Construction method and application of blood sinus networked liver organoid

Abstract

The invention provides a construction method and application of a blood sinus networked liver organ. According to the method, pluripotent stem cells are co-differentiated into HAND1 < + > mesoderm and endoderm, a front intestine rear endoderm containing KDR < + > mesoderm progenitor cells is generated, a liver endoderm with CD34 < + > / CD31 < + > endothelial progenitor cells is generated, and the blood sinus networked liver organ is formed. The liver organ has a functional blood sinus endothelial network penetrating through the liver cluster, and has the characteristic functions of low-density lipoprotein uptake, human serum albumin removal, mediated secretion of blood coagulation factor 8 and the like. The method provides a new-generation highly bionic research tool for liver disease mechanism analysis, drug screening and regeneration repair.

Description

Technical Field

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

[0002] The technology of hepatic lineage induction differentiation of human induced pluripotent stem cells (iPSC) has experienced rapid development: from the induction differentiation of iPSC into 2D hepatocyte-like cells, 3D hepatobiliary symbiotic organoids (with two cell types of hepatocytes and cholangiocytes), to the successful generation of 3D complex liver organoids with hepatocytes, cholangiocytes, hepatic stellate cells, and hepatic macrophages. With the continuous advancement of the refinement level of liver induction technology, these highly biomimetic human-derived organoids are gradually becoming new precision tools for liver disease deciphering, drug development, and regenerative repair at the application end.

[0003] Currently, liver organoids still lack important liver physiological-related cell types such as functional hepatic sinusoidal endothelial cells. As a unique vascular endothelial type in the liver, hepatic sinusoidal endothelium plays important functions in liver embryonic development, adult homeostasis maintenance, and disease development. For example, during embryonic development, the interaction between hepatic sinusoidal endothelium and hepatic progenitor cells is a necessary condition for the metabolic maturation of this organ; under physiological conditions, sinusoidal endothelium acts as a scavenger in the liver, selectively absorbing nutrients in the blood flow through its connection with the hepatic portal vein and central vein, and is responsible for "cleaning up" redundant lipoproteins, albumin and other metabolic wastes; when exposed to a pathological environment, sinusoidal endothelium directly or indirectly mediates the formation of fibrosis, the exacerbation of inflammatory responses, and the occurrence of cancer. Currently, no other technology can achieve the construction of sinusoid-networked liver organoids. Therefore, the absence of this liver characteristic 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 sinusoid-networked liver organoids and its application. This method differentiates pluripotent stem cells into HAND1 + mesoderm and endoderm, generates posterior foregut endoderm containing KDR + mesoderm progenitor cells, generates hepatic endoderm with CD34 + / CD31 + endothelial progenitor cells, and forms sinusoid-networked liver organoids. The liver organoids generated by this method have a functional sinusoidal endothelial network running through the liver clusters, presenting characteristic functions such as low-density lipoprotein uptake, human serum albumin clearance, and mediating the secretion of coagulation factor 8.

[0005] To achieve the above object, the technical solution of the present invention is realized as follows: A method for constructing a sinusoid-networked liver organoid, the method comprising the following steps: S1. Induce PSCs with a medium containing BMP4 and bFGF to simultaneously generate HAND1 + mesoderm and FOXA2 + endoderm; S2. Continue to induce with a medium containing BMP4, bFGF, and a Wnt agonist to generate HHEX containing KDR + mesoderm progenitor cells, HNF4α + HNF4α + posterior foregut endoderm; and the usage concentrations of BMP4 and bFGF are greater than those in S1. S3. Enzymatically digest and centrifuge the derivatives obtained in S2 to collect them, resuspend the single cells with a medium containing VEGF, EGF, and bFGF, then add COL1 and LN411 for 3D repolymerization; After culturing until the medium is changed, continue to use the medium at this stage to induce the generation of a hepatic endoderm containing CD31 + / CD34 + nascent endothelial cells, and then no longer add COL1 and LN411, and the usage concentration of bFGF is less than that in S2. S4. Continue to induce with a medium containing FSK and a cAMP agonist to generate a sinusoid-networked liver organoid.

[0006] In some embodiments, in S1, culture with a conventional medium on the first day; culture with a medium containing BMP4 and bFGF on the second and third days, and the BMP4 concentration used on the second and third days is lower than that used on the first day, and the medium is changed every 24 h.

