A method for inducing differentiation of human induced pluripotent stem cells to establish liver organoids

Abstract

This invention relates to a method for inducing differentiation of human induced pluripotent stem cells (iPSCs) to establish liver organoids. The method includes: human iPSC culture, directed endoderm induction, foregut endoderm induction, FG cell 3D expansion, and liver organoid induction culture and maturation. This method overcomes the shortcomings of existing technologies. First, human iPSCs can be induced from human blood cells, skin cells, etc., solving the problem of cell source. Second, due to the pluripotency of iPSCs, liver organoids differentiated from iPSCs can generate liver organoids containing multiple cellular components. Furthermore, the method for generating organoids from iPSCs allows for gene editing and labeling of stem cells, and enables large-scale, standardized differentiation, thus possessing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of cell culture, specifically relating to a method for inducing differentiation of human induced pluripotent stem cells to establish liver organoids. Background Technology

[0002] The liver is the most important metabolic organ in the human body, playing a vital role in maintaining physiological homeostasis. The liver is composed of different cell types, including parenchymal hepatocytes and non-parenchymal bile duct cells, Kuff cells, and hepatic stellate cells. Liver diseases caused by various factors have become one of the leading causes of death worldwide. Therefore, a deep understanding of the physiological functions of liver cells is crucial for revealing the mechanisms of liver diseases and providing effective treatments. However, current liver research is largely constrained by the availability of suitable liver research models. Primary human hepatocytes are difficult to obtain, and it is impossible to culture them in vitro for extended periods and maintain their cellular structure and function. Animal models commonly used to study liver diseases, such as rats and mice, differ significantly from humans in their genetic and metabolic mechanisms due to species differences, and thus cannot accurately reflect the true state of human diseases. Therefore, there is an urgent need for a liver disease model that can accurately reflect the true physiological functions of the human liver for experimental and clinical research.

[0003] Current technologies often use human liver-resident stem cells, i.e., adult stem cells (including tumor cells), as the source for constructing liver organoids. Although liver organoids differentiated from human liver-resident stem cells have a short generation cycle, high maturity, and stable genome, cell acquisition is relatively difficult, requiring liver tissue to be obtained through liver biopsy or surgical resection. Moreover, for some severe liver diseases, such as end-stage liver disease, it is even impossible to obtain effective source cells. In addition, due to the limited differentiation potential of adult stem cells, liver organoids differentiated from adult stem cells have a single cell composition, containing only hepatoid cells or bile duct-like cells, and therefore cannot well simulate and reflect liver function. These factors restrict the research and application of liver organoids derived from adult stem cells.

[0004] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a method for inducing differentiation of human induced pluripotent stem cells to establish liver organoids. This method overcomes the aforementioned shortcomings. First, human induced pluripotent stem cells can be induced from human blood cells, skin cells, etc., solving the problem of cell source. Second, due to the pluripotency of pluripotent stem cells, liver organoids differentiated from pluripotent stem cells can generate liver organoids containing multiple cellular components. Furthermore, the method for generating organoids from pluripotent stem cells allows for gene editing and labeling of stem cells, and enables large-scale, standardized differentiation, thus possessing broad application prospects.

[0006] The present invention provides a method for inducing differentiation of human induced pluripotent stem cells to establish liver organoids, the method comprising the following steps:

[0007] (1) Human pluripotent stem cell culture:

[0008] (1) Human induced pluripotent stem cells were cultured in mTeSR1 medium on a culture plate coated with low growth factor Matrigel. The culture plate was placed in a cell culture incubator at 36-38℃ and 4-5% CO2. The culture medium was changed every day. When the cell density reached 75%-85%, the cells were digested into single cells with Accutase enzyme.

[0009] (2) According to approximately 1×10 5 / cm 2 Cells were passaged at a low density and seeded onto new Matrigel-coated culture plates with low growth factor. 10 μM of rock inhibitor (Y-27632) was added to mTeSR1 medium, and the cells were cultured in a cell culture incubator at 36–38°C and 4–5% CO2 for 24 hours. The medium was then replaced and the cells were cultured again. The addition of rock inhibitor during passage was to promote cell adhesion and increase cell viability.

[0010] (II) Induction of directional endoderm (differentiation days 1-3):

[0011] (S1) After passage for about 48 hours, the cells will grow to a density of about 70-90%. If there is no spontaneous differentiation at this time, differentiation can be started (differentiation day 1). Discard the old mTeSR1 medium, wash once with pre-warmed PBS, and add RPMI1640 medium (source: Solarbio, catalog number 31800, the same below) with 50 ng / mL recombinant human bone morphogenetic protein-4 (BMP4) and 80 ng / mL recombinant human activin A (Activin A). Place the culture plate in a cell culture incubator at 36-38℃ and 4-5% CO2.

