5, 0.7, 0.9, 1, 1.1, 1.3, 1.5, 1.7, 1.9, or 2, etc. The concentration of basic fibroblast growth factor is 2.5 to 10 ng / mL, which can be, for example, 2.5 ng / mL, 5 ng / mL, 7.5 ng / mL, or 10 ng / mL, etc. The concentration of vascular endothelial growth factor is 5 to 20 ng / mL, which can be, for example, 5 ng / mL, 7.5 ng / mL, 10 ng / mL, 12.5 ng / mL, 15 ng / mL, 17.5 ng / mL, or 20 ng / mL, etc. The concentration of epidermal growth factor is 10 to 40 ng / mL, which can be, for example, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, or 40 ng / mL, etc. The concentration of GSK3β inhibitor is 1 to 5 μM, which can be, for example, 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM, etc. The concentration of TGF-β type I receptor inhibitor is 0.1 to 4 μM, which can be, for example, 0.1 μM, 0.25 μM, 1 μM, 2 μM, or 4 μM, etc. The concentration of ascorbic acid is 20 to 200 μg / mL, which can be, for example, 20 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL, 125 μg / mL, 150 μg / mL, 175 μg / mL, or 200 μg / mL, etc. The concentration of the antibiotic is 0.5 to 2%, which can be, for example, 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, or 2%, etc.

[0043] Preferably, the GSK3β inhibitor is CHIR99021.

[0044] Preferably, the TGF-β type I receptor inhibitor is A83-01.

[0045] Preferably, the antibiotic is penicillin and streptomycin.

[0046] Preferably, the time for the second-stage differentiation culture is 3 to 5 days, which can be, for example, 3, 4, or 5, etc.

[0047] Preferably, in (S5), the components of the differentiation medium three include: Advanced DMEM / F12 medium, B27 supplement, N2 supplement, GlutaMAX supplement, retinoic acid, and antibiotic.

[0048] Preferably, in the differentiation medium three, the concentration of the B27 supplement is 0.5× to 2×, the concentration of the N2 supplement is 0.5× to 2×, the concentration of the GlutaMAX supplement is 0.5× to 2×, the concentration of retinoic acid is 1 to 5 μM, and the concentration of the antibiotic is 0.5 to 2%.

[0049] Among them, 0.5× to 2× can be, for example, 0.5, 0.7, 0.9, 1, 1.1, 1.3, 1.5, 1.7, 1.9 or 2, etc. The concentration of retinoic acid is 1 to 5 μM, which can be, for example, 1 μM, 2 μM, 3 μM, 4 μM or 5 μM, etc. The concentration of the antibiotic is 0.5 to 2%, which can be, for example, 0.5%, 1%, 1.5% or 2%, etc.

[0050] Preferably, the antibiotic is penicillin and streptomycin.

[0051] Preferably, the time for the third-stage differentiation culture is 3 to 5 days, which can be, for example, 3, 4 or 5, etc.

[0052] Preferably, in (S6), the composition of the maintenance and maturation medium includes: hepatocyte medium, hepatocyte growth factor, dexamethasone, tumor growth factor and antibiotic.

[0053] In the present invention, the hepatocyte medium is from Lonza, with the catalog number CC-3198 and without epidermal growth factor.

[0054] Preferably, in the maintenance and maturation medium, the concentration of hepatocyte growth factor is 5 to 20 ng / mL, the concentration of dexamethasone is 0.05 to 0.2 μM, the concentration of tumor growth factor is 10 to 40 ng / mL, and the concentration of the antibiotic is 0.5 to 2%.

[0055] Among them, 5 to 20 ng / mL can be, for example, 5 ng / mL, 10 ng / mL, 15 ng / mL or 20 ng / mL, etc. 0.05 to 0.2 μM can be, for example, 0.05 μM, 0.1 μM, 0.15 μM or 0.2 μM, etc.

[0056] Preferably, the antibiotic is penicillin and streptomycin.

[0057] Preferably, in (S6), the time for differentiation and maturation is 5 to 15 days, which can be, for example, 5, 7, 9, 10, 11, 13 or 15, etc.

[0058] Preferably, in (S1) to (S6), the culture conditions are 36 to 38 °C, 4 to 6% CO2. 36 to 38 °C can be, for example, 36 °C, 37 °C or 38 °C, etc., and 4 to 6% can be, for example, 4%, 5% or 6%, etc.

[0059] In a second aspect, the present invention provides a liver organoid derived from human induced pluripotent stem cells, and the liver organoid is prepared by the method for differentiating human induced pluripotent stem cells into liver organoids described in the first aspect.