[0007] In some embodiments, the conventional medium comprises 10 - 75 ng / ml BMP4, 100 - 150 ng / ml Activin A; the medium used on the second and third days comprises 100 - 150 ng / ml Activin A, 1 - 10 ng / ml BMP4, 50 - 200 ng / ml bFGF.

[0008] In some embodiments, the medium used in S2 comprises 10 - 50 ng / ml BMP4, 200 - 500 ng / ml bFGF, 1 - 5 μM Wnt agonist, and the Wnt agonist includes CHIR99021, Wnt3a, Wnt7a. The number of days of culture in S2 is 3 - 4 days, and the medium is changed daily.

[0009] 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.

[0010] In some embodiments, the culture medium used in S3 comprises 50 - 150 ng / ml VEGF, 5 - 25 ng / ml EGF, 10 - 50 ng / ml bFGF. The number of days for culturing in S3 is 4 - 5 days, and the culture medium is changed every 48 h.

[0011] In some embodiments, the culture medium used in S3 further comprises 1 - 10 μM Wnt agonist and 1 - 10 μM TGFβ inhibitor. The Wnt agonists include CHIR99021, Wnt3a, and Wnt7a, and the TGFβ inhibitors include A83 - 01 and SB431542.

[0012] In some embodiments, the culture medium used in S4 comprises 1 - 10 μM FSK and 0.25 - 1 mM cAMP agonist. The cAMP agonist is 8 - Br - cAMP. The number of days for culturing in S4 is 20 - 25 days, and the culture medium is changed every 72 h.

[0013] In some embodiments, the culture medium used in S4 further comprises 5 - 25 ng / ml HGF, 10 - 25 ng / ml OSM, and 0.1 - 0.5 μM DEX.

[0014] The present invention also provides a culture medium for constructing a sinusoid - networked hepatic organoid, which comprises a first culture medium, a second culture medium, a third culture medium, a fourth culture medium, and a fifth culture medium; The first culture medium comprises BMP4 and bFGF for co - differentiating mesoderm while inducing the generation of endoderm in S1; The second culture medium comprises components for inducing the generation of KDR + mesoderm progenitor cells and HHEX + HNF4α + BMP4, bFGF, and Wnt agonist for the posterior - side foregut endoderm; The third culture medium comprises VEGF, EGF, bFGF for promoting angiogenesis, and COL1 and LN411 for forming a soft scaffold; The fourth culture medium comprises VEGF, EGF, bFGF for promoting angiogenesis; The fifth culture medium comprises FSK and cAMP agonist for promoting the generation of a sinusoid network.

[0015] The present invention also provides a sinusoid-networked hepatic organoid constructed by the above-described method for constructing sinusoid-networked hepatic organoids. This hepatic organoid has a functional sinusoidal endothelial network running through the hepatic cluster, exhibits functions such as low-density lipoprotein uptake, human serum albumin clearance, and mediating the secretion of coagulation factor VIII, and also contains CK7 + cholangiocytes, CD68 + hepatic macrophages and VIM + hepatic stellate cells.

[0016] The present invention also provides an application of the sinusoid-networked hepatic organoid as described above in the analysis of liver disease mechanisms, drug screening, and regeneration and repair.

[0017] Compared with the prior art, the method for constructing sinusoid-networked hepatic organoids and the application thereof according to the present invention have the following advantages: (1) The method for constructing sinusoid-networked hepatic organoids according to the present invention establishes hepatic organoids with a functional sinusoidal network. This hepatic organoid has 5 cell types related to liver physiology, such as sinusoidal endothelial cells, hepatocytes, cholangiocytes, hepatic stellate cells, and hepatic macrophages. And this hepatic organoid exhibits characteristic functions such as low-density lipoprotein uptake, human serum albumin clearance, and mediating the secretion of coagulation factor VIII. This method provides a new generation of highly biomimetic research tools for the analysis of liver disease mechanisms, drug screening, and regeneration and repair.