[0012] (S2) 24 hours later (day 2 of differentiation), discard the old culture medium and add RPMI1640 medium supplemented with 80 ng / mL Activin A and 0.2 vol% serum substitute (KSR). Place the culture plate in a cell culture incubator at 36-38℃ and 4-5% CO2 for further culture.

[0013] (S3) 24 hours later (day 3 of differentiation), discard the old culture medium and add RPMI1640 medium supplemented with 80 ng / mL Activin A and 2 vol% KSR. Place the culture plate in a cell culture incubator at 36-38℃ and 4-5% CO2 for further culture.

[0014] Activin A regulates the Nodal signaling pathway, inducing differentiation of the endoderm and mesoderm. BMP4, a member of the BMP family, participates in the induction and differentiation of the endoderm and mesoderm. The low concentration of Activin A (80 ng / mL) was chosen to reduce its stimulation of cells and decrease cell death.

[0015] (III) Induction of foregut endoderm (FG) (differentiation days 4-6):

[0016] On day 4 of differentiation, the cells had been induced to differentiate into directed endoderm. Immunofluorescence staining at this stage revealed that the vast majority of cells expressed FoxA2 and SOX17. Next, the cells were differentiated into foregut endoderm. The old culture medium was discarded, and high-glucose DMEM medium (source: SC102-02, catalog number SC102-02) supplemented with 50 ng / mL recombinant human fibroblast growth factor 10 (FGF-10), 3 μM CHR99021, 10 vol% KSR, 1% non-essential amino acids (NEAA), and 1% penicillin / streptomycin antibiotics was added. The culture plates were placed in a cell culture incubator at 36–38°C and 4–5% CO2 for further culture. The culture medium was changed after 24 and 48 hours. CHR99021 is a GSK-3α / β inhibitor, a hydrochloride compound that can stimulate the WNT signaling pathway in vivo.

[0017] It is important to emphasize that in this step, high-glucose DMEM medium combined with KSR and non-essential amino acids was used to provide the necessary nutrients for cell growth. Compared to the Advanced DMEM medium combined with N2 and B27 supplements commonly used in other literature, this method reduces economic costs. Furthermore, mitotic growth factor FGF10 was selected to promote cell proliferation and division. Compared to FGF4 and FGF2, which are commonly used in other differentiation methods, the differentiated liver organoids showed richer expression of liver-related genes. Specifically... Figure 5 As shown, by Figure 5 It is evident that fibroblast growth factor (FGF) is required during the differentiation of directional endoderm cells into foregut endoderm. Differentiation was performed using FGF4 (500 ng / ml), FGF2 (10 ng / ml), and FGF10 (50 ng / ml), respectively. By comprehensively comparing the expression of related genes in liver organoids, FGF10 was finally selected as the differentiation factor.

[0018] Differentiation from directional endoderm to foregut endoderm generally uses a combination of fibroblast growth factors FGF4 / FGF2 / FGF10 and CHIR99021 / BMP4 / R-spondin. Mitotic growth factor FGF can promote cell division, and this invention ultimately selected FGF10 through testing; CHIR99021 / BMP4 / R-spondin are all WNT signaling agonists, and this invention selected the low-cost compound CHIR99021.

[0019] (IV) FG cell 3D expansion and liver organoid induction culture (differentiation days 7-13):

[0020] (SS1) On day 7, most cells are induced to foregut endoderm. Microscopic observation at this point reveals a three-dimensional structure formed by a monolayer of cells on the culture plate, including attached and floating spheres. Immunofluorescence staining of the foregut endoderm cells shows that the vast majority express FoxA2 and CDX2. On this day, FG cells need to be transferred from a 2D culture environment to a 3D culture environment for expansion and induction into liver organoids. The old culture medium is discarded, and the cells are digested into single cells with Accutase, resuspended in liquid Matrigel, and added to the culture plate at 75 μl / drop for immobilization. Then, add high-glucose DMEM medium supplemented with 3 μM CHR99021, 5 ng / mL recombinant human fibroblast growth factor-2 (FGF2), 0.5 μM A83-01 (an inhibitor of TGF-β receptor), 20 ng / mL EGF epidermal growth factor, 10 vol% KSR, 1% NEAA and 1% penicillin / streptomycin antibiotics. The culture plates are placed in a cell culture incubator at 36-38℃ and 4-5% CO2 for further culture.

