Methods and applications of constructing liver organoids with sinusoidal networks

Abstract

The present invention provides a method for constructing a sinusoidal network liver organoid and its application, wherein pluripotent stem cells are co-differentiated into HAND1 + Mesoderm and endoderm produce KDR + Mesodermal progenitors give rise to the posterior foregut endoderm, which has CD34 + / CD31 + Endothelial progenitor cells form hepatic endoderm and form a sinusoidal network in liver organoids. These liver organoids possess a functional sinusoidal endothelial network throughout the liver clusters, exhibiting characteristic functions such as low-density lipoprotein uptake, human serum albumin clearance, and secretion of coagulation factor 8. This method provides a new generation of highly biomimetic research tools for understanding liver disease mechanisms, drug screening, and regenerative repair.

Description

Technical Field

[0001] The present invention belongs to the field of cell culture technology, and in particular relates to a method for constructing a sinusoidal networked liver organoid and its application. Background Art

[0002] The technology for liver-directed differentiation of human induced pluripotent stem cells (iPSCs) has experienced rapid advancements, from the induction of iPSCs into 2D hepatocyte-like cells, to 3D hepatobiliary organoids (combining both hepatocytes and cholangiocytes), and finally to the successful generation of complex 3D liver organoids containing hepatocytes, cholangiocytes, hepatic stellate cells, and hepatic macrophages. With the continuous advancement of liver-directed differentiation technology, these highly biomimetic human organoids are becoming new precision tools for liver disease investigation, drug development, and regenerative repair.

[0003] Currently, liver organoids lack functional hepatic sinusoidal endothelial cells, important cell types associated with liver physiology. As a unique type of vascular endothelium in the liver, the hepatic sinusoidal endothelium plays a crucial role in liver embryonic development, maintenance of adult homeostasis, and disease progression. During embryonic development, the interaction between the hepatic sinusoidal endothelium and hepatic progenitor cells is essential for the organ's metabolic maturation. Under physiological conditions, the sinusoidal endothelium acts as a scavenger within the liver, selectively absorbing nutrients from the bloodstream through connections to the portal vein and central veins, and clearing away metabolic waste products such as redundant lipoproteins and albumin. However, when exposed to pathological environments, the sinusoidal endothelium directly or indirectly mediates the formation of fibrosis, exacerbated inflammatory responses, and the development of carcinogenesis. Currently, no other technology can achieve the construction of sinusoidal networked liver organoids. Therefore, the lack of this characteristic liver lineage / structure hinders the accuracy of this tool model. Summary of the Invention

[0004] In view of this, the present invention aims to propose a method for constructing a sinusoidal network liver organoid and its application, in which pluripotent stem cells are differentiated into HAND1 + Mesoderm and endoderm produce KDR + Mesodermal progenitors give rise to the posterior foregut endoderm, which has CD34 + / CD31 + Hepatic endoderm formed from endothelial progenitor cells and formed sinusoidal network-forming liver organoids. The liver organoids generated using this method possess a functional sinusoidal endothelial network throughout the liver clusters, exhibiting characteristic functions such as low-density lipoprotein uptake, human serum albumin clearance, and secretion of coagulation factor 8.

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] A method for constructing a sinusoidal networked liver organoid, the method comprising the following steps:

[0007] S1. Use medium containing BMP4 and bFGF to induce PSCs and produce HAND1 + Mesoderm and FOXA2 + Endoderm;

[0008] S2, use culture medium containing BMP4, bFGF, and Wnt agonist to continue inducing KDR + HHEX of mesodermal progenitor cells + HNF4α + The posterior endoderm of the foregut; and the concentrations of BMP4 and bFGF used were greater than those used in S1;

[0009] S3, enzymatically digesting the derivatives obtained in S2, collecting them by centrifugation, and resuspending the single cells in a medium containing VEGF, EGF, and bFGF, and then adding COL1 and LN411 for 3D repolymerization;

[0010] After the culture medium was replaced, the culture medium was used to induce the production of CD31 + / CD34 + Hepatic endoderm of primary endothelial cells, and no COL1 and LN411 were added thereafter, and the concentration of bFGF used was lower than that used in S2;

[0011] S4. Use culture medium containing FSK and cAMP agonist to continue inducing the generation of sinusoidal network liver organoids.