[0018] (2) The sinusoid-networked hepatic organoids according to the present invention are fully endogenously differentiated, and these organoids are completely derived from a single iPSC without the addition of any exogenous cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings: Figure 1 is a flow chart of the method for constructing sinusoid-networked hepatic organoids; Figure 2 is a representative immunofluorescence staining of S1 derivatives. HAND1 is a mesoderm marker, and FOXA2 is an endoderm marker; scale bar = 200 μm; Figure 3 is a representative immunofluorescence staining of S2 derivatives. A is an immunofluorescence co-staining of the hematopoietic mesoderm marker KDR and the posterior foregut endoderm marker HHEX; B is an immunofluorescence co-staining of the posterior foregut endoderm marker HHEX and HNF4α; KDR is a hematopoietic mesoderm marker; both HHEX and HNF4α are posterior foregut endoderm markers; scale bar = 200 μm; Figure 4Representative immunofluorescence staining of S3 derivatives. A: Immunofluorescence co-staining of vascular endothelial progenitor cell marker CD34 and hepatic progenitor cell marker AFP; B: 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 cholangiocyte marker CK19 and hepatic progenitor cell marker AFP; Scale bar = 200 μm; Figure 5 Characterization of S4 derivatives. A: Immunofluorescence staining of sinusoidal endothelial cell marker LYVE1 and hepatocyte marker ALB; B: Immunofluorescence staining of cholangiocyte markers CK7 / CK19, macrophage marker CD68, and hepatic stellate cell marker VIM; C: Flow cytometry analysis of sinusoidal endothelial cell marker LYVE1 and hepatocyte marker ALB, with biological replicates n = 3; LYVE1, sinusoidal endothelial cell marker; ALB, HNF4α, hepatocyte markers; CK7, cholangiocyte-specific marker; CK19, cholangiocyte marker; CD68, macrophage marker; VIM, hepatic stellate cell marker; Scale bar = 50 μm; Figure 6 Functional analysis of sinusoidal endothelium. A: Analysis of the clearance efficiency of LYVE1 + cells for low-density lipoprotein (AcLDL-AF594), B: Analysis of the clearance efficiency of LYVE1 + cells for formaldehyde-treated serum albumin (FSA-FITC), with biological replicates n = 4; *, P < 0.05; ***, P < 0.001; C: ELISA analysis of coagulation factor 8 (F8) in the culture supernatant; Among them, PSC (pluripotent stem cells on day 0 of differentiation), sinusoidal KO liver organoids (i.e., organoids formed by 3D repolymerization after sorting and removing LYVE1 + cells from day 30 differentiated organoids), and HUVEC (human umbilical vein endothelial cells) were used as negative controls; biological replicates n = 4; ***, P < 0.001; N.D., below the limit of detection; Figure 7 Comparison of the results of two induction methods. A: Immunofluorescence identification of mesoderm markers on day 3 of differentiation by two induction methods, Scale bar = 200 μm; B: Flow cytometry analysis of sinusoidal endothelial cells on day 30 of differentiation, with biological replicates n = 3; ***, P < 0.001; Figure 8 Flow cytometry analysis results of day 3 derivatives under different concentrations of bFGF and BMP4 in S1, with biological replicates n = 3; **, P < 0.01; ***, P < 0.001; Figure 9Immunofluorescence identification results of derivatives on day 6 of differentiation under different combinations of inductive factors in S2. Among them, 3F represents the conditions of using this method in the S2 stage, 2F (-BMP4) represents the removal of BMP4, 2F (-CHIR) represents the removal of CHIR99021, 2F (-bFGF) represents the removal of bFGF, 1F (-CHIR -bFGF) represents the simultaneous removal of CHIR99021 and bFGF, scale bar = 200 μm; Figure 10 Immunofluorescence identification results of derivatives on day 10 of differentiation under different combinations of extracellular matrix components COL1 and LN411 in S3. Among them, 2M represents the conditions of using this method in the S3 stage, 1M (-COL1) represents the removal of COL1, 1M (-LN411) represents the removal of LN411, scale bar = 200 μm; Figure 11 Analysis of the expression levels of vascular progenitor cell markers CD34 and CD31 in derivatives on day 10 of differentiation under different combinations of inductive factors in S3. 2F represents CHIR99021 + A83-01; biological replicates n = 3; *, P < 0.05; **P < 0.01; ***, P < 0.001; Figure 12 Analysis of the junction nodes of the blood sinus network in hepatic organoids on day 30 of differentiation under different combinations of FSK and cAMP activators. 3F represents OSM + HGF + DEX; biological replicates n = 8; **P < 0.01; ***, P < 0.001. Detailed implementation manners

[0020] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments may be combined with each other.

