Use of the fusion proteins E-cadherin-Fc, VE-cadherin-Fc and VEGF-Fc

Abstract

The present invention provides the use of E-cad-Fc, VE-cad-Fc and VFGF-Fc to promote differentiation into hepatic-like cells, endothelial-like cells, biliary epithelial-like cells and pancreatic islet-like cells. The present invention provides use of a VE-cadherin-Fc fusion protein for promoting differentiation of stem cells into pancreatic islet-like cells, preferably in combination with an E-cadherin-Fc fusion protein and / or a VEGF-Fc fusion protein, to promote differentiation of stem cells into pancreatic islet-like cells.

Description

Technical Field

[0001] The present invention belongs to the field of cell differentiation, and particularly relates to a novel use of an epithelial cadherin (E-cadherin)-Fc fusion protein, and / or a vascular endothelial cadherin (VE-cadherin)-Fc fusion protein, and / or an endothelial cell growth factor (VEGF)-Fc fusion protein, which promotes the induction of differentiation of stem cells into hepatocyte-like cells, endothelial-like cells, islet-like cells or cholangiocyte-like cells.

Background Art

[0002] Organogenesis in mammals is a very dynamic process influenced by the microenvironment constructed by multiple signaling molecules. In recent years, organoid technology based on three-dimensional stem cell culture is expected to simulate the entire process of organ formation and development in vitro, and has gradually become a new research hotspot in stem cell technology and regenerative medicine. The liver, with its complex three-dimensional structure and functions, is one of the most important endocrine and exocrine organs in the human body, and its in vivo development process is jointly controlled by many factors. Hepatocytes (liver parenchymal cells) and liver non-parenchymal cells (e.g., cholangiocytes, Kupffer cells, NK cells) are the basic units that constitute the structure and functions of liver tissue. Among them, hepatocytes and cholangiocytes play the main functions in the liver and differentiate from hepatoblasts under the control of various cytokines. It has been reported that early liver endothelial cells and their secreted factor TGFβ not only control the differentiation fate of hepatoblasts (differentiation fate into hepatocytes or cholangiocytes) during liver development, but also promote the migration of early liver endothelial cells into the septum transversum mesenchyme and the formation of liver buds during early liver development. Therefore, in the process of inducing the formation of liver organoids in vitro, accurately controlling the self-organization for inducing the differentiation of stem cells into vascular endothelial cells or hepatocytes / cholangiocytes to form hepatocyte aggregates with certain structures and functions has become the main technical bottleneck of this technology.

[0003] Cell sorting out is the first stage of liver organoid formation and is related to cell adhesion molecules on the cell surface. Cadherin (abbreviated as "Cad" in this article) is one of the cell-specific adhesion factors. By the homologous binding of the same subtype of calcium cadherin on the surface of the same type of cells, an adhesion junction is formed between cells, which affects cell differentiation. In addition, cadherin also plays a major role in cell recognition, migration, tissue differentiation during embryonic development, or the composition of adult tissues and organs. Epithelial cadherin (E-cadherin) is the cadherin that is first expressed during mammalian development and has an important impact on the tight junction of embryonic stem cell blastomeres and the differentiation of epithelial cells. It should be noted that vascular endothelial cadherin (VE-cadherin) plays an important role in the differentiation of stem cells / endothelial progenitor cells into endothelial cells and the realization of their functions.

[0004] Multiple cadherin fusion proteins based on the Fc segment have been applied to the research of tissue engineering and regenerative medicine. For example, E-cadherin-Fc, N-cadherin-Fc, etc. are used as one of the modifying components of the extracellular matrix to study their influence on the control of cell behavior. It has been revealed by research that vascular endothelial cadherin protein (VE-cadherin), an important component of the adhesion junction between endothelial cells, plays a very important role in the process of angiogenesis.

[0005] Dr. Du Fengyi and Dr. Xu Ke of Nankai University biosynthesized a fusion protein (hVE-cad-Fc) consisting of the extracellular domain of human endothelial cadherin protein and the Fc domain of immunoglobulin IgG, and investigated and optimized its biological activity in the immobilization on the surface of a polystyrene culture plate and the regulation of the adhesion, proliferation, migration, and differentiation functions of vascular endothelial cells (Du Fengyi, Xu Ke, Doctoral Dissertation, 2011, 2016, Nankai University).

[0006] However, so far, there has been no report in the prior art on the promotion of the differentiation of stem cells into hepatocytes by human endothelial cadherin protein-Fc fusion protein.

Summary of the Invention

[0007] The present invention immobilizes epithelial cadherin-Fc fusion protein (for example, abbreviated as human epithelial cadherin-Fc fusion protein, hE-cad-Fc), vascular endothelial cadherin-Fc fusion protein (for example, abbreviated as human vascular endothelial cadherin-Fc fusion protein, hVE-cad-Fc) and endothelial cell growth factor fusion protein (for example, abbreviated as human VEGF165-Fc fusion protein, hVEGF-Fc) on a substrate, and two-dimensionally cultures stem cells on the fusion protein immobilized on the substrate surface, or three-dimensionally introduces the substrate with the fusion protein immobilized on its surface into the interior of the aggregate of stem cells. By the binding of cadherin on the cell surface and the same type of cadherin-Fc on the substrate surface, the adhesion, migration and sorting of stem cells on the substrate surface are controlled, and further, the secretion function of stem cells is controlled. By the synergistic effect with exogenous cell differentiation factors, a biological extracellular microenvironment is constructed by biotechnology to control the differentiation of stem cells. By immobilizing E-cad-Fc, VE-cad-Fc and VEGF-Fc on the same or different substrates, the differentiation efficiency of stem cells into hepatocyte-like cells, endothelial-like cells, islet-like cells and bile duct epithelial-like cells is improved.

[0008] The fusion protein plays the following main roles in the whole process of differentiation induction. 1. Different subtypes of cadherin fusion proteins immobilized on the surface of the substrate material mediate the formation of aggregates of multiple cells from stem cells, enable cadherin subtype-dependent cell adhesion, and promote the construction of a tissue-specific extracellular microenvironment by stem cells. 2. Also, in cooperation with corresponding exogenous cytokines, it directionally induces stem cell differentiation into endothelium and epithelium. VE-cad-Fc promotes the rapid induction of stem cell differentiation into endothelial-like cells in cooperation with endogenous and exogenous VEGF. At the same time, through the extracellular matrix, cytokines they secreted and the formation of endothelial niche, it further cooperates with E-cad-Fc to induce stem cell differentiation into epithelial-like cells. E-cad-Fc, exogenous added cytokines, endothelial niche and the function of its endogenous TGFβ regulate the differentiation of stem cells into hepatocyte-like cells or cholangiocyte-like cells respectively. This process better mimics the liver development process in vitro through the regulation of mainly the types and concentrations of fusion proteins, which is more bioengineered. 3. During the differentiation process, E-cad-Fc continuously activates the phosphorylation of the EGF receptor of stem cells to replace the use of exogenous EGF. VE-cad-Fc not only continuously upregulates the expression of VEGF in stem cells, but also effectively activates the phosphorylation of the VEGF receptor in stem cells, reducing the dependence on the addition of exogenous VEGF in the directed differentiation of stem cells into endothelial cells.

[0009] 4. E-cad-Fc upregulates the expression of HNF4α in stem cells, promotes the MET transformation of stem cells, and promotes the differentiation of stem cells into hepatocyte-like cells. 5. E-cad-Fc not only suppresses the expression of β-catenin in stem cells, but also promotes the differentiation of stem cells into hepatocyte-like cells by enhancing its localization in the cytoplasm. 6. E-cad-Fc and VE-cad-Fc promote the directed differentiation of stem cells into endothelium and epithelium by regulating the expression of YAP protein and its distribution in cells. 7. E-cad-Fc and VE-cad-Fc improve the stability of the aggregate structure and function of stem cells, mimic the intracellular microenvironment in vivo, and promote the in vitro directed differentiation of stem cells. 8. E-cad-Fc and VE-cad-Fc can form stem cells into aggregates of multiple cells, efficiently control the directed differentiation of stem cells, and increase the differentiation efficiency. They provide new solutions and technologies for the construction of liver, gallbladder, and pancreatic organoids, as well as their use in the fields of regenerative medicine, drug research and development, and the like.

[0010] Specifically, the present invention relates to the use of E-cad-Fc, VE-cad-Fc, and VFGF-Fc in the differentiation of hepatocyte-like cells, endothelial-like cells, cholangiocyte-like cells, and pancreatic islet-like cells. One aspect of the present invention relates to the use of E-cad-Fc for activating EGFR present in cells. One aspect of the present invention relates to the use of E-cad-Fc for promoting the induction of differentiation of stem cells into hepatocyte-like cells, pancreatic-like cells, or cholangiocyte-like cells. In one embodiment of the present invention, the E-cad-Fc is used instead of EGF. In one embodiment of the present invention, the E-cad-Fc is immobilized on a substrate. One aspect of the present invention relates to the use of VE-cad-Fc for promoting the induction of differentiation of stem cells into endothelial-like cells, hepatocyte-like cells, pancreatic-like cells, or cholangiocyte-like cells. In one embodiment of the present invention, the VE-cad-Fc is immobilized on a substrate. In one embodiment of the present invention, the VE-cad-Fc is used in combination with VEGF-Fc. In one embodiment of the present invention, the VE-cad-Fc and the VEGF-Fc are immobilized on a substrate, preferably on the same or different substrates.

[0011] One aspect of the present invention relates to the use of E-cad-Fc and VE-cad-Fc for promoting the induction of differentiation of cells into endothelial-like cells, hepatocyte-like cells, pancreatic-like cells, or cholangiocyte-like cells. In one embodiment of the present invention, the ratio of the E-cad-Fc to the VE-cad-Fc is 3:1 to 1:3, preferably 1:3, 3:1, or 1:1. In one embodiment of the present invention, the VE-cad-Fc is used in combination with a VEGF-Fc fusion protein. In one embodiment of the present invention, the VE-cad-Fc and the VEGF-Fc fusion protein are immobilized on a substrate, preferably on the same or different substrates. One aspect of the present invention relates to a method for producing hepatocyte-like cells, pancreatic-like cells or bile duct epithelial-like cells, characterized by culturing stem cells in the presence of E-cad-Fc and / or VE-cad-Fc.

[0012] One aspect of the present invention relates to a method for producing endothelial-like cells, characterized by culturing stem cells in the presence of VE-cad-Fc. In one embodiment of the present invention, stem cells are cultured with the further addition of a VEGF-Fc fusion protein. In one embodiment of the present invention, the E-cad-Fc and the VE-cad-Fc are immobilized on a substrate, preferably on the same or different substrates. In one embodiment of the present invention, the stem cells are mesenchymal stem cells, iPS cells or embryonic stem cells. In one embodiment of the present invention, the stem cells are derived from mammals, preferably from humans, mice or pigs. In one embodiment of the present invention, the epithelial cadherin is human epithelial cadherin, preferably having the sequence shown in SEQ ID NO:9. In one embodiment of the present invention, the vascular endothelial cadherin is human vascular endothelial cadherin, preferably having the sequence shown in SEQ ID NO:5. In one embodiment of the present invention, the Fc is the Fc of human IgG (preferably IgG1), preferably having the sequence shown in SEQ ID NO:4. In one embodiment of the present invention, the vascular endothelial growth factor is human VEGF165, preferably having the VEGF sequence shown in SEQ ID NO:81.

[0013] One aspect of the present invention relates to a modified substrate for cell culture, comprising an epithelial cadherin-Fc fusion protein and / or a vascular endothelial cadherin-Fc fusion protein. Preferably, the epithelial cadherin is human epithelial cadherin, preferably the sequence shown in SEQ ID NO:9, and the vascular endothelial cadherin is human vascular endothelial cadherin, preferably the sequence shown in SEQ ID NO:5. In an embodiment of the present invention, the substrate is for promoting the differentiation of cells into endothelial-like cells, liver-like cells, pancreatic-like cells or bile duct epithelial-like cells. Preferably, the stem cells are mesenchymal stem cells, iPS cells or embryonic stem cells, and more preferably, the stem cells are derived from mammals, preferably from humans, mice or pigs. In an embodiment of the present invention, when the modified substrate contains a vascular endothelial cadherin-Fc fusion protein, it further contains a VEGF-Fc fusion protein. Preferably, the VEGF is human VEGF165, and more preferably, the sequence of VEGF is SEQ ID NO:81.

[0014] Another aspect of the present invention relates to the use of the modified substrate for promoting the differentiation of cells (preferably stem cells, more preferably mesenchymal stem cells, iPS cells or embryonic stem cells) (preferably into endothelial-like cells, liver-like cells, pancreatic-like cells or bile duct epithelial-like cells). Another aspect of the present invention relates to a method for preparing a modified substrate, which includes mixing an epithelial cadherin-Fc fusion protein and / or a vascular endothelial cadherin-Fc fusion protein with a substrate to modify the substrate. In an embodiment of the present invention, the substrate is a substrate for promoting the differentiation of cells into endothelial-like cells, liver-like cells, pancreatic-like cells or bile duct epithelial-like cells. Preferably, the stem cells are mesenchymal stem cells, iPS cells or embryonic stem cells, and more preferably, the stem cells are derived from mammals, preferably from humans, mice or pigs. In an embodiment of the present invention, when modifying a substrate using a vascular endothelial cadherin-Fc fusion protein, the substrate is further modified using a VEGF-Fc fusion protein. Preferably, the VEGF is human VEGF165, and more preferably the sequence of VEGF is SEQ ID NO:81.

[0015] In the various embodiments of the present invention described above, the substrate is selected from the group consisting of a cell culture plate, a cell culture dish, a hydrogel, a porous scaffold (preferably a PLGA scaffold or a PGL scaffold) or microbeads. Preferably, the particle diameter of the microbeads is 10 - 50 μm (preferably 15 - 30, 15 - 20 μm), preferably hydrophobic microbeads or hydrophilic microbeads, more preferably the microbeads are PLGA microbeads, and even more preferably PLGA microbeads with a particle diameter of 10 - 50 μm (preferably 15 - 30, 15 - 20 μm). Alternatively, the microbeads are polystyrene beads, and more preferably polystyrene microbeads with a particle diameter of 10 - 50 μm (preferably 15 - 30, 15 - 20 μm).