[0012] In some embodiments, in S1, regular culture medium is used for culture on day 1; culture medium containing BMP4 and bFGF is used for culture on days 2-3, and the BMP4 concentration used on days 2-3 is lower than that used on day 1, and the culture medium is replaced every 24 hours.

[0013] In some embodiments, the regular culture medium includes 10-75 ng / ml BMP4 and 100-150 ng / ml Activin A; the culture medium used on days 2-3 includes 100-150 ng / ml Activin A, 1-10 ng / ml BMP4, and 50-200 ng / ml bFGF.

[0014] In some embodiments, the culture medium used for S2 includes 10-50 ng / ml BMP4, 200-500 ng / ml bFGF, and 1-5 μM Wnt agonist, including CHIR99021, Wnt3a, and Wnt7a. S2 is cultured for 3-4 days, and the culture medium is changed daily.

[0015] In some embodiments, in S3, the concentration of COL1 is 0.25-1.25 mg / ml, and the concentration of LN411 is 1-5 μg / ml.

[0016] In some embodiments, the culture medium used for S3 includes 50-150 ng / ml VEGF, 5-25 ng / ml EGF, and 10-50 ng / ml bFGF. The S3 culture is performed for 4-5 days, and the culture medium is replaced every 48 hours.

[0017] In some embodiments, the culture medium used for S3 further comprises 1-10 μM Wnt agonist, 1-10 μM TGFβ inhibitor, Wnt agonists include CHIR99021, Wnt3a, Wnt7a, and TGFβ inhibitors include A83-01 and SB431542.

[0018] In some embodiments, the culture medium used for S4 includes 1-10 μM FSK, 0.25-1 mM cAMP agonist, the cAMP agonist is 8-Br-cAMP, the S4 culture period is 20-25 days, and the culture medium is replaced every 72 hours.

[0019] In some embodiments, the culture medium used for S4 further includes 5-25 ng / ml HGF, 10-25 ng / ml OSM, and 0.1-0.5 μM DEX.

[0020] The present invention also provides a culture medium for constructing sinusoidal networked liver organoids, the culture medium comprising a first culture medium, a second culture medium, a third culture medium, a fourth culture medium and a fifth culture medium;

[0021] The first culture medium includes BMP4 and bFGF for inducing the generation of endoderm in S1 and co-differentiating into mesoderm;

[0022] The second culture medium includes a KDR + Mesodermal progenitor cells and HHEX + HNF4α + BMP4, bFGF, and Wnt agonists in the posterior foregut endoderm;

[0023] The third culture medium includes VEGF, EGF, bFGF that promote angiogenesis, and COL1 and LN411 that form a flexible scaffold;

[0024] The fourth culture medium includes VEGF, EGF, and bFGF that promote angiogenesis;

[0025] The fifth culture medium includes FSK and cAMP agonists that help promote the growth of sinusoidal networks.

[0026] The present invention also provides a sinusoidal network liver organoid constructed by the above-mentioned sinusoidal network liver organoid construction method, which has a functional sinusoidal endothelial network running through the liver clusters, exhibits the functions of low-density lipoprotein uptake, human serum albumin clearance, and mediating the secretion of coagulation factor 8, and also contains CK7 + Cholangiocytes, CD68 + Hepatic macrophages and VIM + Hepatic stellate cells.

[0027] The present invention also provides an application of the sinusoidal networked liver organoid as described above in liver disease mechanism analysis, drug screening, and regeneration and repair.

[0028] Compared with the existing technology, the sinusoidal network liver organoid construction method and application described in the present invention have the following advantages:

[0029] (1) The method for constructing a sinusoidal network liver organoid described in the present invention establishes a liver organoid with a functional sinusoidal network. The liver organoid possesses five cell types related to liver physiology, such as sinusoidal endothelial cells, hepatocytes, bile duct cells, hepatic stellate cells, and hepatic macrophages. The liver organoid also exhibits characteristic functions such as low-density lipoprotein uptake, human serum albumin clearance, and mediated secretion of coagulation factor 8. This method provides a new generation of highly biomimetic research tools for analyzing liver disease mechanisms, drug screening, and regenerative repair.