[0021] (SS2) On day 9, discard the old culture medium and add high-glucose DMEM medium supplemented with 3 μM CHR99021, 5 ng / mL LFGF2, 0.5 μM A83-01, 20 ng / mL EGF epidermal growth factor, 2 μM retinoic acid (RA), 10 vol% KSR, 1% NEAA and 1% penicillin / streptomycin antibiotics. The culture plates are placed in a cell culture incubator at 36-38℃ and 4-5% CO2 for further culture.

[0022] (SS3) On the 11th day, discard the old culture medium and then add high-glucose DMEM medium supplemented with 2 μM RA, 10 vol% KSR, 1% NEAA and 1% penicillin / streptomycin antibiotics. Place the culture plate in a cell culture incubator at 36-38℃ and 4-5% CO2 for further culture.

[0023] It is important to emphasize that in this step, high-glucose DMEM medium was used in conjunction with KSR and non-essential amino acids to provide the necessary nutrients for cell growth, further reducing economic costs. Additionally, CHR99021 and FGF2 were used to further promote the differentiation of foregut endoderm cells into liver progenitor cells, while A83-01 and EGF were added to promote cell proliferation. Furthermore, the addition of RA on day 9 of differentiation induced some cells to differentiate into mesodermal mesenchymal cells, ultimately forming hepatic stellate cells and Kupffer cells in liver organoids.

[0024] The differentiation of foregut cells into hepatic progenitor cells is a crucial step in the differentiation of mesenchymal cells. Several other factors aim to improve differentiation efficiency and increase the number of liver organoids. This invention has conducted controlled experiments, such as... Figure 8 As shown, Figure 8 The left image shows a culture medium with only RA added. Figure 8 The right figure shows the culture medium with added RA, CHR99021, FGF2, A83-01 and EGF. The comparison clearly shows that the addition of CHR99021, FGF2, A83-01 and EGF increases the number of organoids generated and improves the differentiation efficiency.

[0025] (V) Liver organoid maturation (13 days after differentiation):

[0026] On day 13 of differentiation, cell clusters of varying sizes, i.e., liver organoids, were visible in the Matrigel. From this day onwards, the culture medium was changed to hepatocyte culture medium supplemented with 100 nM dexamethasone, 20 ng / mL recombinant human tumor suppressor M, and 10 ng / mL recombinant human hepatocyte growth factor (HGF) to induce liver organoid maturation. The medium was changed every 3 days during passage. On day 16, the solid Matrigel gel was separated into a solution by pipetting, and the organoid / Matrigel mixture was transferred to an ultra-low adsorption culture plate, which was then returned to the incubator for further culture. From day 25 of differentiation, the liver organoids could be used for functional analysis or other subsequent applications, or they could be further cultured to allow for further maturation.

[0027] The beneficial effects of this invention are as follows:

[0028] The method for inducing differentiation of human induced pluripotent stem cells to establish liver organoids described in this invention overcomes many shortcomings of the prior art. First, human induced pluripotent stem cells can be induced from human blood cells, skin cells, etc., solving the problem of cell source. In addition, due to the pluripotency of pluripotent stem cells, liver organoids differentiated from pluripotent stem cells can generate liver organoids containing multiple cellular components. Furthermore, the scheme for generating organoids from pluripotent stem cells allows for gene editing and labeling of stem cells, and can be differentiated in large quantities and in a standardized manner, thus having broad application prospects. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 It is a flowchart and morphological diagram of each stage of the differentiation of human induced pluripotent stem cells into liver organoids.

[0031] Figures 2A to 2D This is a graph showing the detection of cell markers at different stages of differentiation, including:

[0032] Figure 2A This is a graph showing the detection of pluripotency markers (NANOG, OCT4) in human induced pluripotent stem cells.

[0033] Figure 2B This is a detection image of directional endoderm cell markers (FOXA2, SOX17);

[0034] Figure 2C This is a detection image of foregut endoderm cell markers (FOXA2 CDX2);

[0035] Figure 2D This is a graph showing the detection of markers for hepatocytes (HNF4α, CK18), bile duct cells (CK19), Kupffer cells (CD68), and hepatic stellate cells (VIM, DES, COL1A1) in liver organoids.

[0036] Figures 3A-3B This is a structural identification diagram of liver organoids, in which:

[0037] Figure 3A This is a light micrograph of a liver organoid;

[0038] Figure 3BThis image shows immunofluorescence staining of paraffin sections of liver organoids with antibodies against epithelial cell marker E-cadherin, hepatocyte marker hepatocyte nuclear factor (HNF4α), and cytoplasmic tight junction protein (ZO-1).