[0021] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0022] The reagents used in the examples and comparative examples are shown in Table 3-4.

[0023] The present invention provides a method for constructing a blood sinus networked hepatic organoid, and the method includes the following steps: S1. Use a culture medium containing BMP4 and bFGF to induce PSC to simultaneously generate HAND1 + mesoderm and FOXA2 + endoderm.

[0024] Specifically, S1 includes the following steps: S11. Culture for 1 day in a conventional medium, which includes a basal medium and additives. The basal medium is RPMI-1640 containing 2% B27 by volume, and 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.

[0025] S12. Culture for 2 - 3 days in a first medium, which includes a basal medium and additives. The basal medium is RPMI-1640 containing 2% B27 by volume, and the additives include 100 - 150 ng / ml Activin A, 1 - 10 ng / ml BMP4, and 50 - 200 ng / ml bFGF. Specifically, 100 ng / ml Activin A, 5 ng / ml BMP4, and 50 ng / ml bFGF can be selected. At this stage, the concentrations of BMP4 and bFGF need to be within the required range, and exceeding the range will affect the final differentiation efficiency. The medium is changed every 24 h.

[0026] S2. Use a medium containing BMP4, bFGF, and a Wnt agonist to continue inducing the generation of HHEX + mesoderm progenitor cells expressing KDR + HNF4α + posterior foregut endoderm; and the usage concentrations of BMP4 and bFGF are greater than those in S1. The medium used in this stage is a second medium, which includes a basal medium and additives. The basal 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 agonist. The Wnt agonist includes CHIR99021, Wnt3a, and Wnt7a. The culture time in S2 is 3 - 4 days, and the medium is changed daily.

[0027] Specifically, the additives can be selected as 10 ng / ml BMP4, 500 ng / ml bFGF, and 3 μM Wnt agonist CHIR99021. Moreover, the concentrations of the additives are not limited to this, and can be achieved within the above range. The Wnt agonist is not limited to the listed CHIR99021, Wnt3a, and Wnt7a, as long as the object of the present invention can be achieved, which is not listed one by one here, including single use or mixed use of multiple ones, and preferably single use of one of them.

[0028] S3. Digest the derivative obtained in S2 by enzymatic digestion, collect by centrifugation, resuspend the single cells with a medium containing VEGF, EGF, and bFGF, add COL1 and LN411, mix well, and inoculate into a 96-well U-shaped culture plate at an inoculation density of 10,000 cells / well for 3D repolymerization; The medium used is the third medium, which includes a basal medium and additives. The basal medium includes 2% (by volume) B27, 23% (by volume) StemPro34, and 75% (by volume) 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, 0.25 - 1.25 mg / ml COL1, 1 - 5 μg / ml LN411. The Wnt agonists include CHIR99021, Wnt3a, and Wnt7a, and the TGFβ inhibitors include A83 - 01 and SB431542.

[0029] Specifically, the additives can be selected as 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. Moreover, the concentrations of the additives are not limited to this, and can be achieved within the above range. The Wnt agonists are not limited to the listed CHIR99021, Wnt3a, and Wnt7a, and the TGFβ inhibitors are not limited to the listed A83 - 01 and SB431542, as long as the object of the present invention can be achieved, which are not listed one by one here, including single use or mixed use of multiple ones, and preferably single use of one of them.

[0030] When culturing until the first medium change, continue to use the medium at this stage to induce the generation of intrahepatic endoderm containing CD31 + / CD34 + nascent endothelial cells, and then no longer add COL1 and LN411; Specifically, the medium used is the fourth medium, which includes a basal medium and additives. The basal medium includes 2% (by volume) B27, 23% (by volume) StemPro34, and 75% (by volume) 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. The Wnt agonists include CHIR99021, Wnt3a, and Wnt7a, and the TGFβ inhibitors include A83 - 01 and SB431542.

[0031] Specifically, the additives can be selected from 100 ng / ml VEGF, 10 ng / ml EGF, 10 ng / ml bFGF, 3 μM CHIR99021, and 2.5 μM A83-01.

[0032] The culture time is 4 - 5 days, and the culture medium is changed every 48 h.

[0033] S4. Continue to induce the generation of sinusoid-networked liver organoids using a culture medium containing FSK and cAMP agonist.