[0030] (2) The sinusoidal networked liver organoids described in the present invention are fully endogenously differentiated. The organoids are completely derived from a single iPSC without the need for any exogenous cell addition. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0032] Figure 1 Flowchart of the method for constructing sinusoidal networked liver organoids;

[0033] Figure 2 Representative immunofluorescence staining of S1 derivatives, HAND1 is a mesoderm marker, and FOXA2 is an endoderm marker; scale bar = 200 μm;

[0034] Figure 3 Representative immunofluorescence staining of S2 derivatives. A is immunofluorescence co-staining of KDR, a hematopoietic mesoderm marker, and HHEX, a posterior foregut endoderm marker. B is immunofluorescence co-staining of HHEX, a posterior foregut endoderm marker, and HNF4α. KDR is a hematopoietic mesoderm marker; HHEX and HNF4α are both posterior foregut endoderm markers. Scale bar = 200 μm.

[0035] Figure 4 Representative immunofluorescence staining of S3 derivatives, A: immunofluorescence co-staining of vascular endothelial progenitor cell marker CD34 and hepatic progenitor cell marker AFP; B: immunofluorescence co-staining of vascular progenitor cell markers CD34 and CD31; C: immunofluorescence co-staining of sinusoidal endothelial cell marker LYVE1 and hepatic progenitor cell marker AFP; D: immunofluorescence co-staining of bile duct cell marker CK19 and hepatic progenitor cell marker AFP; scale bar = 200 μm;

[0036] Figure 5 Characterization analysis of S4 derivatives. A: Immunofluorescence staining of LYVE1, a marker of sinusoidal endothelial cells, and ALB, a marker of hepatocytes. B: Immunofluorescence staining of CK7 / CK19, a marker of cholangiocytes, CD68, a marker of macrophages, and VIM, a marker of hepatic stellate cells. C: Flow cytometric analysis of LYVE1, a marker of sinusoidal endothelial cells, and ALB, a marker of hepatocytes. Biological replicates (n=3). LYVE1: a marker of sinusoidal endothelial cells. ALB and HNF4α: markers of hepatocytes. CK7: a marker specific to cholangiocytes. CK19: a marker of cholangiocytes. CD68: a marker of macrophages. VIM: a marker of hepatic stellate cells. Scale bar = 50 μm.

[0037] Figure 6 For functional analysis of sinusoidal endothelium, A is LYVE1 + Analysis of the clearance efficiency of low-density lipoprotein (AcLDL-AF594) by cells, B is LYVE1 + Analysis of the cell clearance efficiency of formaldehyde-treated serum albumin (FSA-FITC), biological replicates n=4; *, P < 0.05; ***, P < 0.001; C is the ELISA analysis of coagulation factor 8 (F8) in the culture supernatant; PSC (pluripotent stem cells on differentiation day 0), sinusoidal KO liver organoids (i.e., LYVE1 was removed from the organoids sorted on differentiation day 30) + Organoids formed by 3D reaggregation of cells), and HUVECs (human umbilical vein endothelial cells) as negative controls; biological replicates, n = 4; ***, P < 0.001; ND, not reaching the limit of detection;

[0038] Figure 7Comparison of the results of the two induction methods. A is the immunofluorescence identification of mesoderm markers on differentiation day 3 of the two induction methods, scale bar = 200 μm; B is the flow cytometric analysis of sinusoidal endothelial cells on differentiation day 30, biological replicates n = 3; ***, P < 0.001;

[0039] Figure 8 Flow cytometric analysis of S1 derivatives at day 3 of differentiation under different bFGF and BMP4 concentrations, biological replicates n = 3; **, P < 0.01; ***, P < 0.001;

[0040] Figure 9 The immunofluorescence identification results of differentiation day 6 derivatives under different induction factor combinations in S2, where 3F represents the use of this method at the S2 stage, 2F (-BMP4) represents the withdrawal of BMP4, 2F (-CHIR) represents the withdrawal of CHIR99021, 2F (-bFGF) represents the withdrawal of bFGF, and 1F (-CHIR -bFGF) represents the simultaneous withdrawal of CHIR99021 and bFGF. Scale bar = 200 μm.

[0041] Figure 10 The results of immunofluorescence identification of differentiation day 10 derivatives under different combinations of extracellular matrix components COL1 and LN411 in S3, where 2M represents the use of this method at the S3 stage, 1M (-COL1) represents the withdrawal of COL1, and 1M (-LN411) represents the withdrawal of LN411. Scale bar = 200 μm.