[0016] In the various embodiments of the present invention described above, the porous scaffold, film or microbeads are hydrophilic or hydrophobic. In one embodiment, when the porous scaffold, film or microbeads are hydrophilic porous scaffold, film or microbeads, the epithelial cadherin-Fc fusion protein or the vascular endothelial cadherin-Fc fusion protein is linked to the porous scaffold, film or microbeads via a linker (preferably, an Fc-binding peptide). Preferably, the Fc-binding peptide is selected from the group consisting of CHWRGWV (SEQ ID NO:93), HYFKFD (SEQ ID NO:94, see REFERENCES 3, 4), HFRRHL (SEQ ID NO:95, see REFERENCES 3, 4), FYWHCLDE (SEQ ID NO:96, see REFERENCES 1, 2) or SpA (staphylococcal protein A, see REFERENCES 1, 2).

[0017] In the various embodiments of the present invention described above, preferably, the hydrogel is a hyaluronic acid hydrogel, and more preferably, an acrylated hydrazide hyaluronic acid hydrogel or a PAMAM dendrimer / thiolated hyaluronic acid hydrogel. In an embodiment of the present invention, the acrylated hydrazide hyaluronic acid hydrogel is prepared by modifying hyaluronic acid with adipic dihydrazide and N-acryloxysuccinimide.

[0018] In an embodiment of the present invention, the PAMAM dendrimer / thiolated hyaluronic acid hydrogel is prepared by a Michael addition reaction of a PAMAM dendrimer and thiolated hyaluronic acid.

[0019] REFERENCES 1. Biomimetic design of affinity peptide ligands for human IgG based on protein A-IgG complex, Biochemical Engineering Journal, 88(2014)(1 - 11). 2. FYWHCLDE-based affinity chromatography of IgG: Effect of ligand density and purifications of human IgG and monoclonal antibody, Journal of Chromatography A, 1355(2014)107 - 114. 3. Performance of hexamer peptide ligands for affinity purification of immunoglobulin G from commercial cell culture media, Journal of Chromatography A, 1218(2011)1691 - 1700. 4. Purification of human immunoglobulin G via Fc-specific small peptide ligand affinity chromatography, Journal of Chromatography A 1216(2009)910 - 918.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0029] Hereinafter, the present invention will be described in detail with reference to examples and drawings. As can be understood by those of ordinary skill in the art, the following embodiments are for illustrative purposes only, and the present invention should not be construed as being limited thereto. The protection scope of the present invention is limited by the claims described below.

[0030] Example 1: Construction and Expression of Human hVE-cad-Fc Fusion Protein Refer to Fengyi Du, doctoral thesis, Nankai University, November 2011. The main content is as follows.

[0031] 1.1 Cloning and sequence analysis of the extracellular domain gene VE-cad of vascular endothelial cadherin Based on the protein sequence and functional domains of human VE-cadherin published in the UniProt database, specific PCR primers were designed with reference to the gene sequence (NCBI Reference Sequence: NM_001795.3) published in GenBank to amplify the extracellular domain (EC1-EC5) of the hVE-cadherin protein. Forward primer (P1): 5′-CCG GATATC ATGCAGAGGCTCATGATGCTCC-3′ (SEQ ID NO:1), incorporating an EcoRV enzyme digestion site (underlined), reverse primer: (P2) 5′-AA GCGGCCGC TCTGGGCGGCCATATC-3′ (SEQ ID NO:2), incorporating a NotI enzyme digestion site (underlined). The synthesis and sequence analysis of the primers were commissioned to Invitrogen Ltd. for implementation.

[0032] Extraction of total mRNA from human umbilical vein endothelial cells (HUVEC, ScienCell, USA): mRNA was extracted according to the conventional method in "Molecular Cloning Experiment Guide" (3rd Edition). The O.D. value was measured to quantify the purity and concentration of RNA. Reverse transcription was performed using the TaqMan@MicroRNA Assays kit purchased from BD, and the following reverse transcription system was used:

[0033]

Table 1

[0034] The reverse transcription program is as follows.

[0035]

Table 2

[0036] Using the cDNA reverse transcribed from HUVEC mRNA as a template, the gene fragment of VE-cad was amplified. The PCR reaction system is as follows.

[0037]

Table 3

[0038] The amplification conditions were: denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, a total of 35 cycles, and finally extension at 72°C for 10 minutes. 380 μL of ddH2O was added to the reaction solution, and extraction was performed once with an equal volume of phenol / chloroform / isoamyl alcohol. 1 / 10 volume of 3M NaAc (pH 5.0) and 2 volumes of absolute ethanol were added, and it was left at -20°C for 1 h. Centrifugation was performed at 4°C and 12,000 rpm for 10 minutes. The DNA precipitate was washed twice with 70% ethanol and vacuum dried. The precipitate was dissolved in an appropriate amount of TE.

[0039] 1.2 Construction of the eukaryotic expression vector pcDNA 3.1-hVE-cad-Fc (1) PCR product purified by double digestion with EcoRV and NotI The enzyme digestion system is as follows.

[0040]

Table 4

[0041] Reaction was carried out overnight at 37°C, the enzyme was inactivated at 65°C for 15 minutes, 350 μL of ddH2O was added to the reaction solution, and extraction was performed once with an equal volume of phenol / chloroform / isoamyl alcohol. 1 / 10 volume of 3M NaAc (pH 5.0) and 2 volumes of absolute ethanol were added, and it was left at -20°C for 1 h. Centrifugation was performed at 4°C and 12,000 rpm for 10 minutes. The DNA precipitate was washed twice with 70% ethanol and vacuum dried. The precipitate was dissolved in 10 μL of TE.

[0042] (2) EcoRV and NotI enzyme digestion of pcDNA / 3.1: The double enzyme digestion system (3 × 50 μL) of pcDNA / 3.1 (Thermo Fisher Scientific, USA, product number V79020) is as follows.

[0043] [Table 5]

[0044] The reaction was carried out overnight at 37°C. The enzyme digestion products were separated by electrophoresis on a 1% agarose gel. The target fragment was cut out under a UV lamp and recovered using a DNA agarose gel recovery kit (TaKaRa, Japan, product number 9762). The recovered fragment was dissolved in 25 μL of ddH2O.

[0045] (3) Ligation reaction and transformation reaction between vector and target fragment The reaction system is as follows.

[0046] [Table 6]

[0047] The reaction was carried out at 16°C for 16 h. Then, it was transformed into competent cell BL21(DE3) with CaCl2 and cultured overnight at 37°C for 16 - 18 h. The transformed cells were picked up, and a small amount of plasmid was extracted and detected. The extracellular domain of hVE-cadherin, which is the target gene recovered, and pcDNA 3.1, a vector with an Fc fragment, were fractionated and digested with double enzymes (EcoRV and NotI) at a constant temperature of 37°C. They were recovered by electrophoresis, the recovered products were mixed, and overnight ligation was performed at 16°C using T4 DNA ligase as a catalyst. The ligation product was used to transform E. coli DH5α competent cells, and screening based on resistance was carried out using ampicillin (Amp+). After the plasmid was extracted, it was identified by double enzyme digestion. For those approved as correct recombinant plasmids in the initial identification, the DNA sequence was analyzed. The constructed recombinant plasmid was named pcDNA 3.1 / hVE-cad-Fc (shown in Figure 1A). The accuracy of the sequence was confirmed by sequence analysis. The hVE-cad-Fc fusion protein sequence is shown in SEQ ID NO:3. The sequence of Fc is shown in SEQ ID NO:4, and the sequence of hVE-cad is shown in SEQ ID NO:5.

[0048] 1.3 Cell Transfection and Protein Purification 293F cells (Cell Bank of the Committee for the Depository of Typical Cultures of the Chinese Academy of Sciences) were transfected with pcDNA 3.1 / hVE-cad-Fc. Due to the specific binding between the Fc segment of immunoglobulin and rProtein A, the target protein was purified using a Hitrap rProtein A FF column from GE Healthcare.

[0049] 1.4 Western Blot Analysis The purified hVE-cad-Fc fusion protein was electrophoresed on 10% SDS-PAGE and transferred to a PVDF membrane. It was blocked with 5% non-fat milk for 2 hours and incubated overnight at 4°C with the primary antibody, a rabbit anti-human VE-cadherin extracellular domain monoclonal antibody (RD, USA, diluted at 1:400). It was then incubated for 1 h at room temperature with the HRP-labeled goat anti-rabbit secondary antibody (Abcam, USA, diluted at 1:10000). The membrane was washed with TBST, exposed to DAB reagent, developed and fixed for analysis. When detecting the dimer consisting of Fc, the loading buffer did not contain β-mercaptoethanol. The results are shown in Figure 1B. As can be seen from Figure 1B, under non-reducing conditions, one band was observed at approximately 240 KD, and under reducing conditions, one band was observed at approximately 120 KD. These results suggest that the hVE-cad-Fc fusion protein exists as a dimer.

[0050] Example 2: Construction and expression of human hE-cad-Fc fusion protein Reference was made to the doctoral thesis of Xu Jianbin, Nankai University, December 2013. The main content of this example is as follows.

[0051] 2.1 Cloning and sequence analysis of E-cad, the extracellular domain gene of epithelial cadherin Based on the protein sequence and functional domains of human E-cadherin published in the UniProt database, with reference to the gene sequence (NCBI Reference Sequence: NM 004360.3) published in GenBank, specific PCR primers were designed to amplify the extracellular domain of the E-cadherin protein. Forward primer (P1): 5’-CGCAAGCTTATGGGCCCTTG-GAGCCGCAGC-3’, SEQ ID NO:6; Reverse primer (P2): 5’-TTGCGGCCGCAGGCAGGAATTTGCAATCCTGC-3’, SEQ ID NO:7. The synthesis and sequence analysis of the primers were commissioned to Invitrogen Ltd. for implementation.

[0052] Extraction of total mRNA from L-02 cells (Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection): mRNA was extracted according to the conventional method in "Molecular Cloning Experiment Guide" (3rd edition). The O.D. value was measured to quantify the purity and concentration of RNA. Reverse transcription was performed according to the TaqMan@MicroRNA Assays kit purchased from BD company. The reverse transcription system is as follows.

[0053]

Table 7

[0054] The reverse transcription program is as follows.

[0055]

Table 8

[0056] Using the cDNA reverse transcribed from L-02 mRNA as a template, the gene fragment of E-cad was amplified. The PCR reaction system is as follows.

[0057]

Table 9

[0058] The amplification conditions were: denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles, and finally, extension at 72°C for 10 minutes. 380 μL of ddH2O was added to the reaction solution, and extraction was performed once with an equal volume of phenol / chloroform / isoamyl alcohol. 1 / 10 volume of 3M NaAc (pH 5.0) and 2 volumes of absolute ethanol were added, and it was left at -20°C for 1 h. Centrifugation was performed at 4°C, 12000 rpm for 10 minutes. The DNA precipitate was washed twice with 70% ethanol and vacuum dried. The precipitate was dissolved in an appropriate amount of TE.

[0059] 2.2 Construction of the eukaryotic expression vector pcDNA 3.1-hE-cad-Fc (1) PCR products purified by double digestion with Hind III and NotI The enzyme digestion system is as follows.

[0060]

Table 10

[0061] React overnight at 37°C, inactivate the enzyme at 65°C for 15 minutes, add 350 μL of ddH2O to the reaction solution, extract once with an equal volume of phenol / chloroform / isoamyl alcohol, add 1 / 10 volume of 3M NaAc (pH 5.0) and 2 volumes of absolute ethanol, and leave at -20°C for 1 h. Centrifuge at 4°C, 12,000 rpm for 10 minutes, wash the DNA precipitate twice with 70% ethanol, and dry in vacuo. Dissolve the precipitate in 10 μL of TE.

[0062] (2) Hind III and NotI enzyme digestion of pcDNA / 3.1

[0063]

Table 11

[0064] React overnight at 37°C. The enzyme digestion products were separated by electrophoresis on a 1% agarose gel. The target fragment was cut out under a UV lamp and recovered using a DNA agarose gel recovery kit (TaKaRa). The recovered fragment was dissolved in 25 μL of ddH2O.

[0065] (3) Ligation reaction and transformation reaction between the vector and the target fragment The reaction system is as follows.

[0066]

Table 12

[0067] React at 16°C for 16 h. Then, transform competent cell BL21(DE3) with CaCl2 and culture overnight at 37°C for 16 - 18 h. Pick up the transformed cells, extract a small amount of plasmid and detect it. The extracellular domain of E-cadherin, which is the target gene recovered, and the vector pcDNA 3.1 with an Fc fragment were digested with double enzymes (Hind III and NotI) at a constant temperature of 37°C respectively. After recovery by electrophoresis, the recovered products were mixed and ligated overnight at 16°C using T4 DNA ligase as a catalyst. After the ligation product transformed E.coli DH5α competent cells, screening by resistance was performed using ampicillin (Amp+). After extracting the plasmid, it was identified by double enzyme digestion. The recombinant plasmid approved as correct in the preliminary identification was analyzed for DNA sequence (shown in Figure 2A). The constructed recombinant plasmid was named pcDNA 3.1 / hE-cad-Fc. The accuracy of the sequence was confirmed by sequence analysis. The hE-cad-Fc fusion protein sequence is shown in SEQ ID NO:8. Among them, the sequence of hE-cad is shown in SEQ ID NO:9, and the sequence of Fc is shown in SEQ ID NO:4.

[0068] 2.3 Cell Transfection and Protein Purification Transfect 293F cells (Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection) with pcDNA 3.1 / hE-cad-Fc. Purify the target protein with a Hitrap rProtein A FF column from GE Healthcare by the specific binding between the Fc segment of immunoglobulin and rProtein A.