[0034] The culture medium used in this stage is the fifth culture medium. The fifth culture medium includes a basal medium and additives. The basal medium includes 7.5% FBS by volume, 17.5% StemPro34 by volume, and 75% HCM by volume; the additives include 5 - 25 ng / ml HGF, 10 - 25 ng / ml OSM, 1 - 10 μM FSK, 0.25 - 1 mM cAMP agonist, 0.1 - 0.5 μM DEX. The cAMP agonist is 8-Br-cAMP. The number of days for S4 culture is 20 - 25 days, and the culture medium is changed every 72 h.

[0035] Specifically, the additives can be selected from 10 ng / ml HGF, 20 ng / ml OSM, 1 μM FSK, 500 μM cAMP agonist 8-Br-cAMP, and 0.5 μM DEX. Moreover, the concentration of the additives is not limited to this, and it can be achieved within the said range.

[0036] Example 1. Construction of sinusoid-networked liver organoids The differentiation process of these organoids is as Figure 1 shown.

[0037] When the confluence of hPSC reaches 80 - 95%, initiate differentiation.

[0038] S1 (Differentiation days 1 - 3): Induce PSC to simultaneously generate HAND1 + mesoderm and FOXA2 + endoderm.

[0039] On differentiation day 1: The culture medium used is RPMI-1640 containing 2% B27 by volume supplemented with 50 ng / ml BMP4 and 100 ng / ml Activin A.

[0040] On differentiation days 2 - 3: Use the first culture medium: RPMI-1640 containing 2% B27 by volume supplemented with 100 ng / ml Activin A, 5 ng / ml BMP4, and 50 ng / ml bFGF. The culture medium is changed every 24 h.

[0041] S2 (Differentiation day 4 - 6): Generate HHEX-expressing + mesoderm progenitor cells + HNF4α + posterior foregut endoderm.

[0042] Use the second medium: Add 10 ng / ml BMP4, 500 ng / ml bFGF, and 3 μM CHIR99021 to IMDM containing 2% (v / v) B27, 23% (v / v) StemPro34, and 75% (v / v) IMDM, and change the medium daily.

[0043] S3 (Differentiation day 7 - 10): Generate CD31-expressing + / CD34 + intrahepatic endoderm of nascent endothelial cells.

[0044] S31. 3D repolymerization based on extracellular matrix (ECM) components - COL1 and LN411: Digest the derivatives on differentiation day 6 by enzymatic digestion, collect by centrifugation, resuspend single cells using the third medium, and add COL1 at a final concentration of 0.25 mg / ml and LN411 at 1 μg / ml to the third medium; after mixing, inoculate into a 96-well U-bottom culture plate at an inoculation density of 10,000 cells / well for 3D repolymerization.

[0045] The third medium used is: Add 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 to IMDM containing 2% (v / v) B27, 23% (v / v) StemPro34, and 75% (v / v) IMDM.

[0046] S32. After culturing until the first medium change, continue to use the fourth medium to induce the generation of CD31-expressing + / CD34 + intrahepatic endoderm of nascent endothelial cells, and the fourth medium does not contain COL1 and LN411.

[0047] The fourth medium used is: Add 100 ng / ml VEGF, 10 ng / ml EGF, 10 ng / ml bFGF, 3 μM CHIR99021, and 2.5 μM A83 - 01 to IMDM containing 2% (v / v) B27, 23% (v / v) StemPro34, and 75% (v / v) IMDM.

[0048] Change the medium every 48 h in S3.

[0049] S4 (Differentiation day 11 - 30): Generation of sinusoid - ized hepatic organoids.

[0050] Use the fifth culture medium: Add 10 ng / ml HGF, 20 ng / ml OSM, 1 μM FSK, 500 μM 8 - Br - cAMP, 0.5 μM DEX to HCM containing 7.5% FBS by volume, 17.5% StemPro34 by volume, and 75% HCM by volume. Replace the culture medium every 72 h.

[0051] By differentiation day 30, sinusoid - networked hepatic organoids are formed; they present a sinusoidal endothelial network that runs through clusters of hepatocytes and also contain cholangiocytes, macrophages, and hepatic stellate cells.

[0052] The products at each differentiation stage were examined, and the results are as follows.