[0042] Figure 11 Analysis of the expression levels of vascular progenitor cell markers CD34 and CD31 in S3 derivatives at differentiation day 10 under different induction factor combinations; 2F represents CHIR99021+A83-01; biological replicates, n=3; *, P<0.05; **, P<0.01; ***, P<0.001;

[0043] Figure 12 Analysis of sinusoidal network intersection nodes in liver organoids at differentiation day 30 under different combinations of FSK and cAMP activators. 3F represents OSM+HGF+DEX; biological replicates, n=8; **P<0.01; ***, P<0.001. DETAILED DESCRIPTION

[0044] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0045] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0046] The reagents used in the examples and comparative examples are shown in Table 3-4.

[0047] The present invention proposes a method for constructing a sinusoidal network liver organoid, which comprises the following steps:

[0048] S1. Use medium containing BMP4 and bFGF to induce PSCs and produce HAND1 + Mesoderm and FOXA2 + Endoderm.

[0049] Specifically, S1 includes the following steps:

[0050] S11. Culture in conventional culture medium for 1 day. Conventional culture medium includes basal culture medium and additives. The basal culture medium is RPMI-1640 containing 2% B27 by volume. The additives include 10-75 ng / ml BMP4 and 100-150 ng / ml Activin A. Specifically, 50 ng / ml BMP4 and 100 ng / ml Activin A can be selected.

[0051] S12. Culture cells for 2-3 days in the first culture medium, which includes basal medium (RPMI-1640 containing 2% B27 by volume) and supplements (100-150 ng / ml Activin A, 1-10 ng / ml BMP4, and 50-200 ng / ml bFGF). Specifically, consider 100 ng / ml Activin A, 5 ng / ml BMP4, and 50 ng / ml bFGF. During this stage, the concentrations of BMP4 and bFGF must be within the specified range; exceeding these limits will affect the final differentiation efficiency. Change the culture medium every 24 hours.

[0052] S2, use culture medium containing BMP4, bFGF, and Wnt agonist to continue inducing KDR + HHEX of mesodermal progenitor cells + HNF4α + The posterior endoderm of the foregut; and the concentrations of BMP4 and bFGF used were greater than those used in S1;

[0053] The culture medium used in this stage is the second culture medium, which includes a basal culture medium and additives. The basal culture medium includes 2% B27 by volume, 23% StemPro34 by volume, and 75% IMDM by volume; the additives include 10-50 ng / ml BMP4, 200-500 ng / ml bFGF, and 1-5 μM Wnt agonists. Wnt agonists include CHIR99021, Wnt3a, and Wnt7a. The S2 culture period is 3-4 days, and the culture medium is replaced daily.

[0054] Specifically, the additives may include 10 ng / ml BMP4, 500 ng / ml bFGF, and 3 μM Wnt agonist CHIR99021. Furthermore, the concentrations of the additives are not limited to these and can be achieved within the stated range. The Wnt agonist is not limited to the listed CHIR99021, Wnt3a, and Wnt7a. Any Wnt agonist that can achieve the objectives of the present invention can be used alone or in combination, and the use of any one of these is preferred.

[0055] S3. The derivatives obtained in S2 were enzymatically digested and collected by centrifugation. Single cells were resuspended in a medium containing VEGF, EGF, and bFGF, and COL1 and LN411 were added. After mixing, the cells were seeded into a 96-well U-shaped culture plate at a seeding volume of 10,000 cells / well for 3D repolymerization.

[0056] The culture medium used is the third culture medium, which includes a basal culture medium and additives. The basal culture medium includes 2% B27 by volume, 23% StemPro34 by volume, and 75% IMDM by volume; the additives include 50-150ng / ml VEGF, 5-25ng / ml EGF, 10-50ng / ml bFGF, 1-10μM Wnt agonist, 1-10μM TGFβ inhibitor, 0.25-1.25mg / ml COL1, 1-5μg / ml LN411, Wnt agonists include CHIR99021, Wnt3a, Wnt7a, and TGFβ inhibitors include A83-01 and SB431542.

[0057] Specifically, additives may include 100 ng / ml VEGF, 10 ng / ml EGF, 10 ng / ml bFGF, 3 μM CHIR99021, 2.5 μM A83-01, 0.25 mg / ml COL1, and 1 μg / ml LN411. Furthermore, the concentrations of the additives are not limited to these and can be achieved within the stated range. Wnt agonists are not limited to the listed CHIR99021, Wnt3a, and Wnt7a, and TGFβ inhibitors are not limited to the listed A83-01 and SB431542. Any additive that can achieve the objectives of the present invention may be used alone or in combination, and the use of any one of these additives is preferred.