[0039] Figures 4A-4B This is a graph showing the expression of liver-related proteins in liver organoids; among which:

[0040] Figure 4A Immunofluorescence staining was performed on liver organoids to detect the expression of liver-related proteins, including albumin (ALB), α-antitrypsin (AAT1), alpha-fetoprotein (AFP), cytokeratin 18 (CK18), alcohol dehydrogenase (ADH), alkaline phosphatase (ALP), and cytochrome P450 family member 2E1 (CYP2E1).

[0041] Figure 4B The method involves extracting proteins from liver organoids and performing Western blot analysis on liver-related proteins AAT1, ALB, CK18, CYP2E1, HNF4α, and ADH.

[0042] Figure 5 This is the FGF optimized screening chart.

[0043] Figures 6A-6B It is the identification of specific liver functions; among which:

[0044] Figure 6A This is a periodic acid-Schiff staining image of a paraffin section of a liver organoid.

[0045] Figure 6B This is a diagram illustrating the uptake and excretion process of indocyanine green (ICG) in liver organoids.

[0046] Figure 7 This is a diagram for identifying the maturity of liver organoids.

[0047] Figure 8 These are morphological diagrams of liver organoids differentiated into different culture media. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0049] Example

[0050] This embodiment provides a method for inducing differentiation of human induced pluripotent stem cells to establish liver organoids, the method comprising the following steps:

[0051] (1) Human pluripotent stem cell culture:

[0052] (1) Human induced pluripotent stem cells were cultured in mTeSR1 medium on a culture plate coated with low growth factor Matrigel. The culture plate was placed in a cell culture incubator at 37°C and 5% CO2. The culture medium was changed every day. When the cell density reached 75%, the cells were digested into single cells with Accutase enzyme.

[0053] (2) According to 1×10 5 / cm 2 Cells were passaged at a low density and seeded onto new Matrigel-coated culture plates with low growth factor. 10 μM of rock inhibitor was added to mTeSR1 medium, and the cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The medium was then changed and the cells were cultured for another 24 hours.

[0054] (II) Induction of directional endoderm (differentiation days 1-3):

[0055] (S1) After passage for 48 hours, the cells reach a density of about 70%. If there is no spontaneous differentiation, differentiation can begin (day 1 of differentiation). Discard the old mTeSR1 medium, wash once with pre-warmed PBS, and add RPMI1640 medium (source: Solarbio, catalog number 31800, the same below) containing 50 ng / mL recombinant human bone morphogenetic protein-4 (BMP4) and 80 ng / mL recombinant human activin A (Activin A). Place the culture plate in a cell culture incubator at 37°C and 5% CO2.

[0056] (S2) 24 hours later (day 2 of differentiation), discard the old culture medium and add RPMI1640 medium supplemented with 80 ng / mL Activin A and 0.2 vol% serum substitute (KSR). Place the culture plate in a cell culture incubator at 37°C and 5% CO2 for further culture.

[0057] (S3) 24 hours later (day 3 of differentiation), discard the old culture medium and add RPMI 1640 medium with 80 ng / mL Activin A and 2 vol% KSR. Place the culture plate in a cell culture incubator at 37°C and 5% CO2 and continue to culture.

[0058] (III) Induction of foregut endoderm (FG) (differentiation days 4-6):

[0059] On day 4 of differentiation, the cells had been induced to differentiate into directed endoderm. Immunofluorescence staining of these cells at this stage revealed that the vast majority expressed FoxA2 and SOX17. Next, the cells were differentiated into foregut endoderm. The old culture medium was discarded, and high-glucose DMEM medium (source: Scivine, catalog number SC102-02, hereinafter the same) supplemented with 50 ng / mL recombinant human fibroblast growth factor 10 (FGF-10), 3 μM CHR99021, 10 vol% KSR, 1% non-essential amino acids (NEAA), and 1% penicillin / streptomycin antibiotics was added. The culture plates were placed in a cell culture incubator at 37°C and 5% CO2 and cultured for another 24 and 48 hours.