[0060] Thirdly, the present invention provides an application of the method for differentiating human induced pluripotent stem cells into hepatic organoids as described in the first aspect in the culture of hepatic organoids.

[0061] The method for differentiating human induced pluripotent stem cells into hepatic organoids of the present invention significantly improves cell viability, organoid size, and the proportion of hepatocytes, and can be applied to the construction of in vitro fibrosis models.

[0062] The numerical ranges described in the present invention include not only the above-listed point values, but also any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of brevity, the present invention does not exhaustively list the specific point values included in the described ranges.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] Existing hepatic organoid technologies have problems of poor repeatability and low efficiency, and in the cultured hepatic organoids, the proportion of hepatocytes is low and the proportion of other stromal cells is large. By adding the small molecule compound Y-27632 to the culture system and using it only on the first day of the culture system, the present invention finds that this improved strategy significantly improves cell survival rate, promotes the differentiation of target hepatocytes, and simultaneously inhibits the differentiation of non-target cells, thereby obtaining higher-quality liver organoids. While the experimental method of the present invention is simple, it significantly improves the culture efficiency and quality of organoids, providing new possibilities for in vitro research on liver diseases and applications in regenerative medicine. Description of the Drawings

[0065] Figure 1 are the microscope bright field detection results of Comparative Example 1 and Comparative Example 2 on the 10th and 16th days.

[0066] Figure 2 are the microscope bright field detection results of Example 1 and Comparative Example 1 on the 1st day.

[0067] Figure 3 are the microscope bright field detection results of Example 1 and Comparative Example 1 on the 10th and 12th days.

[0068] Figure 4 are the fluorescence microscope detection results of the main markers of hepatic organoids in Example 1 and Comparative Example 1.

[0069] Figure 5 are the fluorescence microscope detection results of multiple markers of hepatic organoids in Example 1 and Comparative Example 1.

[0070] Figure 6 are the microscope bright field detection results of Example 2 and the comparative example on the 1st and 5th days.

[0071] Figure 7It is the bright-field microscopic detection result on the 10th day of the scheme of Example 3 and Comparative Example 3.

[0072] Figure 8 Example 4 is about liver organoids simulating liver fibrosis disease. Detailed implementation manners

[0073] The technical solutions of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0074] For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels of commercial purchase.

[0075] The sources of experimental materials in the following specific implementation manners are as follows:

[0076] B27 supplement: Stemcell, 5711.

[0077] N2 supplement: Stemcell, 7152.

[0078] Accutase solution: Stemcell, 07920.

[0079] GlutaMAX supplement: Gibco, 35050061.

[0080] Comparative Example 1

[0081] Day 0: Start culturing and treating. Seed the cells into a 6-well plate pre-coated with Matrigel (diluted 1:100), about 1×10 6 cells per 10 cm 2 well area. Use RPMI1640 medium (Roswell Park Memorial Institute 1640), supplemented with 100 ng / mL activin A and 50 ng / mL bone morphogenetic protein 4 (BMP4). Incubate the cells in an incubator at 37°C and 5% CO2.

[0082] Day 1: Aspirate the medium added on Day 0. Add fresh RPMI1640 medium, supplemented with 100 ng / mL activin A and 0.2% fetal bovine serum (FBS). Return it to the incubator and continue culturing at 37°C and 5% CO2.

[0083] Day 2: Replace with RPMI 1640 medium containing 100 ng / mL activin A and 2% fetal bovine serum. Continue culturing in the incubator.

[0084] Days 3 to 5: Replace with differentiation medium: Starting from Day 3, replace with Advanced DMEM / F12 medium and add the following components: 1× B27 supplement, 1× N2 supplement, 500 ng / mL fibroblast growth factor 4 (FGF4), 3 μM GSK3β inhibitor (CHIR99021). Replace with fresh medium every day until the end of this stage.

[0085] Days 5 to 9: Single-cell digestion and re-seeding. Single-cell digestion: Gently digest the cells from the well plate using 1× Accutase solution to form a single-cell suspension. Centrifuge at 300 g for 2 minutes to collect the cells. Embed the cells in Matrigel: Seed 1×10 5 cells into 50 μL of Matrigel. Use Advanced DMEM / F12 as the basal medium and add the following components: 1× B27 supplement, 1× N2 supplement, 1× GlutaMAX supplement, 5 ng / mL basic fibroblast growth factor (FGF2), 10 ng / mL vascular endothelial growth factor (VEGF), 20 ng / mL epidermal growth factor (EGF), 3 μM GSK3β inhibitor (CHIR99021), 0.5 μM TGF-β type I receptor inhibitor (A83-01), 50 μg / mL ascorbic acid (VC), penicillin and streptomycin (1% P / S), and continue culturing in the incubator.