[0058] After the first culture medium change, continue to use this stage culture medium to induce CD31 + / CD34 + Hepatic endoderm of primary endothelial cells, and no COL1 and LN411 were added thereafter;

[0059] Specifically, the culture medium used is the fourth culture medium, which includes a basal culture medium and additives. The basal culture medium includes 2% by volume of B27, 23% by volume of StemPro34, and 75% by volume of IMDM; the additives include 50-150 ng / ml VEGF, 5-25 ng / ml EGF, 10-50 ng / ml bFGF, 1-10 μM Wnt agonist, 1-10 μM TGFβ inhibitor, Wnt agonists include CHIR99021, Wnt3a, Wnt7a, and TGFβ inhibitors include A83-01 and SB431542.

[0060] Specifically, the additives may include 100 ng / ml VEGF, 10 ng / ml EGF, 10 ng / ml bFGF, 3 μM CHIR99021, and 2.5 μM A83-01.

[0061] The culture time is 4-5 days, and the culture medium is replaced every 48 hours.

[0062] S4. Use culture medium containing FSK and cAMP agonist to continue inducing the generation of sinusoidal network liver organoids.

[0063] The culture medium used in this stage is the fifth culture medium, which includes basal culture medium and additives. The basal culture medium includes 7.5% FBS by volume, 17.5% StemPro34 by volume, and 75% HCM by volume; the additives include 5-25ng / ml HGF, 10-25ng / ml OSM, 1-10μM FSK, 0.25-1mM cAMP agonist, and 0.1-0.5μM DEX. The cAMP agonist is 8-Br-cAMP. The S4 culture period is 20-25 days, and the culture medium is replaced every 72h.

[0064] Specifically, the additives may be 10 ng / ml HGF, 20 ng / ml OSM, 1 μM FSK, 500 μM cAMP agonist 8-Br-cAMP, and 0.5 μM DEX. Furthermore, the concentrations of the additives are not limited thereto and can be achieved within the above range.

[0065] Example 1 Construction of sinusoidal networked liver organoids

[0066] The differentiation process of the organoid is as follows Figure 1 shown.

[0067] When the confluence of hPSCs reaches 80-95%, differentiation is initiated.

[0068] S1 (differentiation days 1-3): Induce PSCs and produce HAND1 + Mesoderm and FOXA2 + Endoderm.

[0069] Differentiation Day 1: The culture medium used was RPMI-1640 containing 2% B27 by volume plus 50 ng / ml BMP4 and 100 ng / ml Activin A.

[0070] Differentiation Days 2-3: Use the first culture medium: add 100 ng / ml Activin A, 5 ng / ml BMP4, and 50 ng / ml bFGF to RPMI-1640 containing 2% B27 by volume, and change the culture medium every 24 hours.

[0071] S2 (differentiation day 4-6): generate KDR + HHEX of mesodermal progenitor cells + HNF4α + Endoderm posterior to the foregut.

[0072] The second culture medium was used: 10 ng / ml BMP4, 500 ng / ml bFGF, and 3 μM CHIR99021 were added to a medium containing 2% B27 by volume, 23% StemPro34 by volume, and 75% IMDM by volume. The culture medium was changed daily.

[0073] S3 (differentiation day 7-10): generate CD31 + / CD34 + Primary endothelial cells of the hepatic endoderm.

[0074] S31. 3D repolymerization based on extracellular matrix (ECM) components COL1 and LN411:

[0075] Differentiation day 6 derivatives were enzymatically digested and collected by centrifugation. Single cells were resuspended in a third culture medium supplemented with COL1 at a final concentration of 0.25 mg / ml and LN411 at 1 μg / ml. After mixing, the cells were seeded into a 96-well U-shaped culture plate at a seeding size of 10,000 cells / well for 3D repolymerization.

[0076] The third culture medium used was: 2% by volume B27, 23% by volume StemPro34, and 75% by volume IMDM supplemented with 100 ng / ml VEGF, 10 ng / ml EGF, 10 ng / ml bFGF, 3 μM CHIR99021, 2.5 μM A83-01, 0.25 mg / ml COL1, and 1 μg / ml LN411.

[0077] S32, after the first culture medium change, continue to use the fourth culture medium to induce the production of CD31 + / CD34 + The fourth culture medium does not contain COL1 and LN411.