[0053] 1. The immunofluorescence and quantitative analysis results at the S1 stage are as Figure 2 shown. The positive rate is generated by quantitatively analyzing the overlapping ratio of each marker with the cell nucleus (DAPI) using Image J.

[0054] It can be seen from the figure that the derivatives simultaneously possess 7.2% HAND1 + mesodermal cells and 87.7% FOXA2 + endodermal cells, and there is almost no overlap between the signals of the two, confirming the efficient co - differentiation of meso - endoderm.

[0055] 2. The immunofluorescence results at the S2 stage are as Figure 3 shown. The emergence of KDR + hematopoietic mesodermal progenitor cells was identified ( Figure 3 A). In parallel, the endodermal part of the derivatives has differentiated into HHEX + HNF4α + posterior foregut endoderm (the starting site for the development of liver epithelial cells, Figure 3 B).

[0056] 3. The immunofluorescence results at the S3 stage are as Figure 4 shown. At the end of the differentiation stage, the derivatives have aggregated into compact 3D spheres. At this time, CD34 + vascular endothelial progenitor cells appear in multiple parts of the spheres, and new lumen - like structures are formed ( Figure 4 A); some of them have differentiated into CD31 + ( Figure 4 B), but the sinusoidal endothelial cell markers are still negative ( Figure 4C); These phenomena indicate that the derivatives are in the angiogenesis stage. Parallelly, the posterior foregut cell population in the derivatives has undergone further specialization, and most of them exhibit AFP + CK19 + molecular phenotype, indicating the formation of hepatic progenitor cells with bipotential differentiation ability (i.e., expressing both hepatic progenitor cell markers and cholangiocyte markers; Figure 4 D).

[0057] 4. The identification results at the S4 stage are as Figure 5 shown, indicating that a large area of LYVE1 + hepatic sinusoidal endothelial cells has appeared in the derivatives on day 30 of differentiation and formed a tubular network that runs through the ALB + hepatocyte clusters ( Figure 5 A), with proportions of 8.22% and 76.49% respectively ( Figure 5 C). In addition, the derivatives at this stage also contain CK7 + cholangiocytes, CD68 + hepatic macrophages (i.e., Kupffer cells), and VIM + hepatic stellate cells ( Figure 5 B). These results indicate that sinusoidalized hepatic organoids have been formed, which possess at least 5 cell types related to liver physiology.

[0058] Functional analysis was performed on the obtained sinusoidal networked hepatic organoids, and the results are as follows.

[0059] At 37 °C, LYVE1 + cells (sorted from LYVE1 + cells of the hepatic organoids on day 30 of differentiation), PSC (pluripotent stem cells on day 0 of differentiation, negative control), and LSEC (primary hepatic sinusoidal endothelial cells, positive control) were incubated with acetylated low-density lipoprotein AcLDL-AF594 and formaldehyde-treated serum albumin FSA-FITC for 15 min, and quantitative analysis was performed by flow cytometry.

[0060] The clearance experiment showed that compared with the negative control - PSC (pluripotent stem cells on day 0 of differentiation), the LYVE1 + cells sorted from the hepatic organoids on day 30 of differentiation had significant ability to clear acetylated low-density lipoprotein and human serum albumin ( Figure 6 A and Figure 6 B), although the efficiency was still lower than that of primary hepatic sinusoidal endothelial cells (LSEC). More importantly, supernatant ELISA analysis showed that the hepatic organoids on day 30 of differentiation could secrete coagulation factor 8 (F8), while after sorting and removing LYVE1 + , the 3D reaggregated hepatic organoids immediately lost the secretion ability ( Figure 6C), these results confirmed the maturity and functionality of the sinusoidal endothelium in the organoids.

[0061] In summary, the sinusoidal network in the day 30 liver organoids after differentiation has shown mature functions.

[0062] Effect of the combination of BMP4 and bFGF in Comparative Example 1 S1 The comparative method used the classical definitive endoderm induction method (Teo A K K, Valdez I A, Dirice E, et al. Comparable generation of activin-induced definitive endoderm via additive Wnt or BMP signaling in absence of serum[J]. Stem cell reports, 2014, 3(1): 5-14.): Day 1 of differentiation: The medium used was RPMI-1640 containing 2% B27 by volume supplemented with 50 ng / ml BMP4 and 100 ng / ml Activin A.