[0086] Days 9 to 13: Replace with Advanced DMEM / F12 medium containing the following components: 1× B27 supplement, 1× N2 supplement, 1× GlutaMAX supplement, 2 μM retinoic acid, 1% penicillin and streptomycin (1% P / S), and continue culturing until the end of this stage.

[0087] Days 13 to 23: Maintenance and maturation medium. The composition of the maintenance and maturation medium includes: hepatocyte medium (Lonza, CC-3198, without epidermal growth factor), 10 ng / mL hepatocyte growth factor (HGF), 0.1 μM dexamethasone, 20 ng / mL oncostatin M (OSM), 1% penicillin and streptomycin (1% P / S). This stage aims to promote further differentiation and maturation of the cells until the end of the experiment.

[0088] Comparative Example 2

[0089] Day 0: Start culturing and processing. Inoculate the cells into a 6-well plate pre-coated with Matrigel (2% dilution), with approximately 1×10 6 cells per 10 cm 2 well area. Use DMEM / F12 medium supplemented with 1× insulin, transferrin, and selenium additive (ITS), 1× non-essential amino acids (NEAA), 1 mM sodium pyruvate, 100 ng / mL Activin A, 10 ng / mL bone morphogenetic protein 4 (BMP4), and 10 μM ROCK inhibitor (Y-27632). Incubate the cells in an incubator at 37 °C and 5% CO2.

[0090] Day 3: Change the medium. Change the medium to RPMI1640 medium supplemented with 1× B27 supplement, 2% Matrigel, and 50 ng / mL fibroblast growth factor 10 (FGF10). Continue to incubate the cells in an incubator at 37 °C and 5% CO2.

[0091] Day 6: Change the medium. Change the medium to RPMI1640 medium supplemented with 1× B27 supplement (SM1), 2% Matrigel, 10 ng / mL fibroblast growth factor 10 (FGF10), and 10 ng / mL bone morphogenetic protein 4 (BMP4). Continue to incubate the cells in an incubator at 37 °C and 5% CO2.

[0092] Day 9: Change the medium. Change the medium to hepatocyte medium (from Lonza), supplemented with 1% Matrigel, 50 ng / mL hepatocyte growth factor (HGF), 50 ng / mL oncostatin M, and 10 μM dexamethasone. At this time, no expected organoids were floating in the Jet low-attachment culture plate (non-cell culture treated, NON-TREATED). Therefore, the cells were dispersed by mechanical pipetting and transferred to an ultra-low attachment 6-well plate and continued to be cultured on a shaker.

[0093] Figure 1 Microscopic bright-field detection results on days 10 and 16 for the protocols of Comparative Example 1 and Comparative Example 2.

[0094] On the 10th day of culture, an organoid structure in the form of vesicles appeared in the protocol of Comparative Example 1, while no organoids were formed in the protocol of this comparative example, which was less effective than the original literature (possible reasons include culture consumables and cell lines, etc.). After the cells were formed into a three-dimensional structure by mechanical separation in the protocol of this comparative example, it was also observed that the protocol of this comparative example could form three-dimensional spherical organoids more easily, but it was not easy to control the diameter, and the spheres were too large. Generally speaking, comparing the two protocols, the protocol of Comparative Example 1 is better than that of Comparative Example 2. However, there are still problems in Comparative Example 1 such as slower growth of organoids and more other miscellaneous cells.

[0095] Example 1

[0096] The difference between this example and Comparative Example 1 is that Y-27632 was added on Day 0. The specific steps are as follows:

[0097] Day 0: Start culturing and treating. Inoculate the cells into a 6-well plate pre-coated with Matrigel (diluted 1:100), and inoculate about 1×10 6 cells / 10 cm 2 well area. Use RPMI1640 medium (Roswell Park Memorial Institute 1640), add 100 ng / mL activin A and 50 ng / mL bone morphogenetic protein 4 (BMP4). Place the cells in an incubator at 37 °C and 5% CO2 for culture. In this step, 10 μM ROCK inhibitor (Y-27632) was added simultaneously.