[0069] 2.4 Western Blot Analysis The purified hE-cad-Fc fusion protein was electrophoresed on 10% SDS-PAGE and transferred to a PVDF membrane. It was blocked with 5% skim milk for 2 h and incubated overnight at 4°C with a monoclonal antibody against the extracellular domain of rabbit anti-human E-cadherin (RD, USA, diluted 1:400), which is the primary antibody. It was incubated for 1 h at room temperature with an HRP-labeled goat anti-rabbit secondary antibody (Abcam, USA, diluted 1:10,000). The membrane was washed with TBST, exposed to DAB reagent, developed and fixed for analysis. When detecting the dimer consisting of Fc, the loading buffer does not contain β-mercaptoethanol. The results are shown in Figure 2B. As can be seen from Figure 2B, under non-reducing conditions, one band was observed at approximately 240 KD, and under reducing conditions, one band was observed at approximately 120 KD. These results suggest that hE-cad exists as a dimer.

[0070] Unless otherwise specified, the PCR reaction according to the following examples is carried out as follows: Using a PCR kit provided by Transgen Biotech Co., LTD, according to the following table, the following components were added to PCR tubes free of RNase / DNase, respectively:

[0071]

Table 13

[0072] PCR machine setting program: 1 cycle at 95°C for 5 min, 95°C for 5 min, 1 min at the annealing temperature, 45 s at 72°C, repeat 35 cycles, 1 cycle at 72°C for 10 min, and keep warm at 4°C. The expression status of the PCR product was detected by electrophoresis on a 1% agarose gel.

[0073] Example 3. Detection of cytokine and extracellular matrix secretion of hMSCs on the surfaces of substrates with different modifications Type I collagen (collagen, BD, USA, product number 354249), hE-cad-Fc, hVE-cad-Fc, and a mixed solution of hE-cad-Fc / hVE-cad-Fc (the ratio of the two fusion proteins was 1:1) were each diluted to a final concentration of 10 μg / mL with 0.01 M PBS (pH = 7.2). 1.5 mL of the diluted collagen solution and the mixed solution of hE-cad-Fc / hVE-cad-Fc were separately added to a 6-well cell culture plate, placed in a cell incubator, and incubated at 37°C for 2 h. After removal, the supernatant was discarded and washed three times with 0.01 M PBS (pH = 7.2).

[0074] Subsequently, hMSCs (Cyagen Biosciences Inc., China) were seeded at a cell density of 105 cells / well onto TCPS plates and culture plates after incubation with the above solution. The cells were cultured in a cell incubator (37°C, 5% CO2) using DMEM / Ham's F12 1:1 (DF12, BI, USA) medium containing 10% fetal bovine serum (FBS, BI, USA). After culturing individually for 24 h and 48 h, the medium was discarded, and the cells were washed three times with 0.01 M PBS (pH = 7.2). 1 mL of Trizol (Invitrogen, USA, product number 15596026) was added to each well, and RNA was extracted according to the product manual. The concentration of the RNA sample was measured using a Biodrop. 2 μg of RNA was taken and reverse transcribed using the random primers included in the Roche reverse transcription kit (product number 4655877001) according to the kit manual to obtain cDNA (PCR machine setting program: 55°C for 30 minutes for 1 cycle, 85°C for 5 minutes for 1 cycle, incubated at 4°C). The gene expression level was analyzed using a Roche fluorescence quantitative PCR kit (product number 4914058001). The sequences and annealing temperatures of the primers used are shown in Table 1 below (PCR machine setting program: 95°C for 5 minutes for 1 cycle, 95°C for 5 minutes, 1 minute at the annealing temperature, 45 s at 72°C for 35 cycles repeated, 72°C for 10 minutes for 1 cycle, incubated at 4°C). The above samples were mixed well and detected using a fluorescence quantitative PCR machine (Biorad). The obtained data were processed by the 2-ΔΔCt method using the TCPS group as the control group. The detection results of the 24-h culture are shown in Figure 3, and the detection results of the 48-h culture are shown in Figure 4.

[0075] As can be seen from the PCR results, the combined use of the two fusion proteins, hE-cad-Fc and hVE-cad-Fc, significantly improves the self-secretory ability of hMS extracellular matrix proteins and cytokines, contributing to the directed differentiation induction of hMSCs by the action of exogenous cytokines.

[0076]

Table 14

[0077] Example 4. Evaluation of the activation time of EGF receptor on the substrate surface with different modifications A 6-well cell culture plate was treated by the method of Example 3, and hMSCs were separately inoculated onto the surfaces of culture plates modified with type I collagen, hE-cad-Fc, and hVE-cad-Fc at a cell density of 105 cells / well. The cells were cultured in a maintenance medium in a cell incubator (37 °C, 5% CO2) for 4 h. The cells inoculated on the substrate surface modified with type I collagen were cultured in a maintenance medium containing 10 ng / mL EGF (Peprotech, USA, product number AF-100-15), and the cells inoculated on the substrate surfaces separately modified with hE-cad-Fc and hVE-cad-Fc were cultured in a maintenance medium (without EGF). At 0 min, 10 min, 30 min, 60 min, 90 min, and 120 min after the start of culture, the cells were lysed with 100 μL of cell lysis buffer (Beyotime Biotechnology), and the proteins were extracted as follows. For different samples, the expression status of total EGF receptor (total EGF receptor, CST, USA, diluted 1:1000) and phospho-EGF receptor Tyr 1068 (phosphorylated at position 1068, CST, USA, diluted 1:1000) at different time points was detected by Western blot. The results are shown in Figure 5.

[0078] Protein extraction: [1] The lysis buffer was added at a ratio of adding 100 μL of lysis buffer to 2×106 cells. The cells were further lysed with a cell scraper to release all the proteins in the cells, and centrifuged at 4 °C and 13,000 rpm for 10 min to obtain the supernatant; [2] The protein content in the supernatant was detected using a BCA kit (Beyotime Biotechnology, product number P0010); [3] 5× Loading Buffer (Beyotime Biotechnology, product number P0015) was added to the supernatant at a ratio of 1:5 and boiled for 5 minutes. The boiled protein was stored alone in a -80°C refrigerator to achieve an injection of 20 μg of protein per well, completing the extraction of all cellular proteins.

[0079] As can be seen from Figure 5, upon stimulation by EGF added to the medium, phosphorylation of the EGF receptor of hMSCs on the substrate modified with collagen maintained an activated state within 60 minutes. After 60 minutes, the activation of EGF receptor phosphorylation began to disappear. Also, phosphorylation of the hMSC EGF receptor on the surface of the substrate modified with hE-cad-Fc maintained an activated state for up to 2 hours. Phosphorylation of the EGF receptor of hMSCs on the surface of the substrate modified with hVE-cad-Fc did not become activated. This indicated that hE-cad-Fc could specifically and continuously activate the EGF receptor and its phosphorylation.

[0080] Example 5. Influence of different concentrations of EGF on the directional differentiation of hMSCs on the surface of the substrate modified with hE-cad-Fc hMSCs were seeded at a cell density of 105 cells / well on the surface of a 6-well plate modified with hE-cad-Fc prepared as described in Example 3. After overnight culture in maintenance medium, 1.5 mL of hepatocyte-directed differentiation medium (DF12 + 2% FBS + 10 ng / mL FGF4 + 20 ng / mL HGF + 1*ITS, where FGF4 was purchased from Peprotech, USA, product number 100-31; HGF was purchased from Peprotech, USA, product number 100-39H; ITS was purchased from Sigma, USA, product number I3146) containing different concentrations (0 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, and 25 ng / mL) of EGF was added to different wells. The medium was changed every two days. After culturing for 7 days, proteins were extracted and the expression levels of Oct4 (Abcam, USA, diluted 1:1000), total EGF receptor (CST, USA, diluted 1:1000), phosphorylated-EGF receptor Tyr 1068 (CST, USA, diluted 1:1000), CK18 (Abcam, USA, diluted 1:1000), and CK19 (Abcam, USA, diluted 1:1000) in different samples were detected by Western blot. The results are shown in Figure 6.

[0081] As can be seen from Figure 6, one week after adding the differentiation medium, the test group with the differentiation medium without EGF had the highest efficiency of inducing hepatocyte-directed differentiation. This is because the hE-cad-Fc substrate replaced the related function of EGF during the process of hepatocyte-directed differentiation from hMSCs, continuously stimulated the phosphorylation of the EGF receptor, and increased the differentiation induction efficiency from hMSCs to hepatocytes.

[0082] Example 6. Effect of different concentrations of hVEGF-Fc immobilized on the surface of a substrate modified with hVE-cad-Fc on the directed differentiation of hMSCs hVE-cad-Fc solution (final concentration 10 μg / mL) (where the sequence of hVEGF is SEQ ID NO: 81) was prepared to contain hVEGF-Fc (SEQ ID NO: 26, synthesized by GenScript) at different final concentrations of 0 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, 2 μg / mL, and 2.5 μg / mL. hMSCs were seeded in culture plates at a cell density of 105 cells / well and cultured overnight in maintenance medium, then switched to endothelial cell differentiation medium (DF12 + 2% FBS + 10 ng / mL FGF2, FGF2, from Peprotech, USA, product number 100 - 18B) and cultured with medium changes every two days. After culturing for 7 days, the expression levels of Oct4 (Abcam, MA, USA, diluted 1:1000), total VEGF receptor (CST, USA, diluted 1:1000), phosphorylated-VEGF receptor Tyr 1175 (CST, USA, diluted 1:1000), CD31 (CST, USA, diluted 1:1000), and hVE-cadherin (Abcam, USA, diluted 1:1000) in each sample were detected by Western blot. The results are shown in Figure 7. As can be seen from Figure 7, one week after adding the differentiation medium, when the fixed amount of hVEGF-Fc is 2 μg / mL, the differentiation efficiency of hMSCs into vascular endothelial-like cells is the highest. This proves that in the process of inducing differentiation of hMSCs into vascular endothelial-like cells, a certain concentration of hVE-cad-Fc substrate and hVEGF-Fc fusion protein play a cooperative role in regulating their differentiation, and they can continuously stimulate the phosphorylation of VEGF receptor, increasing the efficiency of inducing differentiation of hMSCs into vascular endothelial-like cells.

[0083] The most preferred components of the differentiation medium for use on substrate surfaces with different modifications are as follows. (1) Collagen: DF12 + 2% FBS + 10 ng / mL FGF-4 + 10 ng / mL FGF-2 + 20 ng / mL HGF + 1*ITS + 20 ng / mL EGF + 40 ng / mL hVEGF (2) hE-cad-Fc: DF12 + 2% FBS + 10 ng / mL FGF-4 + 10 ng / mL FGF-2 + 20 ng / mL HGF + 1*ITS + 40 ng / mL hVEGF (3) hE-cad-Fc / hVE-cad-Fc / hVEGF-Fc: DF12+2%FBS+10ng / mL FGF-4+10ng / mL FGF-2+20ng / mL HGF+1*ITS.

[0084] Example 7. Detection of induction of differentiation from hMSCs to hepatocytes on substrate surfaces with different modifications According to the method of Example 3, 6-well cell culture plates were prepared, each modified with collagen solution and hE-cad-Fc. 6-well cell culture plates were prepared, each modified with a mixed solution of hE-cad-Fc (final concentration: 10 μg / mL), hVE-cad-Fc (final concentration: 10 μg / mL), and hVEGF-Fc (final concentration: 2 μg / mL). hMSCs were seeded on the surface of the culture plate at a cell density of 105 cells / well, cultured overnight in a maintenance medium, and then replaced with a specific differentiation medium (the components of the medium refer to the medium (1)-(3) in Example 6). The medium was replaced every two days, and the culture was continued for 28 days. The morphology of the cells was observed on days 0, 7, 14, 21, and 28. As can be seen from FIG. 8, after the addition of the differentiation medium, the morphology of the hMSCs on the different substrate surfaces all began to change, and the cells contracted at the first differentiation cycle, and the cells became oval at the fourth differentiation cycle. Throughout the differentiation process, the number of cells on the surface of the fusion protein substrate was significantly higher than that of the collagen group, and the morphological changes occurred more quickly. This demonstrated that the fusion protein could promote the directed differentiation of hMSCs and improve the differentiation efficiency. At the same time, the cell RNA of each group was extracted and reverse transcribed at the above time points, and the expression status of related germ layer genes, hepatocyte genes, liver polarity protein genes, metabolic enzyme genes and endothelial cell genes was detected by PCR. The primer sequences and annealing temperatures of the related genes are shown in the table below.

[0085] PCR amplification results of Oct4, FoxA2, Sox17 and β-actin (internal standard), and the relative expression levels of mRNA according to the differentiation time are shown in Fig. 9 (unless otherwise specified, in the following examples, C represents cells cultured on the collagen surface, E represents cells cultured on the hE-cad-Fc surface, and E+V represents cells cultured on the hE-cad-Fc / hVE-cad-Fc / hVEGF-Fc surface). PCR amplification results of CK18, CK19, AFP, ALB and β-actin (internal standard), and the relative expression levels of mRNA according to the differentiation time are shown in Fig. 10. PCR amplification results of OATP, MRP2 and β-actin (internal standard), and the relative expression levels of mRNA according to the differentiation time are shown in Fig. 11. PCR amplification results of G6Pase, α1-AT and β-actin (internal standard), and the relative expression levels of mRNA according to the differentiation time are shown in Fig. 12. PCR amplification results of CD31, VE-cadherin, VEGFR2 and β-actin (internal standard), and the relative expression levels of mRNA according to the differentiation time are shown in Fig. 13.