[0063] Days 2-3 of differentiation: The medium used was RPMI-1640 containing 2% B27 by volume supplemented with 100 ng / ml Activin A. The medium was changed every 24 h.

[0064] Vascular endothelial cells develop from the mesoderm. Therefore, co-differentiating the mesoderm while inducing the definitive endoderm at stage S1 is a prerequisite for subsequent sinusoid formation. Using the classical definitive endoderm induction method for backward differentiation, there was almost no HAND1 positive signal at stage S1 ( Figure 7 A), resulting in the proportion of sinusoidal endothelial cells at the final stage S4 dropping to less than 0.5% ( Figure 7 B).

[0065] In the medium at stage S1, the combination and concentration of BMP4 and Activin A used on day 1 of differentiation are common induction methods and have been reported. Based on this, co-generation of the endoderm and mesoderm can be achieved by combining BMP4 (1-10 ng / ml) + bFGF (50-200 ng / ml) on days 2-3 of differentiation, and a high cell viability can be ensured. As Figure 8 shown by the effect of the concentrations of bFGF and BMP4 on the co-differentiation efficiency of the mesoderm, when BMP4 or bFGF is removed or the use concentrations of BMP4 and bFGF are not within the range, the mesoderm differentiation efficiency will be reduced.

[0066] Effect of Inducing Factors BMP4, Wnt Agonist, and bFGF in Comparative Example 2 S2 To study the effects of BMP4, Wnt agonist, and bFGF on the co-differentiation of mesoderm progenitor cells and posterior foregut cells, BMP4, CHIR99021, bFGF were removed respectively, and CHIR99021 and bFGF were removed simultaneously for comparison. Immunofluorescence identification was performed on the derivatives at differentiation day 6, and the results are as Figure 9 shown.

[0067] As can be seen from the figure, the three inducing factors BMP4, CHIR (CHIR99021), and bFGF involved in the S2-stage medium are necessary for the generation of KDR + mesoderm progenitor cells and HHEX + HNF4α + posterior foregut cells. Removal of BMP4 will significantly reduce the number of KDR + mesoderm progenitor cells; removal of CHIR or bFGF will affect the expression of posterior foregut markers such as HNF4α and HHEX.

[0068] Comparative Example 3 Effects of Extracellular Matrix Components COL1 and LN411 To study the effects of extracellular matrix components COL1 and LN411 on endothelial neovascularization, COL1 and LN411 were removed respectively, and immunofluorescence identification was performed on the derivatives at differentiation day 10, and the results are as Figure 10 shown.

[0069] After the S2 stage, the soft scaffold formed by the combination of ECM components COL1 and LN411 has a significant effect on promoting angiogenesis and sinusoid formation in subsequent stages. As can be seen from the figure, removal of COL1 or LN411 will lead to a significant decrease in CD31 + endothelial progenitor cells of the derivatives at differentiation day 10, resulting in almost no identifiable positive signal.

[0070] Comparative Example 4 Effects of Inducing Factors VEGF, EGF, and bFGF in S3 To study the effects of inducing factors VEGF, EGF, and bFGF on endothelial neovascularization, the following Table 1 was designed, and the expression levels of vascular progenitor cell markers CD34 and CD31 in the derivatives at differentiation day 10 were analyzed, and the results are as Figure 11 shown.

[0071] Table 1 Different Inducing Factor Combination Schemes

[0072] As can be seen from the figure, on the basis of 2F (CHIR99021 + A83-01), the addition of only VEGF 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.

[0073] Effect of FSK and cAMP activator in Comparative Example 5 S4 To study the effect of FSK and cAMP activator on the sinusoid network formation, the following Table 2 protocol was designed, and the sinusoid network intersection nodes of liver organoids on day 30 of differentiation were analyzed. The results are as Figure 12 shown.

[0074] Table 2 Combination protocol of FSK and cAMP activator

[0075] As can be seen from the figure, on the basis of 3F (OSM + HGF + DEX), the simultaneous use of FSK and 8-Br-cAMP results in a higher proportion of sinusoid network intersection nodes, which helps to promote the generation of the sinusoid network.