[0060] (IV) FG cell 3D expansion and liver organoid induction culture (differentiation days 7-13):

[0061] (SS1) On day 7, most cells are induced to foregut endoderm. Microscopic observation at this point reveals a three-dimensional structure formed by a monolayer of cells on the culture plate, including attached and floating spheres. Immunofluorescence staining of the foregut endoderm cells shows that the vast majority express FoxA2 and CDX2. On this day, FG cells need to be transferred from a 2D culture environment to a 3D culture environment for expansion and induction into liver organoids. The old culture medium is discarded, and the cells are digested into single cells with Accutase, resuspended in liquid Matrigel, and added to the culture plate at 75 μl / drop for immobilization. Then, high-glucose DMEM medium containing 3 μM CHR99021, 5 ng / mL recombinant human fibroblast growth factor-2 (FGF2), 0.5 μM A83-01 kinase inhibitor, 20 ng / mL EGF epidermal growth factor, 10 vol% KSR, 1% NEAA and 1% penicillin / streptomycin antibiotics was added, and the culture plates were placed in a cell culture incubator at 37°C and 5% CO2 for further culture.

[0062] (SS2) On day 9, discard the old culture medium and add high-glucose DMEM medium supplemented with 3 μM CHR99021, 5 ng / mL LFGF2, 0.5 μM A83-01, 20 ng / mL EGF epidermal growth factor, 2 μM retinoic acid (RA), 10 vol% KSR, 1% NEAA and 1% penicillin / streptomycin antibiotics. The culture plate was placed in a cell culture incubator at 37°C and 5% CO2 for further culture.

[0063] (SS3) On day 11, discard the old culture medium and add high-glucose DMEM medium supplemented with 2 μM RA, 10 vol% KSR, 1% NEAA and 1% penicillin / streptomycin antibiotics. Place the culture plate in a cell culture incubator at 37°C and 5% CO2 for further culture.

[0064] It is important to emphasize that in this step, high-glucose DMEM medium was used in conjunction with KSR and non-essential amino acids to provide the necessary nutrients for cell growth, further reducing economic costs. Additionally, CHR99021 and FGF2 were used to further promote the differentiation of foregut endoderm cells into liver progenitor cells, while A83-01 and EGF were added to promote cell proliferation. Furthermore, the addition of RA during differentiation induced some cells to differentiate into mesodermal mesenchymal cells, ultimately forming hepatic stellate cells and Kupffer cells in liver organoids.

[0065] (V) Liver organoid maturation (13 days after differentiation):

[0066] On day 13 of differentiation, cell clusters of varying sizes, i.e., liver organoids, were visible in the Matrigel. From this day onwards, the culture medium was changed to hepatocyte culture medium supplemented with 100 nM dexamethasone, 20 ng / mL recombinant human tumor suppressor M, and 10 ng / mL recombinant human hepatocyte growth factor (HGF) to induce liver organoid maturation. The medium was changed every 3 days during passage. On day 16, the solid Matrigel gel was separated into a solution by pipetting, and the organoid / Matrigel mixture was transferred to an ultra-low adsorption culture plate, which was then returned to the incubator for further culture. From day 25 of differentiation, the liver organoids could be used for functional analysis or other subsequent applications, or they could be further cultured to allow for further maturation.

[0067] Experimental Results and Analysis:

[0068] To illustrate the differentiation process and its corresponding effects, the inventors tracked the entire cell differentiation process, as follows:

[0069] (1) Figure 1 This demonstrates the process by which human induced pluripotent stem cells differentiate into desiccated organoids, and the cell morphology shows that each stage of differentiation was successfully carried out.

[0070] (2) Furthermore, different specific protein markers are expressed within cells at different differentiation stages, and these specific protein markers at different differentiation stages were detected by immunofluorescence staining. Figure 2A It is evident that the pluripotency markers NANOG and OCT4 were detected in human induced pluripotent stem cells; Figure 2B It was observed that the markers FOXA2 and SOX17 were detected in the directional endoderm cells; Figure 2C It was observed that the markers FOXA2 and CDX2 were detected in foregut endoderm cells; Figure 2D As can be seen, hepatocyte markers HNF4α and CK18, bile duct cell marker CK19, Kupffer cell marker CD68, and hepatic stellate cell markers VIM, DES, and COL1A1 were detected in liver organoids. This also indicates that differentiation at each stage proceeded successfully and that cell function was fully developed. Figures 2A to 2C The specimen was a cell smear. Figure 2D (The specimen is a paraffin section)

[0071] The immunofluorescence staining method is as follows:

[0072] Cell slide staining: Remove the cell slides and fix them with 4% paraformaldehyde at room temperature for 10-15 min. Aspirate the fixative, wash three times with 1×PBS for 3-5 min each time; then permeabilize on ice with 0.1% Triton X-100 for about 10 min. Aspirate the permeabilization buffer, wash three times with 1×PBS for 3-5 min each time; block with 1% BSA / PBS at room temperature for 1 h. Discard the blocking buffer, add primary antibody diluted with the blocking buffer, and incubate overnight at 4°C. Aspirate the primary antibody, wash three times with 1×PBS for 2-5 min each time; then add secondary antibody diluted with the blocking buffer, and incubate at room temperature in the dark for 1-3 h. Aspirate the secondary antibody, wash three times with 1×PBS in the dark for 3-5 min each time, add DAPI staining solution, and incubate in the dark for 5 min. Discard the DAPI staining solution, add anti-fluorescence quenching mounting medium, and image and photograph under a fluorescence inverted microscope for storage.