[0086] From the results of PCR, culturing hMSCs on the substrate surface co-modified with hE-cad-Fc / hVE-cad-Fc / hVEGF-Fc and the substrate surface modified with hE-cad-Fc can significantly improve the expression of AFP and ALB, which are hepatocyte-like cell markers of hMSCs, and CD31, which is a vascular endothelial-like cell marker, promote the expression and maintenance of OATP and MRP2, which are cell polarity proteins, and promote the expression of G6Pase and α1-AT, which are several metabolic enzymes related to liver function by the cells. Also, it was found that the differentiation induction efficiency on the co-modified substrate surface is the best. Also, from Fig. 13, the expression of CD31, a marker of vascular endothelial-like cells, starts at the 1-week time point, and its expression time is earlier than that of ALB and AFP, which are hepatocyte-like cell markers (Fig. 10), and is similar to the in vivo development process of the liver (that is, first, endothelial cells are generated and formed, and then epithelial cells are generated and formed). Therefore, on the substrate surface of such a fusion protein, hMSCs can regulate the functions of hMSCs such as autocrine and paracrine by the fusion protein, bioengineer the external microenvironment of the cells, mimic the in vivo development process of hepatocytes, increase the differentiation efficiency, shorten the differentiation time, and it was proved that the obtained cells can better maintain and express the functions as hepatocyte-like cells and vascular-like endothelial cells.

[0087] At the above time point, the cell supernatant was collected, and the expression levels of ALB and Urea were detected by ELISA. The results of ELISA are shown in Fig. 14. As can be seen from Fig. 14, for the secretion of albumin and the synthesis of Urea by the differentiated cells detected by ELISA, the expression levels increased step by step from day 14 of differentiation, and it was found that the expression levels in the hE-cad-Fc / hVE-cad-Fc / hVEGF-Fc group were consistently higher than those in the hE-cad-Fc and collagen groups.

[0088]

Table 15

[0089] Example 8. Detection of the directed differentiation induction of hMSCs into hepatocytes on the substrate surface with different modifications - Immunofluorescence staining The laser confocal cell culture dish was processed according to the method of Example 7, and hMSCs were induced to differentiate in different differentiation induction media. After continuous culture for 28 days, immunofluorescence staining was performed. The cells were fixed, perforated, blocked, and then 100 μL of diluted ALB antibody (Abcam, USA) was added at a dilution ratio of 1:500 and incubated overnight at 4°C. Then, 100 μL of FITC-labeled goat anti-rabbit IgG antibody (Thermo Fisher Scientific, USA, product number A27034) was added at a dilution ratio of 1:1000 and incubated at 37°C for 2 h and then at room temperature. Then, an anti-fading agent containing DAPI (Anti-Fade Mouting Medium, SouthernBiotech, USA, product number 0100-20) was added and stored at 4°C in the dark. Photographs were taken with a laser confocal microscope (Leica). The results are shown in Fig. 15.

[0090] As can be seen from Fig. 15, at the time point of 4 weeks of continuous differentiation of hMSCs on different substrate surfaces, from the results of immunofluorescence staining, on the substrate surface co-modified with hE-cad-Fc / hVE-cad-Fc / hVEGF-Fc, the cells expressed more ALB (indicated by arrows), which is a marker of hepatocyte-like cells. This proved that the efficiency of directional differentiation of hMSCs into hepatocyte-like cells on such a substrate surface was the highest, and the expression of its liver function was also the strongest.

[0091] The process of immunofluorescence staining is as follows. [1] Discard the medium, wash the cells 3 times with cold 0.01 M PBS, discard the PBS, add freshly prepared 4% paraformaldehyde, and incubate at room temperature for 10 minutes; [2] Discard the paraformaldehyde, wash the cells 3 times with cold 0.01 M PBS. If the target protein is in the cytoplasm, it is necessary to incubate with a PBS solution containing 0.1% Triton X-100 at room temperature for 10 minutes and wash the cells 3 times with cold 0.01 M PBS. If the target protein is in the cell film, this step is not required; [3] Incubate with 10% goat serum at room temperature for 30 minutes, discard the supernatant, dilute the primary antibody (ALB antibody) with 0.01 M PBS solution at a ratio of 1:500, then add it to the cell culture dish and incubate overnight at 4°C; [4] Discard the supernatant, wash the cells 3 times with 0.01 M PBS, 5 minutes each time; [5] Dilute the secondary antibody (goat anti-rabbit IgG antibody) with 0.01 M PBS solution at a ratio of 1:500, then add it to the cell culture dish and incubate at 37°C in the dark for 2 h; [6] Discard the supernatant, wash the cells 3 times with 0.01 M PBS in the dark, 5 minutes each time; [7] Discard the supernatant, add a fluorescence quenching inhibitor containing DAPI, and store at 4°C in the dark. Take pictures with a laser confocal microscope (Leica).

[0092] Example 9. Investigation of the mechanism of cell directed differentiation induction on the surface of substrates with different modifications - cytokines, secretion of substrate proteins, and expression of proteins related to the regulation of differentiation Cultivate cells according to the method of Example 7, induce differentiation, and extract RNA and proteins on day 0, day 7, day 14, day 21, and day 28. Reverse transcribe the extracted RNA and detect the expression status of related cytokines and substrate proteins by fluorescence quantitative PCR (see Example 3 for primer sequences and annealing temperatures). The obtained data was processed by the 2-ΔΔCt method with the collagen group as the control group; the extracted proteins were detected for the expression status of β-catenin (BD, USA, diluted 1:1000), α-catenin (BD, USA, diluted 1:1000), total VEGF receptor 2 (CST, USA, diluted 1:1000), phosphorylated-VEGF receptor (phosphorylated at Tyr 1175) (CST, USA, diluted 1:1000), total EGF receptor (CST, USA, diluted 1:1000), and phosphorylated-EGF receptor (further phosphorylation at Tyr 1068) (CST, USA, diluted 1:1000) by Western blot.

[0093] As can be seen from FIG. 16, during the differentiation process, the expression of cellular β-catenin continuously decreases. Also, on the surface of the substrate co-modified with hE-cad-Fc and hVE-cad-Fc / hVEGF-Fc, the level of β-catenin expressed by the cells is the lowest, which contributes to the differentiation of hMSCs into epithelial cells. At the same time, when exogenous soluble EGF and VEGF are not added, compared with the collagen group and the hE-cad-Fc modified group, the continuous positive expression of phosphorylation of the EGF receptor and the VEGF receptor in the cells of the co-modified group with hE-cad-Fc / hVE-cad-Fc / hVEGF-Fc is due to hE-cad-Fc / hVE-cad-Fc / hVEGF-Fc continuously activating the EGF receptor and the VEGF receptor, proving that it contributes to the differentiation of hMSCs into epithelial cells and endothelial cells.

[0094] From FIG. 17, it was found that during the differentiation induction process, compared with the collagen surface, hMSCs cultured on the surface of the fusion protein continuously highly expressed extracellular matrix protein components (fibronectin, collagen, and laminin), highly expressed cytokines (TGFβ and HGF), and IL-6 at the initial stage of differentiation induction, and VEGF was lowly expressed over time. From such results, it was found that the fusion protein substrate has the function of regulating the secretion function of stem cells and reconstructing the cellular microenvironment, which contributes to the control of inducing the directional differentiation of stem cells into epithelial and endothelial cells by biomimetically imitating the development process of the liver.

[0095] Example 10. Investigation of the mechanism of directional differentiation induction of cells on the surface of substrates with different modifications - Expression and distribution of YAP protein The laser confocal cell culture dish was processed in the same manner as in the method of Example 3. After culturing hMSCs in the maintenance medium for 48 hours, immunofluorescence staining was performed by the method of Example 8. 100 μL of diluted YAP antibody (Santa Cruz, USA) was added at a dilution ratio of 1:1000, and cultured overnight at 4°C. 100 μL of FITC-labeled goat anti-mouse IgG antibody (Thermo Fisher Scientific, USA, product number A32723) was added at a dilution ratio of 1:1000, and incubated at 37°C for 2 h at room temperature. An anti-fluorescence quenching agent containing DAPI (SouthernBiotech, USA, product number 0100-20) was added and stored at 4°C in the dark. Photographs were taken with a laser confocal microscope (Leica). The results are shown in Fig. 18.

[0096] As can be seen from Fig. 18, when hMSCs were cultured on the substrate surface modified with the fusion protein, it can be seen that the YAP protein was more distributed in the cytoplasm. When cultured on the TCPS substrate and the collagen substrate surface, the YAP protein was similarly distributed in the cell nucleus and cytoplasm. According to these results, the substrate modified with the fusion protein can regulate the distribution of the YAP protein in cells, indicating that it promotes the induction of differentiation of stem cells into epithelial cells.

[0097] Example 11. Investigation of the mechanism of cell-directed differentiation induction on the substrate surface with different modifications - Expression of genes related to the MET pathway Cells were cultured in the same manner as in the method of Example 7 to induce differentiation. RNA was extracted and reverse transcribed on days 0, 7, 14, 21, and 28. The expression status of related genes (primer sequences and annealing temperatures are shown in the following table) was detected by PCR, and the results are shown in Fig. 19. As can be seen from Fig. 19, since the fusion protein can promote the expression of HNF4α, a hepatocyte nuclear transcription factor, it further promotes the cells to express α-catenin and E-cadherin, suppresses the cells from expressing Snail, vimentin, and Twist, and promotes the cells to undergo MET transformation.

[0098]

Table 16

[0099] Example 12. Immobilization of fusion protein on the surface of PLGA microbeads and detection of stability According to the method of Example 3, mixed solutions were prepared respectively, with a final concentration of 10 μg / mL hE-cad-Fc, 10 μg / mL hVE-cad-Fc, and final concentrations of 0 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, and 25 μg / mL of hVEGF-Fc. 1 mg of PLGA microbead powder (sigma, USA, product number 805114) was immersed in each of the above mixed solutions, shaken well, then placed in a horizontal rotary shaker at 37°C and incubated at 150 rpm for 2 h. The supernatant was discarded, and the mixture was shaken with PBS to wash away the unfixed fusion proteins (3 times). Then, a fixed amount was detected. 300 μL of 5% BSA solution was added, and the mixture was placed on a horizontal rotary shaker at 37°C and blocked at 150 rpm for 2 h. 100 μL of diluted HRP-labeled goat anti-human IgG (Abcam, USA) was added at a dilution ratio of 1:10000, and the mixture was placed on a horizontal rotary shaker at 37°C, blocked at 150 rpm in the dark for 1 h. After washing 5 times with 0.01 M PBS, 300 μL of TMB (Solarbio, product number PR1200) chromogenic solution was added to each tube, and the mixture was placed on a horizontal rotary shaker at 37°C, reacted at 150 rpm in the dark for 30 min. 300 μL of stop solution was added, and 200 μL of the solution was taken and added to a 96-well plate. The absorbance value was measured at 452 nm to assay the maximum immobilization amount when three types of fusion proteins were co-immobilized on the microbead surface. Then, hE-cad-Fc, hVE-cad-Fc, and hVEGF-Fc were co-immobilized on the PLGA surface at the maximum immobilization amount according to the above method, and after immersion in PBS, DF12 medium, and DF12 + 10% FBS for 28 days respectively, the absorbance of the supernatant was detected on days 0, 7, 14, 21, and 28 according to the above method to evaluate the immobilization time of the fusion proteins on the microbead surface.Using the DyLightTM550 Antibody Labeling Kit (ThermoFisher, product number 84530), the DyLightTM488 Microscale Antibody Labeling Kit (ThermoFisher, product number 53025), and the DyLightTM405 Antibody Labeling Kit (ThermoFisher, product number 53020), respectively, hE-cad-Fc, hVE-cad-Fc, and hVEGF-Fc were labeled (the labeling procedure is as follows). According to the maximum fixation amount, three types of fluorescently labeled proteins were co-fixed on the surface of PLGA microbeads, and the distribution of the fusion proteins on the surface of the microbeads was observed with a laser confocal microscope. The results are shown in Figure 20.

[0100] As can be seen from Figure 20, after co-fixing 10 μg / mL hE-cad-Fc and 10 μg / mL hVE-cad-Fc on the surface of the same PLGA microbeads, the absorbance of the whole system increased more and more with the increase in the concentration of VEGF-Fc. It was proved that the three types of fusion proteins were simultaneously fixed on the surface of the microbeads; when immersed in the commonly used PBS, medium, or medium containing serum for a certain period, it was found that the absorbance of the supernatant hardly changed, proving that the fusion proteins could stably exist on the surface of the microbeads for up to 4 weeks; by fixing the fluorescein-labeled fusion proteins on the surface of the microbeads and assaying with a laser confocal microscope, it was found that the three types of fusion proteins could be uniformly distributed on the surface of the microbeads.

[0101] The fluorescence labeling process of the fusion protein is as follows. [1] 40 μL of 0.67 M borate buffer was taken and uniformly mixed with 500 μL of 2 mg / mL fusion protein solution; [2] The above solution was added to a test tube pre-loaded with 50 μg of fluorescent dye, gently vortexed, pipetted with a pipette to mix uniformly, and incubated at room temperature for 60 minutes in the dark; [3] Take 250 μL of the uniformly mixed adsorption resin, put it into a centrifuge tube containing a centrifuge column, centrifuge at 1000 g for 1 minute, discard the supernatant, and transfer the centrifuge column to a new centrifuge tube; [4] Add 250 μL of the solution after the reaction in step [2] to the centrifuge column in step [3], vortex slightly to mix the reaction solution and the adsorption resin; [5] Centrifuge at 1000 g for 1 minute to collect the purified solution, complete the labeling of the fusion protein, and store it at 4 °C or -80 °C in the dark.

[0102] Example 13. Preparation of cell aggregates and selection of the mixing ratio of PLGA microbeads and cells Mix 8×105 hMSC cells and PLGA microbeads at ratios of 3:1, 1:1, and 1:3 respectively, add them to an AggrewellTM (Stemcell Technologies, product number 27845) culture plate, centrifuge at 1000 rpm for 5 minutes, then place them in a cell incubator and culture overnight at 37 °C. Observe the morphology of the cell aggregates under a microscope. The results are shown in Figure 21. As can be seen from Figure 21, when the ratio of microbeads to cells is between 1:1 and 1:3, the aggregates obtained after 18 hours of incubation have a complete structure and uniform size.