[0078] The fourth culture medium used was: 2% by volume of B27, 23% by volume of StemPro34, and 75% by volume of IMDM, supplemented with 100 ng / ml VEGF, 10 ng / ml EGF, 10 ng / ml bFGF, 3 μM CHIR99021, and 2.5 μM A83-01.

[0079] The culture medium was changed every 48 h in S3.

[0080] S4 (Differentiation Days 11-30): Generation of sinusoidal liver organoids.

[0081] The fifth culture medium was used: 10 ng / ml HGF, 20 ng / ml OSM, 1 μM FSK, 500 μM 8-Br-cAMP, and 0.5 μM DEX were added to 7.5% FBS, 17.5% StemPro34, and 75% HCM by volume. The culture medium was changed every 72 h.

[0082] By differentiation day 30, sinusoidal networked liver organoids were formed; they displayed a sinusoidal endothelial network intersecting the hepatocyte clusters, and also contained bile duct cells, macrophages, and hepatic stellate cells.

[0083] The products at each stage of differentiation were assayed and the results are as follows.

[0084] 1. Immunofluorescence and quantitative analysis results at S1 stage Figure 2 As shown, the positive rate was generated by quantitative analysis of the overlap ratio of each marker with the cell nucleus (DAPI) using Image J.

[0085] As can be seen from the figure, the derivative also has 7.2% HAND1 + Mesodermal cells and 87.7% FOXA2 + There was almost no overlap in the signals between the two, confirming the efficient co-differentiation of mesendoderm.

[0086] 2. Immunofluorescence results of S2 stage Figure 3 As shown, KDR was identified + Emergence of hematopoietic mesodermal progenitor cells ( Figure 3 A). In parallel, the endoderm in the derivatives has differentiated into HHEX + HNF4α + Posterior foregut endoderm (the starting point for the development of liver epithelial cells, Figure 3 B).

[0087] 3. Immunofluorescence results of S3 stage Figure 4 As shown, at the end of the differentiation stage, the derivatives have aggregated into compact 3D spheroids. At this time, CD34 + Endothelial progenitor cells and the formation of new lumen-like structures ( Figure 4 A); some of them have differentiated into CD31 + ( Figure 4 B), but the sinusoidal endothelial cell markers are still negative ( Figure 4 C); These phenomena indicate that the derivatives are in the stage of angiogenesis. In parallel, the posterior foregut cell population in the derivatives has undergone further specialization, most of which show AFP + CK19 +The molecular phenotype of the cells indicated that hepatic progenitor cells with bidirectional differentiation potential were formed (i.e., they expressed both hepatic progenitor cell markers and bile duct cell markers; Figure 4 D).

[0088] 4. The results of S4 stage identification are as follows Figure 5 As shown, it shows that the differentiation derivatives have a large area of ​​LYVE1 on day 30. + Hepatic sinusoidal endothelial cells form a tubular network that runs through the ALB + Between hepatocyte clusters ( Figure 5 A), accounting for 8.22% and 76.49% respectively ( Figure 5 C). In addition, this stage derivative also contains CK7 + Cholangiocytes, CD68 + Liver macrophages (Kupffer cells), and VIM + Hepatic stellate cells ( Figure 5 B). These results demonstrate that sinusoidal liver organoids have been formed and contain at least five cell types relevant to liver physiology.

[0089] Functional analysis of the resulting sinusoidal network liver organoids was performed, and the results are as follows.

[0090] LYVE1 was detected by using low-density lipoprotein (AcLDL-AF594) and formaldehyde-treated serum albumin (FSA-FITC) at 37°C. + Cell sorting (LYVE1 sorting of liver organoids from differentiation day 30) + Cells), PSCs (pluripotent stem cells on differentiation day 0, negative control), and LSECs (primary sinusoidal endothelial cells, positive control) were incubated for 15 min and quantified by flow cytometry.

[0091] The clearance experiment showed that compared with the negative control - PSC (pluripotent stem cells on differentiation day 0), LYVE1 + The cells have significant clearance capabilities for acetylated low-density lipoprotein and human serum albumin ( Figure 6 A and Figure 6 B), although the efficiency is still lower than that of primary liver sinusoidal endothelial cells (LSEC). More importantly, ELISA analysis of the supernatant showed that liver organoids at differentiation day 30 could secrete coagulation factor 8 (F8), while LYVE1 was removed during sorting. + After 3D repolymerization, the liver organoids lost their secretory ability ( Figure 6 C) These results confirm the mature functionality of the sinusoidal endothelium in organoids.