[0076] Table 3 Reagent list

[0077] Table 4 Antibody list

[0078] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for constructing a sinusoid networked hepatic organoid, characterized in that: The method comprises the following steps: S1. Induce PSCs to simultaneously generate HAND1 + mesoderm and FOXA2 + endoderm; S2. Continue to induce the generation of HHEX containing KDR mesoderm progenitor cells using a culture medium containing BMP4, bFGF, and Wnt agonist + in the anterior-posterior endoderm of the foregut + , HNF4α + ; S3. Subject the derivative obtained in S2 to enzymatic digestion, collect by centrifugation, resuspend the single cells with a culture medium containing VEGF, EGF, and bFGF, and then add COL1 and LN411 for 3D repolymerization; After culturing until the medium is changed, continue to use the medium at this stage to induce the generation of intrahepatic endoderm containing CD31 + / CD34 + primary endothelial cells, and thereafter, COL1 and LN411 are not added anymore, and the usage concentration of bFGF is lower than that of bFGF in S2; S4. Use a culture medium containing FSK and a cAMP agonist to continue inducing the generation of sinusoid-networked hepatic organoids.

2. The method for constructing a sinusoid-networked hepatic organoid according to claim 1, wherein: In S1, culture with a conventional culture medium on the first day; culture with a culture medium containing BMP4 and bFGF on the second to third days, and the concentration of BMP4 used on the second to third days is lower than that used on the first day, and the culture medium is changed every 24 h.

3. The method for constructing a sinusoid networked hepatic organoid according to claim 2, characterized in that: The conventional culture medium comprises 10 - 75 ng / ml BMP4 and 100 - 150 ng / ml activin A; the culture medium used on the second to third days comprises 100 - 150 ng / ml activin A, 1 - 10 ng / ml BMP4, and 50 - 200 ng / ml bFGF.

4. The method for constructing a sinusoid networked hepatic organoid according to claim 1, wherein: The culture medium used in S2 comprises 10 - 50 ng / ml BMP4, 200 - 500 ng / ml bFGF, and 1 - 5 μM Wnt agonist. The Wnt agonist includes CHIR99021, Wnt3a, and Wnt7a. The number of days of culture in S2 is 3 - 4 days, and the culture medium is changed daily.

5. The method for constructing a sinusoid networked liver organoid according to claim 1, wherein: In S3, the concentration of COL1 is 0.25 - 1.25 mg / ml, and the concentration of LN411 is 1 - 5 μg / ml.

6. The method for constructing a sinusoid networked liver organoid according to claim 1, characterized in that: The culture medium used in S3 comprises 50 - 150 ng / ml VEGF, 5 - 25 ng / ml EGF, and 10 - 50 ng / ml bFGF. The number of days of culture in S3 is 4 - 5 days, and the culture medium is changed every 48 h.

7. The method for constructing a sinusoid-networked liver organoid according to claim 1, wherein: The culture medium used in S4 comprises 1 - 10 μM FSK and 0.25 - 1 mM cAMP agonist. The cAMP agonist is 8-Br-cAMP. The number of days of culture in S4 is 20 - 25 days, and the culture medium is changed every 72 h.

8. A culture medium for constructing sinusoid-networked liver organoids, characterized in that: The culture medium comprises a first culture medium, a second culture medium, a third culture medium, a fourth culture medium, and a fifth culture medium; The first culture medium comprises BMP4 and bFGF for co-differentiating the mesoderm while inducing the generation of the endoderm in S1; The second culture medium includes those for inducing the production of KDR + mesoderm progenitor cells and HHEX + , HNF4α + BMP4, bFGF, and Wnt agonists for the posterior foregut endoderm; The third culture medium comprises VEGF, EGF, bFGF for promoting angiogenesis and COL1 and LN411 for forming a soft scaffold; The fourth culture medium comprises VEGF, EGF, bFGF for promoting angiogenesis; The fifth culture medium comprises FSK and a cAMP agonist for promoting the generation of the sinusoid network.

9. A sinusoid-networked hepatic organoid constructed by the method for constructing a sinusoid-networked hepatic organoid according to any one of claims 1-7, characterized in that: The liver organoid has a functional sinusoidal endothelial network throughout the liver cluster, presenting functions such as low-density lipoprotein uptake, human serum albumin clearance, and mediating the secretion of coagulation factor VIII, and also contains CK7 + cholangiocytes, CD68 + liver macrophages and VIM + hepatic stellate cells.

10. Use of the sinusoid-networked hepatic organoids as described in claim 9 in the analysis of liver disease mechanisms, drug screening, and regeneration and repair.

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