[0103] Subsequently, the aggregates were blown out with a pipette tip with the tip cut off at 1000 μL, placed in a 15 mL centrifuge tube, and the precipitate was collected by natural sedimentation. The precipitate was resuspended with 200 μL of a 2% alginic acid solution, dropped into a 0.1 M CaCl2 solution, and incubated for 10 minutes to form an alginic acid hydrogel. The supernatant was discarded, the alginic acid hydrogel was washed with physiological saline, cell aggregates containing different ratios of microbeads were placed in a 6-well culture plate, and continuously cultured for 7 days in a differentiation medium (DF12 + 2% FBS + 10 ng / mL FGF-4 + 10 ng / mL FGF-2 + 20 ng / mL HGF + 1*ITS + 20 ng / mL EGF + 40 ng / mL hVEGF), and the medium was changed every 2 days. On the 7th day, the alginic acid hydrogel was dissolved with a 0.1 M EDTA solution. The cell aggregates were collected, 1 mL of Trizol was added, and RNA was extracted. According to the method of Example 7, the expression of genes related to epithelial cells and endothelial cells was assayed. The results are shown in Figure 22. As can be seen from Figure 22, when cultured for 1 week with the addition of the directed differentiation induction medium, if the ratio of microbeads to hMSC was 1:3, it was observed that the ability of the aggregates to express hepatocyte-like cell genes (CK18, ALB, and ASGPR) and vascular endothelial-like cell genes (CD31 and VEGFR2) was the strongest. This proved that the differentiation efficiency of the stem cell aggregates into epithelial cells and endothelial cells was the highest here. Therefore, this ratio was selected for the next test.

[0104] Example 14. Selection of different concentrations of fusion protein on the surface of PLGA microbeads hE-cad-Fc and hVE-cad-Fc were diluted to concentrations of 3:1 (hE-cad-Fc: 15 μg / mL, hVE-cad-Fc: 5 μg / mL), 1:1 (hE-cad-Fc: 10 μg / mL, hVE-cad-Fc: 10 μg / mL), and 1:3 (hE-cad-Fc: 5 μg / mL, hVE-cad-Fc: 15 μg / mL). After that, hVEGF-Fc with a final concentration of 2 μg / mL was added, and PLGA microbeads were modified with the fusion proteins at the different mixing ratios respectively by the method of Example 12, and PLGA microbeads were modified with 10 μg / mL collagen. Then, the microbeads and hMSC were mixed at a ratio of hMSC: microbeads = 3:1. Cell aggregates were prepared and induced to differentiate by the method of Example 13. On the 7th day, RNA was extracted and reverse-transcribed in the same manner as in Example 7 to detect the expression of related genes. The results are shown in Figure 23 (here, mcp represents cell aggregates containing collagen-modified microbeads, and E:VE / VEGF-Fc represents cell aggregates containing microbeads modified with two types of fusion proteins at different ratios). From Figure 23, it was observed that when cultured for one week after adding the directed differentiation induction medium, if the ratio of hE-cad-Fc to hVE-cad-Fc on the surface of the same PLGA microbeads was 1:1, the expression ability of the aggregates for hepatocyte-like cell genes (HNF4α, ALB, OATP, α1-AT, and G6Pase) and vascular endothelial-like cell genes (CD31 and VE-cadherin) was the strongest. This proved that the differentiation efficiency of the stem cell aggregates into hepatocyte-like cells and vascular endothelial-like cells was the highest here. Therefore, this ratio was selected for the next test.

[0105] Example 15. Determination of the ratio between microbeads modified with different fusion proteins By the method of Example 12, PLGA microbeads were modified with a 10 μg / mL collagen solution, hE-cad-Fc modified PLGA microbeads were prepared with a 10 μg / mL hE-cad-Fc solution, and hVE-cad-Fc / hVEGF-Fc modified PLGA microbeads were prepared with a 10 μg / mL hVE-cad-Fc and 2 μg / mL hVEGF-Fc solution. Then, the two types of microbeads were mixed at ratios of 3:1, 1:1, and 1:3. hMSCs were mixed with the microbeads at a ratio of hMSC:microbeads = 3:1. Cell aggregates were prepared and induced to differentiate by the method of Example 13. On the 7th day, RNA was extracted and reverse-transcribed in the same manner as in the method of Example 7 to detect the expression of related genes. The results are shown in Fig. 24 (where mcp represents cell aggregates containing collagen-modified microbeads, and mEp:m(VE / VEGF)p represents cell aggregates containing fusion protein-modified microbeads at different ratios). As can be seen from Fig. 24, when the ratio of hE-cad-Fc modified PLGA microbeads to hVE-cad-Fc / hVEGF-Fc modified microbeads cultured for 1 week with the addition of the directed differentiation induction medium is 1:1, the expression ability of the liver-like cell genes (HNF4α, OATP, and ASGPR) and vascular endothelial-like cell gene (CD31) in the aggregates is the strongest, and it was observed that the differentiation efficiency into liver-like cells and vascular endothelial-like cells is the highest. Therefore, this ratio was selected for the next test.

[0106] Example 16. Examination of Directed Differentiation Induction for Aggregates According to the method of Example 12, microbeads modified with different protein solutions (hE-cad-Fc fusion protein, hVE-cad-Fc / hVEGF-Fc fusion protein, or collagen) were prepared. The details of the modification are as follows. (1) PLGA microbeads were modified with 10 μg / mL collagen; (2) The same PLGA microbeads were co-modified with 10 μg / mL hE-cad-Fc, 10 μg / mL hVE-cad-Fc, and 2 μg / mL hVEGF-Fc fusion proteins; (3) Some of the PLGA microbeads were modified with 10 μg / mL hE-cad-Fc, and some other PLGA microbeads were modified with 10 μg / mL hVE-cad-Fc and 2 μg / mL hVEGF-Fc. The two types of microbeads were mixed at an equal ratio.

[0107] By the method of Example 13, cells were co-mixed with microbeads at a ratio of hMSC:microbeads = 3:1 to prepare aggregates containing collagen-modified microbeads (abbreviated as mcp), aggregates containing co-modified microbeads of hE-cad-Fc, hVE-cad-Fc and hVEGF-Fc (abbreviated as (E / VE / VEGF)p), and aggregates containing a mixture of microbeads individually modified with different fusion proteins at an equal ratio (abbreviated as (mE + mVE / VEGF)p). Aggregates of hMSC without microbeads (abbreviated as m) were used as a control and continuously cultured in the following specific differentiation medium for 28 days. RNA was extracted on days 0, 7, 14, 21 and 28 and detected by PCR. The sequences of specific primers are shown in Example 7. The results are shown in Figure 25.

[0108] As can be seen from FIG. 25, FoxA2 and Sox17 were expressed earlier in the aggregates containing the fusion protein. This demonstrated that the fusion protein could initiate the differentiation into endoderm earlier. In the aggregates containing the fusion protein, hepatocyte-related genes such as HNF4α, CK18, AFP, and ALB, and endothelial cell-related genes such as CD31, VE-cadherin, and VEGF receptor 2 were expressed earlier. This demonstrated that the fusion protein could promote the induction of differentiation from stem cells into epithelial cells and endothelial cells. In the aggregates containing the fusion protein, hepatocyte polarity protein genes (MRP2, OATP, and ASGPR) and related genes of related metabolic enzymes (G6Pase and α1-AT) were expressed earlier. This demonstrated that the fusion protein could promote the hepatocyte structure and function. Compared with the 2D culture method of Example 7, the differentiation efficiency of the aggregates was significantly increased, and the required differentiation time was greatly shortened. Compared with 2D culture, 3D culture could significantly improve the expression of OATP and MRP2, which are hepatocyte polarity genes, and the cells were more effective in coordinating with internal and external environmental factors and promoted the directed differentiation into bile duct epithelial cells; In the hMSC aggregates containing the fusion protein co-immobilized on the same PLGA microbead surface, the indicators related to cell differentiation were efficiently expressed, and the induction of directed differentiation of hMSC aggregates was promoted.

[0109] Differentiation medium: m, mcp: DF12 + 2% FBS + 10 ng / mL FGF-4 + 10 ng / mL FGF-2 + 20 ng / mL HGF + 1*ITS + 20 ng / mL EGF + 40 ng / mL hVEGF; m(E / VE / VEGF)p, (mE + mVE / VEGF)p: DF12 + 2% FBS + 10 ng / mL FGF-4 + 10 ng / mL FGF-2 + 20 ng / mL HGF + 1*ITS.

[0110] Example 17. Staining of Organoids By the method of Example 16, four types of aggregates (m, mcp, m(E / VE / VEGF)p, and (mE + mVE / VEGF)p, respectively) were prepared and induced to differentiate. After two weeks of differentiation induction, alginic acid was dissolved with a 0.1 M EDTA solution containing 0.9% NaCl solution, and the precipitate of the aggregate was collected. In all the following steps, operations were carried out with a pipette tip treated with a PBSB solution (0.01 M PBS solution containing 5% BSA). The organoids were washed three times with cold 0.01 M PBS solution, the supernatant was discarded, 300 μL of freshly prepared 4% paraformaldehyde solution was added, and incubated at room temperature for at least 30 minutes. The precipitate was collected, the organoids were washed three times with cold 0.01 M PBS solution, the supernatant was discarded, 300 μL of PBSDT solution (0.01 M PBS + 1% BSA + 1% goat blocking serum + 0.5% Triton X-100 + 1% DMSO) was added, and blocked at 4°C overnight. The supernatant was discarded, 100 μL of the primary antibody diluted at a certain ratio with PBSDT solution (the dilution ratio will be described below) was added, and reacted at 4°C for 24 - 48 h. The supernatant was discarded, the organoids were washed three times with cold 0.01 M PBS solution, the supernatant was discarded, 100 μL of the secondary antibody diluted at a certain ratio with PBSDT solution (the dilution ratio will be described below) was added. The supernatant was discarded, the organoids were washed three times with cold 0.01 M PBS solution, the supernatant was discarded, and 100 μL of the fluorescence quenching inhibitor containing DAPI was added and stored at 4°C in the dark. Photographs were taken and observed with a laser confocal microscope (Leica). The results are shown in Figure 26. From the results of immunofluorescence staining when hMSC aggregates containing different materials were continuously induced to differentiate for two weeks, it was found that on the substrate surface co-fixed with hE-cad-Fc / hVE-cad-Fc / hVEGF-Fc, cells expressed ALB, a marker of more hepatocyte-like cells, and CD31, a marker of endothelial cells (indicated by arrows). It was proved that the differentiation efficiency from hMSC to hepatocyte-like cells and endothelial-like cells was the highest on the surface of the substrate.

[0111]

Table 17

[0112] Example 18. Comparison with Endothelial Cell Co - culture System hMSCs were mixed with human umbilical vein endothelial cells (HUVECs, Saiergen Bio) at a ratio of 2:8, and 8×10⁵ cells were added to an aggrewell culture plate (abbreviated as HM), and compared with m(E / VE / VEGF)p and (mE + mVE / VEGF)p, which are optimized test groups. The cells were continuously cultured in the directed differentiation induction medium for 2 weeks by the method of Example 16. At the 2 - week time point, RNA and culture supernatants of the samples were extracted, and the expression status of related genes (ALB, MRP2, OATP, α1 - AT, CD31, and VE - cadherin) was detected by PCR respectively, and the cell - secreting ability of ALB was detected by ELISA. The results are shown in Figure 27.

[0113] As can be seen from Figure 27, after 2 weeks of adding the orientation induction medium, the following results were shown by PCR. The control group HM expressed CD31 and VE - cadherin, which are related endothelial cell markers, better than the test groups. Also, since CD31 - and VE - cadherin - positive cells appeared in the test groups differentiated for 2 weeks on the surface of the fusion protein, the presence of endothelial - like cells in the aggregates was indicated. Moreover, the expression levels of genes related to liver differentiation function (ALB, MRP2, and α1 - AT) in the test groups were higher than those in the HM group. Furthermore, the secretion of albumin, which indicates liver differentiation function, was detected by ELISA. From the results, the test groups were significantly better than the HM control group, and the m(E / VE / VEGF)p group showed the highest expression level of indicators related to hepatocytes.

[0114] Example 19. Confirmation of the Ability of hMSCs to Differentiate into Pancreatic Islet Cells on Substrate Surfaces with Different Modifications According to the method of Example 7, 6-well cell culture plates with different fusion protein modifications were prepared. hMSCs were seeded on the surface of the culture plates at a cell density of 105 cells / well and cultured overnight in maintenance medium. After that, they were directionally induced for one week in a specific differentiation medium (10 ng / mL EGF, 10 ng / mL FGF-2, 10 mM nicotinamide, 1X ITS-G DMEM differentiation medium), and then the medium was replaced with a DMEM differentiation medium supplemented with 10 ng / mL HGF, 10 ng / mL FGF-2, 10 mM nicotinamide, and 1X ITS-G for two weeks of orientation induction), and the medium was changed every two days, and continuous culture was carried out for 21 days. According to the method of Example 7, RNA was extracted on the 21st day, and the expression status of related genes was detected by PCR. The results are shown in Figure 28. As can be seen from Figure 28, culturing hMSCs on a substrate surface modified with hE-cad-Fc / hVE-cad-Fc / hVEGF-Fc can also significantly improve the differentiation efficiency of hMSCs into islet-like cells and the maturity of islet-like cells. In the process of pancreatic development, signal molecules secreted by epithelial cells and signal molecules secreted by endothelial cells play extremely important roles in the regulation process of endocrine and exocrine cell fates. In addition, in mature islets, insulin secreted by pancreatic islet β cells or glucagon secreted by α cells both need to be mediated through endothelial signals and the vascular network. From the results shown in Figure 28, it is presumed that the presence of the hE-cad-Fc / hVE-cad-Fc / hVEGF-Fc fusion protein, especially in the presence of endothelial signals and endothelial cells, binds to growth factors and promotes the differentiation of cells into different fates. Pancreatic islet cells provide a new solution for maintaining their functions in vitro.