[0073] Organoid staining: Liver organoids were fixed in 4% PFA for 4 h, then dehydrated and embedded in paraffin, and sectioned to a thickness of 4 μm. Paraffin sections were dewaxed with xylene, rehydrated, placed in Tris-EDTA buffer (pH 9.0), and subjected to antigen retrieval by microwave heating for 15 min. After blocking with 1% BSA / PBS for 1 hour, they were incubated with primary antibody, and subsequent procedures were the same as those described for cell slide staining.

[0074] (3) Continue to perform morphological identification on the obtained liver organoids, by Figure 3A As can be seen from microscopic observation, liver organoids contain spherical cell clusters and irregularly structured cell clusters; the black arrows in the image indicate irregular interstitial cell clusters. Immunofluorescence staining further reveals that... Figure 3B It is evident that the spherical structures in liver organoids are hollow sac-like structures composed of hepatocytes, exhibiting distinct cell arrangement polarity, while the irregular cell clusters are composed of non-epithelial mesenchymal cells.

[0075] (4) Continue to detect liver-related proteins in the obtained liver organoids. Figure 4A As can be seen (specimen type: paraffin section), immunofluorescence staining results showed that liver-related proteins albumin (ALB), α-antitrypsin (AAT1), alpha-fetoprotein (AFP), cytokeratin 18 (CK18), alcohol dehydrogenase (ADH), alkaline phosphatase (ALP), and cytochrome P450 family member 2E1 (CYP2E1) were expressed in liver organoids; Figure 4B As can be seen, the immunoblotting results showed that traces of liver-related proteins AAT1, ALB, CK18, CYP2E1, HNF4α and ADH were present in the protein extract of liver organoids, indicating that liver organoids have cellular composition and histological characteristics close to those of human physiological state.

[0076] Western blotting:

[0077] Total protein was extracted from liver organoids at 4°C using cell lysis buffer supplemented with protease inhibitors, and protein concentration was determined using the BCA method. Protein samples were separated by electrophoresis on a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to a PVDF membrane. After blocking with 5% skim milk for 1 hour at room temperature, the PVDF membrane was incubated overnight at 4°C with diluted primary antibody. After washing the membrane three times with PBST, it was incubated for 2 hours at room temperature with peroxidase-conjugated goat anti-rabbit and mouse secondary antibodies. After washing three times with PBST, the protein blots on the PVDF membrane were detected using ECL luminescent solution (EpiZyme) on a ChemiDoc MP electrophoresis imaging system (Bio-Rad).

[0078] (5) Continue to identify liver-specific functions, by Figure 6A As can be seen, the periodic acid Schiff reaction (PAS) test results show purple positive substances in the epithelial cells of liver organoids, indicating the presence of glycogen within the cells (PAS staining interpretation criteria: cell nuclei are blue; cytoplasm is pale red; positive substances such as glycogen, fungi, and plant starch granules are purple-red). Figure 6B As can be seen from the indocyanine green (ICG) uptake and excretion results, ICG was observed in liver organoids after 0.5 h of ICG treatment. Upon transfer to normal culture medium and continued culturing, the ICG uptake by the liver organoids was gradually excreted, and no visible ICG was found in the organoids after 30 h. This indicates that liver organoids contain albumin that binds to ICG and also possess the liver-specific function of excreting ICG.

[0079] Glycogen PAS staining (periodic acid-Schiff staining):

[0080] Paraffin sections of organoids were dewaxed, rehydrated, and rinsed twice with distilled water. Oxidizing agent was added to the sections, and they were incubated at room temperature for 5 minutes, rinsed with tap water for 2 minutes, and rinsed twice with distilled water. Schiff's stain was added to the sections, and they were stained at room temperature in the dark for 15 minutes, followed by rinsing with tap water for 10 minutes. The sections were then stained with hematoxylin for 1 minute, differentiated in acidic differentiation solution (1% hydrochloric acid / ethanol) for 5 seconds, and rinsed with tap water for 10 minutes. The sections were dehydrated with anhydrous ethanol, cleared with xylene, and mounted with neutral resin. The staining results were observed and interpreted under a microscope, and photographs were taken. Interpretation criteria: cell nuclei were blue; cytoplasm was pale red; positive substances such as glycogen, fungi, and plant starch granules appeared purplish-red.