[0092] In summary, the sinusoidal network in liver organoids at differentiation day 30 has shown mature functions.

[0093] Comparative Example 1 Effect of combined use of BMP4 and bFGF in S1

[0094] The comparison method used was the classical endoderm induction method (Teo AKK, Valdez IA, Dirice E, et al. Comparable generation of activin-induced definitive endoderm via additive Wntor BMP signaling in the absence of serum[J]. Stem cell reports, 2014, 3(1): 5-14.):

[0095] Differentiation Day 1: The culture medium used was RPMI-1640 containing 2% B27 by volume plus 50 ng / ml BMP4 and 100 ng / ml Activin A.

[0096] Differentiation Days 2-3: Use RPMI-1640 supplemented with 2% B27 (volume ratio) and 100 ng / ml Activin A. Change the medium every 24 hours.

[0097] Vascular endothelial cells develop from mesoderm. Therefore, when inducing the generation of endoderm at the S1 stage, the co-differentiation of mesoderm is a necessary condition for the subsequent sinusoidal differentiation. When using the classic endoderm induction method for posterior differentiation, there is almost no HAND1 positive signal at the S1 stage ( Figure 7 A), resulting in the proportion of sinusoidal endothelial cells in the final stage S4 dropping to less than 0.5% ( Figure 7 B).

[0098] In the S1 stage culture medium, the combination and concentration of BMP4 and Activin A used on differentiation day 1 are common induction methods and have been reported. Based on this, by combining BMP4 (1-10ng / ml) + bFGF (50-200ng / ml) on differentiation days 2-3, endomesodermal symbiosis can be achieved and a high cell viability can be guaranteed. Figure 8 The effects of bFGF and BMP4 concentrations on mesoderm co-differentiation efficiency are shown. It can be seen that when BMP4 or bFGF is removed or the concentrations of BMP4 and bFGF are not within the range, the mesoderm differentiation efficiency is reduced.

[0099] Comparative Example 2 Effects of inducing factors BMP4, Wnt agonist, and bFGF in S2

[0100] To investigate the effects of BMP4, Wnt agonist, and bFGF on the co-differentiation of mesoderm progenitor cells and posterior foregut cells, BMP4, CHIR99021, bFGF, and CHIR99021 and bFGF were withdrawn, respectively, and the derivatives on day 6 of differentiation were identified by immunofluorescence. Figure 9 shown.

[0101] As can be seen from the figure, the three induction factors involved in the S2 stage culture medium, BMP4, CHIR (CHIR99021), and bFGF, are the factors that produce KDR + Mesodermal progenitor cells and HHEX + HNF4α + Essential for the cells of the posterior foregut. Deletion of BMP4 will significantly reduce KDR + The number of mesoderm progenitor cells; if CHIR or bFGF is removed, the expression of posterior foregut markers such as HNF4α and HHEX will be affected.

[0102] Comparative Example 3: Interaction of Extracellular Matrix Component COL1 with LN411

[0103] To study the effects of extracellular matrix components COL1 and LN411 on endothelial regeneration, COL1 and LN411 were withdrawn, and immunofluorescence identification was performed on the 10-day differentiation derivatives. Figure 10 shown.

[0104] From the S2 stage onwards, the soft scaffold formed by the combination of ECM components COL1 and LN411 has a significant effect on promoting angiogenesis and sinusoidal formation in the subsequent stages. As can be seen from the figure, the removal of COL1 or LN411 will lead to the CD31 + The number of endothelial progenitor cells decreased significantly, resulting in almost no discernible positive signal.

[0105] Comparative Example 4 Effects of Inducing Factors VEGF, EGF, and bFGF in S3

[0106] In order to study the effects of induction factors VEGF, EGF, and bFGF on endothelial regeneration, the scheme shown in Table 1 was designed, and the expression levels of vascular progenitor cell markers CD34 and CD31 of the derivatives on differentiation day 10 were analyzed. Figure 11 shown.

[0107] Table 1 Combination of different induction factors

[0108]

[0109] As can be seen from the figure, on the basis of 2F (CHIR99021+A83-01), the addition of VEGF alone can significantly enhance the expression of vascular progenitor cell markers CD34 and CD31; thereafter, the combined addition of EGF and bFGF will further enhance their expression levels, while the addition of EGF or bFGF alone has no further enhancing effect.