[0115]

Table 18

[0116] Example 20. Examination of directional differentiation induction from aggregates to endothelial cells According to the method of Example 12, hVE-cad-Fc / hVEGF-Fc modified PLGA microbeads were prepared with 10 μg / mL hVE-cad-Fc and 2 μg / mL hVEGF-Fc solutions. Different aggregates were prepared by mixing cells with microbeads at a ratio of hMSC:microbeads = 3:1 according to the method of Example 13. Cell aggregates without microbeads (abbreviated as m) and cell aggregates containing PLGA microbeads (abbreviated as mp) were used as the control group; cell aggregates containing microbeads co-modified with hVE-cad-Fc and hVEGF-Fc (abbreviated as mvp) were used as the research group, and cultured in an endothelial-directed differentiation induction medium (DF12 + 2% FBS + 10 ng / mL FGF-2), with the medium changed every two days, and continuously induced for differentiation and cultured for 7 days. On the 7th day, RNA and protein were extracted, and the expression of related endothelial cell markers (vWF, CD31, and VE-cadherin) by PCR, and the changes in the matrix and factors secreted by the cells (fibronectin, VEGF, IL-8, IL-6, MCP-1, IGFBP-3, and angiopoietin) were detected. Refer to Example 3, Example 7, and the following table for specific primer sequences; the expression status of related endothelial cell markers CD31, VE-cadherin, and vWF (Abcam, USA, diluted at 1:1000) was detected by Western blot. The results are shown in Figure 29.

[0117] As can be seen from Fig. 29A, compared with the m group and the mp group, the mvp group of cell aggregates containing hVE-cad-Fc / hVEGF-Fc modified PLGA microbeads showed a significant improvement in the expression ability of endothelial cell markers such as vWF, CD31, and VE-cadherin at both the gene level and the protein level. Therefore, it was shown that the hVE-cad-Fc / hVEGF-Fc fusion protein can significantly promote the differentiation of hMSCs into vascular endothelial-like cells and the expression of endothelial cell markers. Moreover, as the differentiation induction time was extended, the expression level of endothelial cell markers in the cells of the mvp research group continued to increase compared with the control group. Therefore, it was shown that hVE-cad-Fc / hVEGF-Fc can cooperate with differentiation factors in the medium and continuously promote the differentiation of hMSCs into endothelial cells and the expression of their functions. As can be seen from Fig. 29B, at the initial stage of the differentiation induction process, the expression levels of cytokines such as fibronectin, VEGF, IL-8, IL-6, MCP-1, IGFBP-3, and angiogenin in the cells of the mvp group were higher than those of the two control groups. Therefore, it was shown that the fusion protein can significantly improve the secretion of endothelial cell-specific related cytokines and the extracellular matrix by cells, construct an endothelial cell microenvironment, and promote the differentiation of hMSCs into endothelial cells.

[0118]

Table 19

[0119] Example 21 Functional Modification of PAMAM / Hyaluronic Acid Hydrogel The synthesis of G5-Ac-DVS, G5-Ac-DVS-RGD, and G5-Ac-DVS-Fc-bp is as follows. The 5th generation PAMAM (PAMAM G5) dendrimer (502 mg, 0.0197 mmol, purchased from Dendritech Inc., Midland, MI, USA) was dissolved in methanol (60 mL), and triethylamine (259 mL, 1.846 mmol) was added, followed by stirring at room temperature for 10 minutes. A solution of acetic anhydride (140 mL, 1.477 mmol) in methanol (40 mL) was added dropwise to the reaction mixture. The reaction was carried out overnight, followed by dialysis using a 10,000 molecular weight cut-off film and washing with PBS (10 mL × 3 times) and deionized water (10 mL × 3 times). Freeze-drying yielded the G5-Ac product.

[0120] G5-Ac (333 mg, 0.0116 mmol) was dissolved in deionized water (20 mL). 2-Sub-amino hydrochloride thiol (80.05 mg, 0.582 mmol) was added. The reaction was carried out at room temperature for 24 hours under a nitrogen gas shield. Dialysis was performed using a 10,000 molecular weight cut-off film, and washing was carried out with PBS (10 mL × 3 times) and deionized water (10 mL × 3 times). Freeze-drying yielded the G5-Ac-SH product. G5-Ac-SH (213.4 mg, 0.00661 mmol) was dissolved in deionized water (15 mL). Divinyl sulfone (DVS, 132.5 μL, 1.321 mmol) was added. The reaction was carried out at room temperature for 2 days under a nitrogen gas shield. Dialysis was performed using a 10,000 molecular weight cut-off film, and washing was carried out with PBS (10 mL × 3 times) and deionized water (10 mL × 3 times). Freeze-drying yielded G5-Ac-DVS.

[0121] G5-Ac-DVS (58.5 mg, 0.00166 mmol) was dissolved in deionized water (5 mL). The RGD polypeptide CGRGDS (SEQ ID NO: 92, 6.9 mg, 0.0116 mmol) was dissolved in deionized water (1 mL). The two solutions were mixed. The reaction was carried out at room temperature for 1 hour. Dialysis was performed with a film having a molecular weight cut-off of 10,000, and washing was carried out with PBS (10 mL × 3 times) and deionized water (10 mL × 3 times). Lyophilization was carried out to obtain G5-Ac-DVS-RGD.

[0122] G5-Ac-DVS (164 mg, 0.00513 mmole) and the Fc-binding polypeptide (4.83 mg, 0.00513 mmol, CHWRGWV, SEQ ID NO: 93, hereinafter abbreviated as Fc-bp) were each dissolved in 5 mL and 1 mL of oxygen-free water. Then, the polypeptide solution was slowly added to the dendrimer solution with stirring. The mixed solution was stirred and reacted at room temperature for 1 hour. The reacted mixed solution was filtered through a film having a molecular weight cut-off of 10,000. The residue was washed with PBS (15 mL × 3 times) and water (15 mL × 3 times), respectively. Finally, lyophilization was carried out to obtain a G5-Ac-DVS-Fc-bp product with a yield of 95.9%. The synthetic route diagram is shown in Fig. 30-A. The hydrogen spectrum of nuclear magnetic resonance is shown in Fig. 30-b.

[0123] The functional modification of the hyaluronic acid hydrogel is as follows. First, dissolve 25 mg of G5-Ac-DVS-Fc-bp dendrimer in 500 μL of anaerobic PBS until completely dissolved for use. Take 50 μL of the above solution, add 10 μL of hVE-cad-Fc fusion protein with an initial concentration of 200 μg / mL thereto, and incubate at 37 °C for 1 h. Then, add 50 μL of thiolated hyaluronic acid (Bio Time Inc) and place it in a 96-well plate. In the control group, G5-Ac-DVS dendrimer was used, and the other working steps were the same. After forming a stable hydrogel, add 100 μL of PBS to each well to immerse the hydrogel completely, and take the supernatant on the 1st, 3rd, 5th, 7th, 10th, and 14th days respectively, and detect the released amount of hVE-cad-Fc fusion protein by the ELISA kit (R&D) method. The method is as follows according to the kit instructions: For the standard well: Add 50 μL of diluted standard to each well. As the zero well, add 50 μL of standard / sample diluent, and then add 50 μL of enzyme-labeled reagent. For the sample well: Add 50 μL of the sample, and then add 50 μL of enzyme-labeled reagent. Shake gently, cover with a cover film, and incubate at 37 °C for 60 minutes. Carefully remove the cover film, discard the liquid, shake until dry, pour the washing solution into each well until full, let stand for 30 seconds, discard the washing solution, repeat 5 times, and tap to remove moisture. For each well, first add 50 μL of chromogenic agent A, then add 50 μL of chromogenic agent B, shake gently to mix well, and develop color at 37 °C in the dark for 10 minutes. Add 50 μL of stop solution to each well to stop the reaction. Measure the absorbance at 452 nm. From the results, it was found that hVE-cad-Fc using the connection of Fc-bp had a fixation rate to the hydrogel of about 85% within two weeks, and hVE-cad-Fc not using the connection of Fc-bp had a fixation rate to the hydrogel of only about 40% (Figure 30-C). The precursor solutions of the hydrogels modified by the above two methods were respectively added to a 48-well plate to make them uniform. After forming a stable hydrogel, 2×104 HUVECs (ScienCell, USA) and hMSC cells (Guangzhou Saiye Co., Ltd.) were separately inoculated into each well.After 24 hours, the adhesion and morphological characteristics of the two types of cells were separately observed under a phase-contrast microscope. The results showed that the hydrogel modified by the immobilization means using hVE-cad-Fc with Fc-bp could significantly promote cell adhesion compared to the hydrogel system without Fc-bp (Figure 30-D).

[0124] Example 22 Characterization of the Physicochemical Properties of Hyaluronic Acid Hydrogels Functionally Modified with Fusion Proteins Two types of hydrogels were prepared. One of them is the hVE-cad-Fc fusion protein-modified hydrogel: 25 mg of the G5-Ac-DVS-Fc-bp dendrimer was completely dissolved in 500 μL of anaerobic PBS. For use, 50 μL of the solution was taken, 10 μL of the hVE-cad-Fc fusion protein with an initial concentration of 200 μg / mL was added, incubated at room temperature for 1 h, and 50 μL of thiolated hyaluronic acid was added to form the hVE-cad-Fc fusion protein-modified hydrogel. The other is the unmodified hydrogel. 25 mg of the G5-Ac-DVS dendrimer was completely dissolved in 500 μL of anaerobic PBS. For use, 50 μL of the solution was taken, and 50 μL of thiolated hyaluronic acid was added thereto to form the unmodified hydrogel.

[0125] The degradation test of the hydrogel is as follows. Two types of hydrogels were immersed in PBS, hyaluronidase (Sangon Biotech (Shanghai) Co., Ltd.), supernatant of HUVEC medium (ScienCell company, containing 500 mL of basal medium, 25 mL of FBS, 5 mL of endothelial cell growth factor, 5 mL of penicillin / streptomycin), and supernatant of hMSC medium (consisting of a 1:1 mixture of DMEM medium and F12 medium, HyClone) for 2 weeks respectively. Taking the initial weight of the hydrogel as M0, at the time points of 1, 3, 5, 7, 10, and 14 days, excess PBS was sucked out with absorbent paper respectively. The weight of the remaining hydrogel was measured as Mt, and the formula M% = (M0 - Mt) / M0 was used. From the results, it was found that the hydrogel hardly degraded in PBS, almost completely degraded within 24 hours in the hyaluronidase solution, and degraded to about 70% in the cell culture supernatant. The hydrogel modified with hVE-cad-Fc had no change in degradability (Figure 31-A).

[0126] The swelling test of the hydrogel is as follows. Two groups of different hydrogels were immersed in PBS for 2 weeks to be completely soaked respectively. Taking the initial weight of the hydrogel as M0, at the time points of 1, 3, 5, 7, 10, and 14 days, excess PBS was gently sucked out with absorbent paper respectively. The weight of the remaining hydrogel was measured as Mt, and the calculation method was the same as above. Since the hydrogel hardly swelled in the PBS solution, after being modified with hVE-cad-Fc, the swelling property of the hydrogel did not change (Figure 31-B).

[0127] For the rheology test of the hydrogel, 500 μL of the precursor solution samples of the two hydrogels (fusion protein-modified hydrogel, unmodified hydrogel) were prepared respectively, and the viscoelasticity of the hydrogel was detected with a rheometer (AR 2000ex). First, the air compressor was turned on, and the air tank pressure automatically increased. When the air tank pressure reached 0.5 MP, the output pressure was adjusted clockwise to 0.45 MP, and the pressure regulating valve of the rheometer was checked and stabilized for 5 minutes to 30 Pis. Next, the circulating water was turned on, the safety lock of the air bearing was detected, and a parallel fixture plate was installed. Then, the host computer of the rheometer was turned on, the rheology program was tested on the computer, and the vibration of the instrument was corrected; finally, certain parameters were set according to the test requirements, the sample was prepared, and the test was started. The specific parameter settings were: parallel fixture plate 4 cm, gap setting 500 μm, temperature 25 °C. Dynamic time scan: time 7200 s, one point was taken back every 20 s, the frequency was 1 rad / s, and the stress was 1%; the strain scan range was 0.1 - 10%, and the frequency was 1 rad / s; the dynamic frequency scan range was 0.1 - 100 rad / s, and the stress was 1%. When measuring the sample, 500 μL of the test sample was taken and placed in a tray, the gap between the parallel fixture plates was lowered to 500 μm, and the measurement of the sample was started. From the results, it was found that the hyaluronic acid solution could form a hydrogel, the formed hydrogel had good viscoelasticity and a certain mechanical stress strength, and after hVE-cad-Fc modification, the rheological properties of the hydrogel did not change (Figure 31-C).

[0128] Regarding the surface appearance characteristics of the hydrogel, 200 μL of each of the two groups of hydrogel samples were prepared and freeze-dried under vacuum. The dried porous hydrogel was cut into a cubic shape. Using double-sided tape, the sample was attached to the copper plate of the scanning electron microscope with the observation surface facing up. From the results of scanning with SEM (QUANTA 200, FEI), it was found that the interior of the hydrogel had a structure with a relatively homogeneous porosity, and there were no significant changes in the appearance characteristics of the hydrogel before and after modification (Figure 31-D). Among them, the left 1 is the hydrogel of the control group, the left 2 is its local enlarged view, the left 3 is the hVE-cad-Fc fusion protein-modified hydrogel, and the left 4 is its local enlarged view.