[0098] Day 1: Aspirate the medium added on Day 0. Add fresh RPMI1640 medium, add 100 ng / mL activin A, 0.2% fetal bovine serum (FBS). Place it back in the incubator and continue to culture at 37 °C and 5% CO2.

[0099] Day 2: Replace it with RPMI1640 medium containing 100 ng / mL activin A and 2% fetal bovine serum. Continue to culture in the incubator.

[0100] Day 3 to Day 5: Replace it with differentiation medium. Starting from Day 3, replace it with Advanced DMEM / F12 medium, and add the following components: 1× B27 supplement, 1× N2 supplement, 500 ng / mL fibroblast growth factor 4 (FGF4), 3 μM GSK3β inhibitor (CHIR99021). Replace the fresh medium every day until the end of this stage.

[0101] Day 5 to Day 9: Single-cell digestion and re-seeding. Single-cell digestion: Use 1× Accutase solution to gently digest the cells from the well plate to form a single-cell suspension. Centrifuge at 300 g for 2 minutes to collect the cells. Embed the cells in Matrigel: Seed 1×10 5 cells into 50 μL of Matrigel. Use Advanced DMEM / F12 as the basal medium and add the following components: 1× B27 supplement, 1× N2 supplement, 1× GlutaMAX supplement, 5 ng / mL basic fibroblast growth factor (FGF2), 10 ng / mL vascular endothelial growth factor (VEGF), 20 ng / mL epidermal growth factor (EGF), 3 μM GSK3β inhibitor (CHIR99021), 0.5 μM TGF-β type I receptor inhibitor (A83-01), 50 μg / mL ascorbic acid (VC), penicillin and streptomycin (1% P / S), and continue culturing in an incubator.

[0102] Day 9 to Day 13: Replace with Advanced DMEM / F12 medium containing the following components: 1× B27 supplement, 1× N2 supplement, 1× GlutaMAX supplement, 2 μM retinoic acid, 1% penicillin and streptomycin (1% P / S), and continue culturing until the end of this stage.

[0103] Day 13 to Day 23: Maintenance and maturation medium, switch to hepatocyte medium (except epidermal growth factor EGF, Lonza), and add the following growth factors and inhibitors: 10 ng / mL hepatocyte growth factor (HGF), 0.1 μM dexamethasone, 20 ng / mL oncostatin M (OSM), 1% penicillin and streptomycin (1% P / S). This stage aims to promote further differentiation and maturation of the cells until the end of the experiment.

[0104] In this example, Y-27632 (10 μM) was added to the culture system; on the first day of culture, it was found that the cell viability of this example (+Y-27632) was significantly increased compared with that of Comparative Example 1. Under bright-field microscopy, the number of adherent cells was significantly increased (as Figure 2 shown).

[0105] After differentiation, on the 10th and 12th days of this example (+Y-27632), the hepatic organoids were compared with those of Comparative Example 3. Under bright-field microscopy, the round organoids had a larger volume and fewer heterogeneous cells similar to dendritic branches, as Figure 3 indicated by the blue arrows on the 12th day.

[0106] After maturation culture, detection was carried out by immunofluorescence in the third week. The proportions of hepatocyte markers, hepatocyte nuclear factor 4α (red fluorescence) and albumin (green fluorescence), in the liver organoids of this example (+Y-27632) increased compared with those of Comparative Example 3, while the proportion of other stromal cells (α-smooth muscle actin, magenta) decreased, as Figure 4 shown.

[0107] Meanwhile, after the liver organoids were differentiated and matured, they could express markers of multiple cell types. Through immunofluorescence detection, in addition to expressing the hepatocyte-related markers hepatocyte nuclear factor 4α and albumin, and the stromal cell marker α-smooth muscle actin, they could also express markers of hepatic progenitor cells and cholangiocytes: E-Cadherin and CK19, as Figure 5 shown.

[0108] Example 2

[0109] This example explored whether Y-27632 was helpful for the differentiation of organoids under low-density conditions. A method for differentiating human induced pluripotent stem cells into liver organoids was provided. The difference between this method and that of Example 1 was that the cell seeding density was (0.25-0.4)×10 6 / 10 cm 2 well, and the remaining steps referred to Example 1.

[0110] Comparative Example 3

[0111] Comparative Example 3 provided a method for differentiating human induced pluripotent stem cells into liver organoids. The difference between this method and that of Example 1 was that the cell seeding density was (0.25-0.4)×10 6 / 10 cm 2 well, and Y-27632 was not added.