[0081] Indocyanine Green (ICG) intake and excretion:

[0082] Collect liver organoids from the culture plate and transfer them to 15 mL centrifuge tubes, washing once with PBS. Add complete HCM medium containing 1 mg / m³ ICG (Source Leaf Biotechnology, S46424) and incubate at 37°C for 30 min. Centrifuge at 300×g for 3 min, discard the supernatant, wash three times with PBS, and then add complete HCM medium. Observe and photograph images of organoid cell ICG uptake under a microscope. After photography, continue culturing at 37°C. Observe the cell excretion of ICG under a microscope every few hours until the cells completely excrete ICG and photograph the result.

[0083] (6) The maturity of liver organoids was further assessed by detecting gene expression. Quantitative real-time PCR was used to measure the expression levels of several genes related to the function of hepatocytes, bile duct cells, hepatic stellate cells, and Kupffer cells in liver organoids differentiated for approximately 30 days. These gene expression levels were compared with those in liver tissue from 20-week gestational age and normal adult liver tissue. The relative maturity of the liver organoids was evaluated using the expression level of relevant genes in adult liver tissue as a baseline. Figure 7 As shown, LO represents liver organoids; FL represents fetal liver tissue; and AL represents adult liver tissue.

[0084] Quantitative real-time PCR:

[0085] Total RNA was isolated from liver organoids, adult liver tissue, and fetal liver tissue using the chloroform method. It was then reverse transcribed into cDNA. Quantitative PCR was performed using Ex Taq™ II (TaKaRa) on an Applied Biosystems QuantStudio3 real-time PCR system (Thermo Fisher). Primer information for each target gene was obtained from Primerbank. Reaction conditions: 95°C for 1 minute, 40 cycles of 95°C for 5 seconds each, followed by 60°C for 30 seconds each, and then a melt curve phase. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and three technical replicates were performed for each sample.

[0086] In summary, this invention establishes a differentiation protocol for differentiating human induced pluripotent stem cells into liver organoids. By adding cytokines and small molecule compounds to the culture medium and using Matrigel in 3D culture, liver organoids containing various liver cell components, including hepatoid cells, bile duct-like cells, hepatic stellate cells, and Kuff's cells, can be generated in approximately 20 days. The generated liver organoids have a three-dimensional structure, in which hepatoid cells and bile duct-like cells form spherical, hollow sac-like structures with the polarized connection arrangement characteristics of epithelial cells; while cells expressing relevant markers of hepatic stellate cells and Kuff's cells aggregate into irregular cell clusters, consistent with the characteristics of mesenchymal cells. Immunofluorescence staining and Western blotting experiments confirmed that liver organoids generated from human induced pluripotent stem cells express various liver-specific proteins and proteins related to liver function. PAS staining confirmed that liver organoids have glycogen synthesis and storage functions. ICG experiments confirmed that liver organoids also have indogreen uptake and excretion functions. Quantitative real-time PCR confirmed that the generated liver organoids contained the expression of multiple genes related to liver function. The expression levels of most liver-related genes detected in liver organoids differentiated at around 30 days were between those of normal adult liver tissue and fetal liver tissue at 20 weeks of gestation, suggesting that the maturity of the organoids was no less than that of fetal liver tissue at 20 weeks of gestation.