[0129] Example 23 Examination of the effect of a hydrogel functionally modified with a fusion protein on the survival of HUVEC The preparation of the test samples is as follows. For the test, it was divided into three groups. (1) In the control group, 25 mg of G5-Ac-DVS dendrimer was completely dissolved in 500 μL of anoxic PBS. For use, 50 μL of the solution was taken, and 50 μL of thiolated hyaluronic acid was added thereto to form a single hydrogel system. (2) In the RGD group, 25 mg of G5-Ac-DVS-RGD dendrimer was completely dissolved in 500 μL of anoxic PBS. For use, 50 μL of the solution was taken, and 50 μL of thiolated hyaluronic acid was added thereto to form an RGD-modified hydrogel system. (3) In the hVE-cad-Fc group, 25 mg of G5-Ac-DVS-Fc-bp dendrimer was completely dissolved in 500 μL of anoxic PBS. For use, 50 μL of the solution was taken, and 10 μL of hVE-cad-Fc with an initial concentration of 200 μg / mL was added and incubated at room temperature for 1 h. Then 50 μL of thiolated hyaluronic acid was added thereto to form an hVE-cad-Fc-modified hydrogel system.

[0130] Observation test of cells under an optical microscope: 100 μL of the precursor solutions of three different hydrogels prepared in advance were added to a 96-well plate to make them uniform, and incubated at 37 °C for 10 minutes, waiting until stable hydrogels were formed. 1×104 cells were seeded in each well and cultured in a cell incubator (37 °C, 5% CO2). When the cells were cultured for 72 hours, the external characteristics of the cells were observed with an inverted phase contrast microscope (Olympus, CKX41). From the results, it was found that in the hVE-cad-Fc modified hydrogel group, structures similar to capillary-like structures appeared in the appearance of HUVECs (Figure 32-A).

[0131] The cell viability and death staining test was as follows. When the cells were cultured for 72 hours, the cell culture medium was aspirated, and the cells were washed with PBS to prepare a cell viability and death staining solution: 2 μL of 2 mM EthD-III dopant + 0.5 μL of 4 mM Calcein Am dopant + 1 mL of PBS. 100 μL of the viability and death staining solution was added to each well, left in the cell incubator for 30 minutes, and washed three times with PBS. Observation was performed with a fluorescence microscope. From the results, it was found that the hVE-cad-Fc modified hydrogel significantly enhanced the cell viability compared to the control group (shown in Figure 32-B).

[0132] Cell proliferation test. 100 μL of the precursor solution samples of three different hydrogels prepared in advance were added to a 96-well plate to make them uniform, incubated at 37 °C for 10 minutes, and waited until a stable hydrogel was formed. 1×10⁴ cells were seeded in each well and cultured in a cell incubator (37 °C, 5% CO₂). The proliferation ability of the cells was detected by the CCK-8 test at 4, 24, 48, and 72 hours respectively. The details are as follows. First, the cell culture medium was aspirated, the cells were washed with PBS, and the previously prepared CCK-8 solution (containing 10 μL of CCK-8 dope (CA1210, Solarbio) and 90 μL of ECM medium (ScienCell) per 100 μL) was added to each well at 100 μL and incubated at 37 °C for 4 hours. The cultured CCK-8 solution was transferred to a new 96-well plate, and the absorbance value at 450 nm was measured with a microplate reader. From the results, it was found that the hVE-cad-Fc modified hydrogel could promote cell adhesion and proliferation (Figure 32-C).

[0133] The test for staining the cytoskeleton is as follows. The precursor solutions of three different hydrogels were added to a laser confocal dish to make them uniform, and waited until a stable hydrogel was formed. 3×10⁴ HUVECs were seeded in each dish and the cells were cultured for 72 hours. The cell culture medium, which was the supernatant, was gently aspirated, the cells were washed with PBS preheated to 37 °C, and the cells were fixed with 4% paraformaldehyde for 10 minutes. The cells were subjected to membrane permeabilization treatment with 0.1% Triton x-100 for 10 minutes and blocked with 1% bovine serum albumin at room temperature for 40 minutes. The cytoskeletal microfilaments were stained with FITC-phalloidin (Sigma). Finally, the cell nuclei were stained with DAPI (Sigma). From the results, it was found that the hydrogel functionally modified with hVE-cad-Fc could significantly promote the elongation and spread of the cytoskeleton (Figure 32-D).

[0134] Examination of the effect of hydrogel on the three-dimensional growth of HUVECs. 100 μL of the precursor solution of hydrogel divided into three different groups was taken, and 105 HUVECs were added to each group. After gently pipetting and mixing uniformly, it was added to a 96-well plate. After the hydrogel was formed, ECM medium was added, and the cell proliferation ability was detected by CCK-8 on the 1st, 3rd, 5th, and 7th days, respectively. The test method was the same as described above. From the results, it was found that the hVE-cad-Fc modified hydrogel could significantly promote cell proliferation compared with the control group (Figure 32-E). Also, on the 7th day, a cell viability and death staining test was performed, and the test method was the same as described above. From the results, it was found that the hVE-cad-Fc functionalized modified hydrogel significantly enhanced the proliferation ability of HUVECs compared with the control group (Figure 32-F).

[0135] Example 24 Effect of Hydrogel Functionally Modified with Fusion Protein on the Vascular Functionalization of HUVECs For the pre-solutions of the three different modified hydrogels described in Example 23, 200 μL of each was prepared and added to a 6-well plate to make it uniform. After forming a stable hydrogel, 1×105 HUVECs were seeded in each well, and cell proteins were extracted at 24, 48, and 72 hours after seeding. The details are as follows. The medium was discarded, and the cells were washed with PBS. The cells were lysed on ice with RIPA (Sigma), the lysate was collected at 13,000 rpm, and centrifuged for 10 minutes. The supernatant was collected, loading buffer was added, and it was boiled for 5 minutes and stored at -20°C for use. Electrophoresis was performed on an 8%-10% gel to separate proteins with different molecular weights. The proteins in the gel were electrotransferred to a PVDF membrane by the wet transfer method. The membrane was blocked, and the primary antibodies against vWF and eNOS (Abcam) were added and incubated overnight at 4°C. The membrane was washed with PBST, the secondary antibody IgG-HRP (Santa Cruz) was added, incubated at room temperature for 2 hours, the membrane was washed with PBST, and the protein expression was detected by ECL luminescence. From the results, it was found that the hVE-cad-Fc modified hydrogel could significantly promote the expression of vWF and eNOS proteins in HUVECs compared with the control group and the RGD group (Figs. 33-A, B).

[0136] The pre-solutions of different modified hydrogels were added to a 96-well plate to make it uniform. After forming a stable hydrogel, 1×104 HUVECs cells were seeded in each well, and the NO release amount of the cells was detected at 4 h, 24 h, 48 h, and 72 h after seeding. The details are as follows. The supernatant of the medium was collected from HUVECs and detected according to the guide instructions of the NO kit (Beyotime Biotechnology, China), and the absorbance value was detected at OD540. From the results, it was found that the hydrogel functionally modified with the hVE-cad-Fc fusion protein could significantly promote the release of NO compared with the control group and the RGD hydrogel group (Fig. 33-C).

[0137] The precursor solutions of different modified hydrogels were added to a confocal dish at 100 μL each to make them uniform. After forming stable hydrogels, 2×104 HUVECs were seeded in each dish. After 72 hours, the phagocytosis of cells against low-density lipoprotein (Ac-LDL) was detected. The details are as follows. At 72 hours of cell culture, the original cell culture medium was removed, washed with PBS, and fluorescently tagged low-density lipoprotein (Invirogen, USA) was added to the new cell culture medium, followed by culturing for about 5 hours. The medium was removed, the cells were washed with PBS, fixed with preheated 4% paraformaldehyde for 10 minutes, washed with PBS, stained with DAPI for 10 minutes to stain cell nuclei, washed with PBS, and observed under a laser confocal microscope. From the results, it was found that compared with the control group and the RGD hydrogel group, the hVE-cad-Fc functionalized modified hydrogel significantly enhanced the phagocytic ability of HUVECs against Ac-LDL (Figure 33-D).

[0138] Example 25. Design and synthesis of hydrazide-modified hyaluronic acid By performing two-step modification on the hyaluronic acid chain with ADH (adipic acid dihydrazide) and NHS-AC (N-acryloxysuccinimide), the hyaluronic acid chain was modified with a hydrazide group. The details are as follows.

[0139] Precisely weigh 1 g of hyaluronic acid (HA, Shandong Furi ▲da▼ Biotechnology Co., Ltd.) powder with a molecular weight of 91 KD. Its hydrogen nuclear magnetic resonance spectrum is shown in Figure 34-a. Add 200 mL of ddH2O and stir until it is completely dissolved. Add 18 g of ADH powder to the solution and stir until it is completely dissolved. Further, add 2 g of EDAC (catalyst) powder and stir until it is completely dissolved. Adjust the pH to 4.75, and adjust the pH of the solution to 4.75 after reacting for 10 minutes, 30 minutes, 1 hour, and 2 hours respectively, and react overnight. On the second day, adjust the pH of the solution to 7 to start the dialysis process: the dialysis solutions are 100 mM sodium chloride solution (48 hours), 1 / 5 ethanol solution (48 hours), and ddH2O (48 hours) respectively. After the dialysis is completed, freeze-dry the powder and weigh the mass of the powder. Also, detect the substitution degree of ADH by hydrogen nuclear magnetic resonance spectrum, as shown in A and B of Figure 34-b. Obtain the HA-ADH product.

[0140] Weigh 0.5 g of the HA-ADH product, add 100 ml of ddH2O and dissolve it. Add NHS-AC with a mass ratio of mHA-ADH:mNHS-AC = 4:3 and dissolve it completely. Adjust the pH to 7.20 to start the reaction. Adjust the pH of the solution to 4.75 at 10 minutes, 30 minutes, 1 hour, and 2 hours after the start of the reaction respectively, and react overnight. On the second day, start the dialysis process: dialyze with 100 mM sodium chloride solution for 48 hours and then with ddH2O for 48 hours. After the dialysis is completed, freeze-dry the powder and weigh the mass of the powder. Also, detect the substitution degree of the Ac group (the two peaks C and D in Figure 34c) by hydrogen nuclear magnetic resonance spectrum. The temperature of all reaction steps in this process was controlled at room temperature. Obtain HA-AC.

[0141] Example 26. Modification of Hydrazide-modified Hyaluronic Acid with hVE-cad-Fc Fusion Protein Preparation of hVE-cad / Fc-binding (-) pre-solution: Add 5 μL of 10 mg / mL hVE-cad-Fc fusion protein to every 100 μL of 5% solution, mix well, and react at 37 °C for 30 minutes. Preparation of pre-solution before hVE-cad / Fc binding (+): To 100 μL of 5% solution, 2 μL of Fc-binding polypeptide with a concentration of 4 mg / mL and 5 μL of 10 mg / mL hVE-cad-Fc fusion protein were added, mixed well, and reacted at 37 °C for 30 minutes. The fixed amount of hVE-cad-Fc fusion protein was assayed by ELISA method. The specific method is as follows. 50 μL of each pre-solution was taken, dithiothreitol (DTT) (purchased from Solarbio Life Sciences Co., Ltd.), a crosslinking agent, was added, mixed well, placed in a 96-well plate, and gelled. After the gelation was completed, it was assayed with an hVE-cadherin ELISA test kit (Solarbio Life Sciences Co., Ltd.). 200 μL of PBS solution was added to the hVE-cad-Fc modified hydrazide-modified hyaluronic acid hydrogel and the hydrazide-modified hyaluronic acid hydrogel modified with the combination of hVE-cad-Fc and Fc-bp, respectively, and incubated. At multiple time points of 1, 3, 5, and 7 days, the supernatant was collected from different solutions, and the hVE-cad-Fc fusion protein remaining in the supernatant was identified with the kit, and its long-term stability was detected. As shown in Figure 35, compared with the hydrogel group without the addition of Fc-bp, in the hydrogel group modified with the combination of hVE-cad-Fc and Fc-bp, 90% of hVE-cad-Fc remained until the 7th day, and in the hydrogel group without the addition of Fc-bp, the remaining amount of hVE-cad-Fc decreased to 50% until the 7th day. This proved that the addition of Fc-bp can significantly increase the long-term sustained stability of hVE-cad-Fc in the hydrogel.

[0142] Example 27. Characterization of the Physicochemical Properties of Hydrazide-Modified Hyaluronic Acid Hydrogel A crosslinking agent containing MMPs-responsive cleavage polypeptide with thiolated ends at both ends was designed, and the effects of the concentration of the hydrogel, crosslinking density, and modification factor on the physicochemical character of the hydrogel were investigated. Both the MMP-2 responsive polypeptide (CRGDPQGIWGQDRC, SEQ ID NO:97), a crosslinking agent, and DTT were diluted to the required concentrations (final concentrations of 160 mM and 292 mM, respectively) with oxygen-free sterile PBS separately. To each 100 μL of the solution of hydrazide-modified hyaluronic acid prepared in Example 25, 3 μL and 6 μL of 0.45 g / mL DTT solution were added respectively, and the degrees of crosslinking were fractionated to 50% and 100%. After thorough mixing and reacting at 37°C for 30 minutes, hydrogels with different degrees of crosslinking were prepared. It is a figure that macroscopically observed the gel states before and after gelation of the hydrogel crosslinked by DDT and MMP-2 and took pictures. The left side of Figure 36-A shows the hydrogel crosslinked by DTT; the right side of Figure 36-A shows the hydrogel crosslinked by the MMP-2 responsive polypeptide.

[0143] To the pre-solution of the hydrogel (containing Fc-bp), an MMP-2 responsive polypeptide crosslinking agent (5 μL of a solution with a concentration of 0.25 g / mL was added per 100 μL of the solution of hydrazide-modified hyaluronic acid) and a DTT crosslinking agent (6 μL of a 0.45 g / mL solution was added per 100 μL of hydrazide-modified hyaluronic acid) were added, and the reaction was carried out at 37°C for 30 minutes. Four hydrogels of DTT, DTT + hVE-cad-Fc, MMP, and MMP + hVE-cad-Fc were generated. 1 mL of PBS solution was added to each hydrogel to fully swell the gel. During the swelling process of the gel, the mass of the gel was weighed at different time points to conduct a swelling test. Also, according to the above test procedures, swelling tests of hydrogels were carried out at two types of low and high crosslinking densities (50% and 100% degrees of crosslinking respectively) to observe the influence of crosslinking density on gel stability. Separately, a DTT-crosslinked hydrogel system and an MMP-2 responsive polypeptide-crosslinked hydrogel system were synthesized. Both hydrogels can stably exist in PBS solution for a long time (the left side of Figure 36-B); also, both solutions of hydrazide-modified hyaluronic acid can gel within 30 minutes after adding the DTT or MMP crosslinking agent (the right side of Figure 36-B). Both of the hydrogels crosslinked by DTT and the MMP-responsive polypeptide have good swelling properties, and the addition of hVE-cad-Fc does not affect the swelling property of the hydrogel itself (Figure 36-C).