[0112] The results of Example 2 were as follows Figure 6 shown. It can be seen from the results of Example 2 that when the seeding density was low, adding Y-27632 on the first day could still significantly improve the survival and growth of cells. However, the cells of Comparative Example 3 were still very low in density after the 5th day and could not continue the differentiation experiment.

[0113] Example 3

[0114] This example compared the optimal seeding densities of Y-27632 at high and low seeding densities. A method for differentiating human induced pluripotent stem cells into liver organoids was provided. The difference between this method and that of Example 1 was that the cell seeding density was 0.25×10 6 / 10 cm 2 well, or 1×10 6 / 10 cm 2Pores, and the remaining steps refer to Example 1.

[0115] The results in Example 3 are as follows Figure 7 as shown. From the results of Example 3, it can be seen that when the seeding density is low (0.25×10 6 / 10 cm 2 pores), adding Y-27632 on the first day can still enable the cells to continue to differentiate into organoids. However, on the 10th day, the organoids are smaller in volume and have more heterogeneous cells, indicating that 1×10 6 / 10 cm 2 pores is a better seeding density.

[0116] Example 4

[0117] This example provides an implementation method of an in vitro fibrosis model. In this example, liver organoids are used to simulate the in vitro liver fibrosis model. The method is the same as the first 18 days of Example 1. When the organoids reach the 18th day, the liver organoids are cultured in suspension and treated with TGFβ for 6 days. At the 24th day, the organoids are fixed and immunofluorescently stained. As Figure 8 shown, there is no obvious change in the bright-field morphology, but the fluorescence microscope detects a significant increase in the SMA signal related to liver fibrosis, indicating that the liver organoids obtained by the method provided by the present invention can simulate some phenotypes of human liver fibrosis diseases and have potential applications related to the simulation of human diseases.

[0118] In summary, the present invention provides a method for differentiating human induced pluripotent stem cells into liver organoids. By adding Y-27632 only on the first day, the differentiation efficiency of liver organoids can be enhanced, and multiple markers can be detected. The method can be used for the construction of liver fibrosis disease models and has important application value in the development of drugs for the treatment of liver diseases.

[0119] The applicant declares that the above description is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for differentiating human induced pluripotent stem cells into liver organoids, characterized in that: The method comprises: (S1) Inoculating human induced pluripotent stem cells into Matrigel and culturing them in a medium supplemented with a ROCK inhibitor; (S2) replacing the culture medium with initial culture medium 1 every other day for initial differentiation culture, and replacing the culture medium with initial culture medium 2 every other day for continuing initial differentiation culture; (S3) After the initial differentiation culture is completed, the culture medium is replaced with differentiation medium 1, and fresh culture medium is replaced every day to carry out the first stage of differentiation culture, and the culture is continued until the end of this stage; (S4) digesting the cells after the first stage of differentiation culture, inoculating them into matrix gel after digestion, and using differentiation medium 2 to perform second stage of differentiation culture; (S5) replacing the culture medium with differentiation medium 3, performing the third stage of differentiation culture, and continuing the culture until the end of this stage; (S6) Continue culturing using maintenance and maturation medium to promote further differentiation and maturation of cells until liver organoids are obtained.

2. The method for differentiating human induced pluripotent stem cells into liver organoids according to claim 1, characterized in that: (S1), the composition of the culture medium supplemented with ROCK inhibitor includes: RPMI1640 culture medium, activin A, bone morphogenetic protein 4 and ROCK inhibitor; Preferably, the concentration of activin A in the culture medium supplemented with ROCK inhibitor is 50-200 ng / mL, and the concentration of bone morphogenetic protein 4 is 25-100 ng / mL; Preferably, the concentration of the ROCK inhibitor in the culture medium added with the ROCK inhibitor is 1 to 20 μM, preferably 5 to 15 μM; Preferably, the ROCK inhibitor is Y-27632; Preferably, in (S1), the inoculation amount of the human induced pluripotent stem cells is (0.75-2)×10 5 cells / cm 2 hole.

3. The method for differentiating human induced pluripotent stem cells into liver organoids according to claim 1 or 2, characterized in that: (S2), the composition of the initial culture medium 1 includes: RPMI1640 culture medium, activin A and fetal bovine serum; Preferably, the concentration of activin A in the initial culture medium 1 is 50-200 ng / mL, and the concentration of fetal bovine serum is 0.1-0.3%; Preferably, in (S2), the composition of the initial culture medium 2 includes: RPMI1640 culture medium, activin A and fetal bovine serum; Preferably, the concentration of activin A in the initial culture medium 2 is 50-200 ng / mL, and the concentration of fetal bovine serum is 1-3%.