[0110] Comparative Example 5 Effects of FSK and cAMP Activators in S4

[0111] To study the effects of FSK and cAMP activators on sinusoidal networking, the scheme shown in Table 2 was designed, and the intersection nodes of the sinusoidal network of liver organoids on differentiation day 30 were analyzed. Figure 12 shown.

[0112] Table 2 Combination of FSK and cAMP activators

[0113]

[0114] As can be seen from the figure, based on 3F (OSM+HGF+DEX), the simultaneous use of FSK and 8-Br-cAM can increase the proportion of intersection nodes of the sinusoidal network, which helps promote the growth of the sinusoidal network.

[0115] Table 3 Reagent list

[0116]

[0117] Table 4 Antibody list

[0118]

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for constructing a sinusoidal network liver organoid, characterized by: The method comprises the following steps: S1, culture in a medium containing 50ng / ml BMP4 and 100ng / ml activin A for 1 day, and culture in a medium containing 100ng / ml activin A, 5ng / ml BMP4, and 50ng / ml bFGF for 2-3 days to produce HAND1 + Mesoderm and FOXA2 + Endoderm; S2, continue to induce KDR-containing cells using a culture medium containing 10 ng / ml BMP4, 500 ng / ml bFGF, and 3 μM Wnt agonist. + HHEX of mesodermal progenitor cells + 、HNF4α + posterior foregut endoderm; S3. The derivatives obtained in S2 were enzymatically digested and collected by centrifugation. Single cells were resuspended in a medium containing 100 ng / ml VEGF, 10 ng / ml EGF, 10 ng / ml bFGF, 3 μM Wnt agonist, and 2.5 μM TGFβ inhibitor. 0.25 mg / ml COL1 and 1 μg / ml LN411 were then added for 3D repolymerization. After the culture medium was replaced, the culture medium was used to induce the production of CD31 + / CD34 + Hepatic endoderm of primary endothelial cells, and no COL1 and LN411 were added thereafter; S4. Continue to induce the generation of sinusoidal network liver organoids using culture medium containing 10 ng / ml HGF, 20 ng / ml OSM, 1 μM FSK, 500 μM cAMP agonist, and 0.5 μM DEX.

2. The method for constructing a sinusoidal network liver organoid according to claim 1, characterized in that: In S1, the culture medium was changed every 24 h.

3. The method for constructing a sinusoidal network liver organoid according to claim 1, wherein: The Wnt agonists in the culture medium used for S2 included CHIR99021, Wnt3a, and Wnt7a. The S2 culture period was 3-4 days, and the culture medium was changed daily.

4. The method for constructing a sinusoidal network liver organoid according to claim 1, wherein: The S3 culture period is 4-5 days, and the culture medium is replaced every 48 hours.

5. The method for constructing a sinusoidal network liver organoid according to claim 1, wherein: The cAMP agonist was 8-Br-cAMP, the S4 culture period was 20-25 days, and the culture medium was changed every 72 h.

6. A culture medium for constructing sinusoidal networked liver organoids, characterized by: The culture medium includes culture medium 1, culture medium 2, culture medium 3, culture medium 4 and culture medium 5; Medium 1 includes 100 ng / ml Activin A, 5 ng / ml BMP4, and 50 ng / ml bFGF for inducing the generation of endoderm in S1 and co-differentiating into mesoderm; Culture medium 2 includes the protein for inducing KDR production + Mesodermal progenitor cells and HHEX + 、HNF4α + 10 ng / ml BMP4, 500 ng / ml bFGF, and 3 μM Wnt agonist for the posterior foregut endoderm; Culture medium 3 includes 100 ng / ml VEGF, 10 ng / ml EGF, 10 ng / ml bFGF to promote angiogenesis and 0.25 mg / ml COL1 and 1 μg / ml LN411 to form a soft scaffold; Medium 4 included 100 ng / ml VEGF, 10 ng / ml EGF, and 10 ng / ml bFGF to promote angiogenesis; Medium 5 includes 1 μM FSK and 500 μM cAMP agonist to promote the growth of sinusoidal networks.

7. An application of a sinusoidal networked liver organoid constructed by the sinusoidal networked liver organoid construction method according to any one of claims 1 to 5 in analyzing liver disease mechanisms and drug screening.

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