[0144] Example 28. Immunofluorescence Staining of Bile Duct Epithelial Cells in Organoids Immunofluorescence staining of liver organoids was performed according to the method of Example 17. The liver organoids were blocked, and the primary antibody diluted at a certain ratio with PBSDT solution (the dilution ratio will be described below) was added, and after reacting at 4°C for 24 - 48 h, the supernatant was discarded, and the organoids were washed 3 times with cold 0.01 M PBS solution. The supernatant was discarded, and 100 μL of the secondary antibody diluted at a certain ratio with PBSDT solution (the dilution ratio will be described below) was added. The supernatant was discarded, the organoids were washed 3 times with cold 0.01 M PBS solution, the supernatant was discarded, 100 μL of the fluorescence fading prevention agent containing DAPI was added, and it was stored at 4°C in the dark. Photographs were taken and observed with a laser confocal microscope (Leica). The results are shown in Figure 37. When hMSC aggregates containing different materials were continuously induced to differentiate for 2 weeks, as can be seen from the immunofluorescence staining, in the m(E / VE / VEGF)p group, the cells expressed more CK18, which is a marker of hepatocytes, and CK19, which is a marker of cholangiocyte epithelial cells (indicated by arrows). Also, the cholangiocyte-like epithelial cells could be distributed relatively uniformly around the hepatocyte-like cells. This proves that the differentiation efficiency from hMSC to hepatocyte-like cells and cholangiocyte-like epithelial cells is the highest on such a substrate surface, and when the three types of fusion proteins are uniformly distributed on the surface of the microbeads, the cells showed more biotechnological self-organization behavior. Also, when 2 - 3 liver organoids aggregated, they interacted with each other and connected to form a certain network structure. Such a network structure was more prominent in the m(E / VE / VEGF)p group (Figure 38) and could form a cholangi-like dendritic structure.

[0145]

Table 20

[0146] Example 29 Ratio of Hepatocyte-like Cells and Endothelial-like Cells in Liver Organoids Hepatic organoids induced to differentiate for 2 weeks were collected in 1.5 mL centrifuge tubes blocked with PBSB, and 1 mL of Cell dissociation reagent (Stemcell Technologies, product number 07174) was added to each tube and digested for 10 minutes. Using a 1 mL tip blocked with PBSB, pipetting was repeated until the organoids were dispersed into single cells, and 1 mL of pre-warmed fresh complete medium was added to stop the digestion. Centrifugation was performed at 1000×g for 5 minutes to collect the precipitate. 1 mL of freshly prepared 4% paraformaldehyde was added to the precipitate and fixed at 37°C for 10 minutes. The supernatant was discarded, the cells were washed 3 times with PBSB solution, and centrifuged at 1000×g for 5 minutes. The supernatant was discarded, 1 mL of 90% cold methanol was added to the precipitate, placed on ice and incubated for 30 minutes to perforate; centrifuged at 4°C, 1000×g for 5 minutes, the supernatant was discarded, the cells were washed with PBSB, the process was repeated once, centrifuged and the supernatant was discarded, and blocked with 0.5% BSA solution at room temperature for 10 minutes; at a ratio of 1:100, separately, rabbit anti-human ALB primary antibody (Abcam, USA), mouse anti-human CD31 primary antibody (CST, USA) were added and incubated at room temperature for 1 hour; centrifuged at 1000×g for 5 minutes, the supernatant was discarded, the cells were washed with PBSB solution, the process was repeated once, and then specific Alexa Fluor 488 marker goat anti-mouse secondary antibody or Alexa Fluor 488 marker goat anti-rabbit secondary antibody was added at a ratio of 1:1000 and incubated at room temperature in the dark for 30 minutes; centrifuged at 1000×g for 5 minutes, the supernatant was discarded, the cells were washed with PBSB solution, the process was repeated once; the precipitate was resuspended in 1 mL of 0.01 M PBS, filtered through a mesh into a flow cytometry tube, and the expression level of positive cells in the sample was analyzed with a flow cytometer.

[0147] As shown in Fig. 39, in the m(E / VE / VEGF)p group, the differentiation efficiencies into hepatocyte-like cells and endothelial-like cells were the highest, reaching 77.1% and 14.9% respectively, and were quite close to the percentage contents of the two types of cells in the liver. In the (mE + mVE / VEGF)p group, about 65.5% of the cells were induced to differentiate into hepatocyte-like cells, and 10.6% of the cells were induced to differentiate into endothelial-like cells. The differentiation efficiency was significantly higher than that of the m group and the mcp group. From the results of flow cytometry, it was found that the microbeads modified with the fusion protein as the substrate could improve the differentiation efficiencies into hepatocyte-like cells and endothelial-like cells during the process of differentiating hMSCs aggregates into liver organoids.

[0148] Example 30 Detection of Glycogen Storage and Uptake and Release Abilities of Indocyanine Green in Liver Organoids Paraffin sections were prepared from the liver organoids induced to differentiate for 2 weeks. They were de-waxed with xylene and returned to water with gradient ethanol. The samples were placed in a periodic acid solution, oxidized at room temperature for 15 - 20 minutes, and washed twice with a 70% ethanol solution; Schiff solution was added to the samples, immersed at room temperature in the dark for 10 - 20 minutes for staining; the supernatant was discarded, and the cells were washed twice with a sodium bisulfite solution for 2 minutes each time, and washed with a 70% ethanol solution for 2 minutes; double-stained with hematoxylin staining solution at room temperature for 1 - 2 minutes, induced to differentiate with hydrochloric acid-ethanol solution for 2s, rinsed with tap water for 2 minutes, then washed twice with distilled water, dehydrated with ethanol in gradient and air-dried, and sealed with a cover glass with neutral gum. Observed with a stereomicroscope and photographed.

[0149] To the liver organoids induced to differentiate for 2 weeks, a medium containing 1 mg / mL indocyanine green solution was added and cultured in a cell incubator for 1 h; after washing the cells three times with sterile PBSB, photographs were taken with a phase contrast microscope; PBSB was discarded, and a new medium without indocyanine green was added and cultured overnight in a cell incubator. Then, the cells were washed three times with sterile PBSB, observed with a phase contrast microscope and photographed.

[0150] Glycogen storage and the ability to uptake and release indocyanine green are commonly used detection means for evaluating hepatocyte function. Therefore, the related functions of hepatic organoids were investigated by PAS staining and indocyanine green uptake and release tests. As can be seen from Figure 40A, hepatic organoids containing fusion protein surface-modified PLGA microbeads (mE+mVE / VEGF)p group, m(E / VE / VEGF)p group) can be stained red-purple with PAS dye, which proves that they have better glycogen storage ability. Also, in the (mE+mVE / VEGF)p group and m(E / VE / VEGF)p group, hepatic organoids can uptake more indocyanine green dye and thus show deep green. When cultured after changing to a new medium, hepatic organoids can release indocyanine green (Figure 40B). From the above results, it was proved that hepatic organoids containing fusion protein surface-modified microbeads have the ability of storage, uptake, and release.

[0151] Example 31 Expression of Polar Proteins and Metabolic Enzymes in Hepatic Organoids In hepatic organoids induced to differentiate for 2 weeks, the expression and distribution of MRP2, a protein related to drug metabolism, and metabolic enzyme CYP3A4 were investigated by immunofluorescence staining using the methods of Examples 17 and 28. Observation and photography were performed with a laser confocal microscope under 488 nm, 561 nm, and ultraviolet excitation light. The dilution ratios of the antibodies are shown in the following table.

[0152] As the center of drug metabolism in the human body, liver metabolism of drugs depends on the expression of polar membrane proteins and metabolic enzymes on the surface of hepatocytes. In immunofluorescence staining (Figures 41 and 42), many hepatic-like cells in the m(E / VE / VEGF)p group and (mE+mVE / VEGF)p group were recognized by specific antibodies against MRP2 and CYP3A4. From the above results, it was found that hepatic organoids induced to differentiate from hMSCs aggregates containing fusion protein surface-modified microbeads can express hepatocyte membrane proteins closely related to drug metabolism and CYP3A4, a phase I enzyme, and contribute to cellular drug metabolism.

[0153]

Table 21

[0154] Example 32 Detection of Drug Sensitivity of Liver Organoids In the medium, culture solutions containing different concentrations of acetaminophen and isonicotinic acid hydrazide (0 mM, 0.39 mM, 0.78125 mM, 1.5625 mM, 3.125 mM, 6.25 mM, 12.5 mM, 25 mM, and 50 mM) were prepared respectively. These were separately added to liver organoids containing the fusion protein and cultured for 24 h. The IC50 values of each drug in each group were assayed by the CCK-8 method. Based on the drug concentration of the IC50 value, culture solutions with drug concentrations 2 times and 0.5 times that concentration were prepared with the medium respectively. These were added to liver organoids containing the fusion protein and cultured for 24 h. The supernatant was discarded, and the liver organoids were washed 3 times with 0.01 M sterile PBS. They were incubated at 37 °C for 30 min in the medium containing 100 nM MitoTrackerTM Red CMXRos (Invitrogen, USA, product number M7512) and 5 mM CellROXTM Oxidative Stress Reagent (Invitrogen, USA, product number C10444). The culture supernatant was discarded, washed 3 times with 0.01 M sterile PBS, and then washed 3 times with PBSB after fixation with freshly prepared 4% paraformaldehyde. They were double-stained with a fluorescence fading prevention agent containing DAPI, and the samples were observed with a laser confocal microscope at 488 nm, 561 nm, and ultraviolet excitation light within 24 h to take pictures.

[0155] From the detection results of CCK8, for the cells in the m(E / VE / VEGF)p group, the IC50 value, which is the half-inhibitory concentration of acetaminophen, is 0.537 mmol / L, and the IC50 value to achieve the half-lethal rate of isoniazid or cells is 2.775 mmol / L; for the cells in the (mE+mVE / VEGF)p group, the IC50 value to achieve the half-lethal rate of acetaminophen is 3.273 mmol / L, and the IC50 value, which is the half-inhibitory concentration of isoniazid in cells, is 5.226 mmol / L. It was found that compared with the (mE+mVE / VEGF)p group, in the m(E / VE / VEGF)p group, the IC50 values of acetaminophen and isoniazid both decreased. From this, it is proved that the m(E / VE / VEGF)p group has a higher sensitivity to hepatotoxic drugs, and compared with isoniazid, acetaminophen causes more severe damage to hepatic organoids. Subsequently, the ROS detection kit (Invitrogen, product number C10444) and the Mitro detection kit (Invitrogen, product number M7512) were used to evaluate the generation of ROS in hepatic organoids and the damage to mitochondria in hepatocytes caused by different model drugs respectively. As can be seen from Figure 43 and Figure 44, as the drug concentration increased, the ROS level continued to increase, the intensity of green fluorescence continued to increase, while the mitochondrial membrane potential decreased and the intensity of red fluorescence continued to decrease. This proved that as the drug concentration increased, the damage to hepatocytes also continued to increase, showing a dose-dependence. In addition, hepatic organoids have a certain sensitivity to hepatotoxic model drugs and are useful as an effective means for evaluating drug toxicity.

[0156] Example 33 Study on the Differentiation from Embryonic Stem Cells to Hepatic Organoids Aggregates of different mouse embryonic stem cells (Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection) were prepared by the method of Example 16. That is: aggregates of embryonic stem cells containing collagen-modified microbeads (abbreviated as ecp); aggregates of embryonic stem cells containing microbeads co-modified with hE-cad-Fc, hVE-cad-Fc and hVEGF-Fc (abbreviated as e(E / VE / VEGF)p); and aggregates of embryonic stem cells containing microbeads modified with different fusion proteins each at an equal ratio (abbreviated as e(E+VE / VEGF)p). Aggregates of embryonic stem cells without microbeads (abbreviated as e) were used as a control and continuously cultured in the directed differentiation induction medium shown in the following table for 21 days for differentiation induction. RNA was extracted on the 21st day and a PCR test was performed. The specific primer sequences are shown in the following table. The results are shown in Figure 45.

[0157] As can be seen from Figure 45, the aggregates containing the fusion protein have a significantly higher expression ability of FoxA2, an endoderm cell marker; the expression ability of HNF4α, a hepatocyte-related gene marker; the expression ability of ALB, a mature hepatocyte gene marker; the expression ability of CD31, an endothelial cell-related gene marker; the expression ability of OATP, a hepatocyte polarity protein gene marker; and the expression ability of G6pase, a gene marker related to hepatocyte-related metabolic enzymes, than the other two groups. This proves that the fusion protein can cooperate with cell differentiation factors to promote the differentiation induction of aggregates of embryonic stem cells into liver organoids with certain structures and functions.

[0158]

Table 22

[0159]

Table 23

Claims

**Claim 1** Use of a VE-cadherin-Fc fusion protein for promoting in vitro differentiation induction of stem cells into islet-like cells, wherein the VE-cadherin-Fc fusion protein is used in combination with an E-cadherin-Fc fusion protein and a VEGF-Fc fusion protein, and wherein the stem cells are mesenchymal stem cells. **Claim 2** A method for promoting differentiation induction of stem cells into islet-like cells by culturing the stem cells in the presence of a VE-cadherin-Fc fusion protein, an E-cadherin-Fc fusion protein, and a VEGF-Fc fusion protein, wherein the stem cells are mesenchymal stem cells.

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