4. The method for differentiating human induced pluripotent stem cells into liver organoids according to any one of claims 1 to 3, characterized in that: (S3), the composition of the differentiation medium 1 includes: Advanced DMEM / F12 medium, B27 supplement, N2 supplement, fibroblast growth factor 4 and GSK3β inhibitor; Preferably, the concentration of the B27 supplement in the differentiation medium 1 is 0.5× to 2×, the concentration of the N2 supplement is 0.5× to 2×, the concentration of the fibroblast growth factor is 250 to 1000 ng / mL, and the concentration of the GSK3β inhibitor is 1 to 5 μM; Preferably, the GSK3β inhibitor is CHIR99021; Preferably, the first stage of differentiation culture lasts for 2 to 3 days.

5. The method for differentiating human induced pluripotent stem cells into liver organoids according to any one of claims 1 to 4, characterized in that: In (S4), the digestion solution used in the digestion is 0.5× to 2× Accutase solution; Preferably, in (S4), the inoculation amount of the digested cells is (0.75-1.5)×10 5 cells / cm 2 hole; Preferably, in (S4), the composition of the differentiation medium 2 includes: Advanced DMEM / F12 medium, B27 supplement, N2 supplement, GlutaMAX supplement, basic fibroblast growth factor, vascular endothelial growth factor, epidermal growth factor, GSK3β inhibitor, TGF-β type I receptor inhibitor, ascorbic acid and antibiotics; Preferably, the concentration of the B27 supplement in the differentiation medium 2 is 0.5× to 2×, the concentration of the N2 supplement is 0.5× to 2×, the concentration of the GlutaMAX supplement is 0.5× to 2×, the concentration of the basic fibroblast growth factor is 2.5 to 10 ng / mL, the concentration of the vascular endothelial growth factor is 5 to 20 ng / mL, the concentration of the epidermal growth factor is 10 to 40 ng / mL, the concentration of the GSK3β inhibitor is 1 to 5 μM, the concentration of the TGF-β type I receptor inhibitor is 0.1 to 4 μM, the concentration of ascorbic acid is 20 to 200 μg / mL, and the concentration of the antibiotic is 0.5 to 2%; Preferably, the GSK3β inhibitor is CHIR99021; Preferably, the TGF-β type I receptor inhibitor is A83-01; Preferably, the antibiotics are penicillin and streptomycin; Preferably, the second stage differentiation culture lasts for 3 to 5 days.

6. The method for differentiating human induced pluripotent stem cells into liver organoids according to any one of claims 1 to 5, characterized in that: (S5), the components of the differentiation medium three include: Advanced DMEM / F12 medium, B27 supplement, N2 supplement, GlutaMAX supplement, retinoic acid and antibiotics; Preferably, the concentration of the B27 supplement in the differentiation medium III is 0.5× to 2×, the concentration of the N2 supplement is 0.5× to 2×, the concentration of the GlutaMAX supplement is 0.5× to 2×, the concentration of retinoic acid is 1 to 5 μM, and the concentration of the antibiotic is 0.5 to 2%; Preferably, the antibiotics are penicillin and streptomycin; Preferably, the third stage differentiation culture lasts for 3 to 5 days.

7. The method for differentiating human induced pluripotent stem cells into liver organoids according to any one of claims 1 to 6, characterized in that: (S6), wherein the maintenance and maturation medium comprises: hepatocyte culture medium, hepatocyte growth factor, dexamethasone, tumor growth factor and antibiotics; Preferably, the concentration of hepatocyte growth factor in the maintenance and maturation medium is 5-20 ng / mL, the concentration of dexamethasone is 0.05-0.2 μM, the concentration of tumor growth factor is 10-40 ng / mL, and the concentration of antibiotics is 0.5-2%; Preferably, the antibiotics are penicillin and streptomycin; Preferably, in (S6), the time for differentiation and maturation is 5 to 15 days.

8. The method for differentiating human induced pluripotent stem cells into liver organoids according to any one of claims 1 to 7, characterized in that: In (S1) to (S6), the culture conditions are 36 to 38°C and 4 to 6% CO2.

9. A liver organoid derived from human induced pluripotent stem cells, characterized in that The liver organoids are prepared by the method of differentiating human induced pluripotent stem cells into liver organoids according to any one of claims 1 to 8.

10. Use of the method for differentiating human induced pluripotent stem cells into hepatic organoids according to any one of claims 1 to 8 in hepatic organoid culture.

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