[0087] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for inducing differentiation of human induced pluripotent stem cells to establish liver organoids, characterized in that, The method includes the following steps: (I) Human pluripotent stem cell culture: (1) Human induced pluripotent stem cells were cultured on a culture plate coated with low growth factor matrix using mTeSR1 medium. When the cell density of the human induced pluripotent stem cells reached 75-85%, they were digested into single cells using Accutase enzyme. (2) The single cells were re-inoculated onto another culture plate coated with low growth factor matrix gel and cultured with ROCK inhibitor, the ROCK inhibitor being Y-27632; (II) Induction of directional endoderm, differentiation days 1-3: (S1) On the first day of differentiation, when the cells grow to a cell density of 70-90%, differentiation is carried out. The mTeSR1 medium is discarded and the cells are added to RPMI1640 medium containing recombinant human bone morphogenetic protein-4 and recombinant human activin A for culture. (S2) On the second day of differentiation, after the culture in step (S1) is completed, discard the culture medium in step (S1) and add RPMI1640 medium containing recombinant human activin A and serum substitute to continue the culture. (S3) On the third day of differentiation, after the culture in step (S2) is completed, discard the culture medium in step (S2) and add RPMI1640 medium containing recombinant human activin A and serum substitute to continue the culture until the cells are induced to differentiate into directional endoderm; (III) Induction of foregut endoderm, differentiation days 4-6: Discard the culture medium in step (S3), add high-glucose DMEM medium supplemented with recombinant human fibroblast growth factor-10, CHIR99021, serum substitute, non-essential amino acids and penicillin / streptomycin antibiotics, and culture until it is induced to form foregut endoderm; (IV) 3D expansion of foregut endoderm cells and induction culture of liver organoids, differentiation days 7-13: (SS1) On day 7 of differentiation, discard the culture medium from step (III), digest the cells with Accutase enzyme into single cells, resuspend them in matrix gel, and then drop them into a culture plate for solidification. Then add a culture medium consisting of CHIR99021, recombinant human fibroblast growth factor-2, A83-01, EGF epidermal growth factor, serum substitute, non-essential amino acids, penicillin / streptomycin antibiotics and high glucose DMEM medium, and culture. (SS2) On day 9 of differentiation, after the culture in step (SS1) is completed, discard the culture medium from step (SS1), and then add a culture medium composed of CHIR99021, recombinant human fibroblast growth factor-2, A83-01, EGF epidermal growth factor, retinoic acid, serum substitute, non-essential amino acids, penicillin / streptomycin antibiotics, and high-glucose DMEM medium, and culture. The concentrations of CHIR99021, recombinant human fibroblast growth factor-2, and A83-01 are 3 μM, 20 ng / mL, 2 μM, 10 vol%, 1%, and 1%, respectively. (SS3) On the 11th day of differentiation, after the culture in step (SS2) is completed, discard the culture medium in step (SS2), and then add a culture medium composed of retinoic acid, serum substitute, non-essential amino acids, penicillin / streptomycin antibiotics and high glucose DMEM medium, and culture. (V) Liver organoids mature and differentiate 13 days later: On day 13 of differentiation, when cell clusters appeared during differentiation, the culture medium in step (SS3) was discarded, and then hepatocyte culture medium containing dexamethasone, recombinant human tumor suppressor M and recombinant human hepatocyte growth factor was added to induce liver organoid maturation.

2. The method for inducing differentiation of human induced pluripotent stem cells to establish liver organoids according to claim 1, characterized in that, The culture temperature is 36~38℃, and the CO2 content is 4~5%.

3. The method for inducing differentiation of human induced pluripotent stem cells to establish liver organoids according to claim 1, characterized in that, In step (S1), the concentration of recombinant human bone morphogenetic protein-4 is 50 ng / mL, and the concentration of recombinant human activin A is 80 ng / mL.

4. The method for inducing differentiation of human induced pluripotent stem cells to establish liver organoids according to claim 1, characterized in that, In step (S2), the concentration of recombinant human activin A is 80 ng / mL, and the concentration of the serum substitute is 0.2 vol.

5. The method for inducing differentiation of human induced pluripotent stem cells to establish liver organoids according to claim 1, characterized in that, In step (S3), the concentration of recombinant human activin A is 80 ng / mL, and the concentration of the serum substitute is 2 vol.

6. The method for inducing differentiation of human induced pluripotent stem cells to establish liver organoids according to claim 1, characterized in that, In step (III), the concentration of recombinant human fibroblast growth factor-10 is 50 ng / mL, the concentration of CHIR99021 is 3 μM, the concentration of serum substitute is 10 vol%, the concentration of non-essential amino acids is 1%, and the concentration of penicillin / streptomycin bispecific antibody is 1%.

7. The method for inducing differentiation of human induced pluripotent stem cells to establish liver organoids according to claim 1, characterized in that, In step (SS1), the concentration of CHIR99021 is 3 μM, the concentration of recombinant human fibroblast growth factor-2 is 5 ng / mL, the concentration of A83-01 is 0.5 μM, the concentration of EGF epidermal growth factor is 20 ng / mL, the concentration of serum substitute is 10 vol%, the concentration of non-essential amino acids is 1%, and the concentration of penicillin / streptomycin bispecific antibody is 1%.

8. The method for inducing differentiation of human induced pluripotent stem cells to establish liver organoids according to claim 1, characterized in that, In step (V), the concentration of dexamethasone is 100 nM, the concentration of recombinant human tumor suppressor M is 20 ng / mL, and the concentration of recombinant human hepatocyte growth factor is 10 ng / mL.

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