Polypeptide hydrogel, pancreatic organ constructed by polypeptide hydrogel and application of polypeptide hydrogel

Abstract

The invention provides a polypeptide hydrogel. The polypeptide hydrogel is an AG73-GelMA / AlgMA hydrogel which is prepared by crosslinking an AG73 polypeptide and a GelMA / AlgMA hydrogel. The invention further provides a preparation method of the polypeptide hydrogel. The hydrogels are used to produce pancreatic progenitor cell (PP) organoid and to improve the SC-beta cell differentiation regimen. AG73 can simulate the interaction between cells and a matrix, activate an AKT signal channel, promote the formation of PDX1 + / NKX6.1 + PP organs, and effectively regulate subsequent epithelial-mesenchymal transition (EMT) in an endocrine lineage; the GelMA / AlgMA hydrogel provides optimal mechanical stress and space structure for differentiation of PP (propene polymer) organs by simulating the physiological stiffness of pancreas. The SDC4-ITGAV compound plays a key role in the early stage of pancreatic development, and promotes the formation of SOX9 + / PDX1 + dual-energy PPs. The SC-beta cell derived from the AG73-GelMA / AlgMA hydrogel disclosed by the invention shows that the insulin secretion function is enhanced in vivo, and meanwhile, the hyperglycemia state of a diabetic mouse is more quickly reversed. The invention provides a cost-effective, stable and efficient alternative scheme for the culture of in vitro SC-beta cells by reducing the uncertainty related to the traditional culture method.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue engineering and proteomics, and in particular to a hydrogel and an application thereof in constructing an organoid, and in particular to a polypeptide hydrogel, a pancreatic organoid constructed by the same and an application thereof. Background Art

[0002] For patients with unstable diabetes or severe complications, islet transplantation is the most promising treatment to reverse the natural development of diabetes. However, its clinical application has been greatly hindered by the relative shortage of donors. In recent years, stem cell-derived β (SC-β) cell transplantation for the treatment of diabetes has made significant progress. SC-β cells extracted from patient-induced pluripotent stem cells (iPSCs) are an effective means of disease modeling and autologous diabetic cell replacement therapy. The current research focus and challenge is to regulate the production of pancreatic-specific subpopulation cells, enhance the production of SC-β cells, and reduce excess cells, especially undifferentiated cells with tumorigenicity. In the established multi-step pancreatic development and differentiation system, it is crucial to generate pancreatic progenitor cells (PPs) with an endocrine lineage differentiation trajectory, ultimately leading to functional β cell differentiation. For example, PDX1 + and NKX6.1 + PPs are considered to be progenitor cells with a monohormone lineage that determine the generation of β-cells.

[0003] However, traditional differentiation models ignore the effects of the in vivo extracellular matrix (ECM) microenvironment on cell differentiation, resulting in non-physiological differentiation reactions and reduced differentiation efficiency. In addition, in current traditional research, there are two problems with the culture and differentiation of iPSCs based on culture plates: (1) The kPa-level matrix hardness is considered to be the most ideal state for stimulating cell growth, while the hardness provided by the culture plate is usually at the GPa level, which cannot well simulate the ECM microenvironment for cell growth and development; (2) The culture and differentiation of iPSCs rely on Matrigel matrix glue, but its batch-to-batch instability and the presence of many unknown biological components make it difficult to use for large-scale stem cell culture and differentiation.

[0004] In recent years, engineered hydrogels have been welcomed by researchers for their ability to accurately replicate the ECM microenvironment, matrix mechanics, and for stem cell culture and in vitro differentiation. In addition, hydrogels constructed from specific peptides or proteins can precisely control stem cell behavior and promote their directional differentiation. It has been publicly reported that Supragel hydrogel provides a support stiffness of about 1.5 kPa, thereby more effectively promoting the differentiation of hiPSCs into endoderm and subsequent insulin-secreting cells. In addition, peptides such as RGD, AG73, IKVAV, and GFOGER have been used for research. In the inventor's previous research, it was found that the self-differentiation and maturation of insulin-secreting cells can be promoted by simply changing the ECM. The use of physical ECM signals to induce cell differentiation has recently attracted increasing attention from researchers. Syndecan-4 (SDC4) is a mechanical cue-related protein. As an important cell surface mechanical signal transduction co-receptor, it promotes the formation of intracellular adhesion spots and stress fibers through interaction with integrins. These interactions activate integrins, enabling ECM signals to be transmitted to the cell nucleus. The AG53 peptide can bind to the SDC4 domain through ligand-receptor interaction, effectively allowing cells to accept ECM stimulation.

[0005] Although the establishment of pancreatic organoids has experienced more than a decade of development, it still faces various challenges, such as differences in cell types and proportions in the structure; in addition, there are significant differences in pancreatic organoid culture between different batches and individuals. Therefore, the development of a stable and efficient pancreatic differentiation system is of great significance for the realization of pancreatic islet biomanufacturing. Summary of the invention

[0006] In view of at least one of the shortcomings of the prior art, the purpose of the present invention is to simulate and reconstruct the key signal cues of pancreatic endocrine cell differentiation in vitro and improve the efficiency of pancreatic endocrine cell differentiation. Based on the ligand-receptor interaction between AG73 and SDC4, AG73-GelMA / AlgMA hydrogel was constructed to induce PDX1 + / NKX6.1 + Differentiation of pancreatic endocrine organoids. Experimental verification results showed that AG73-GelMA / AlgMA hydrogel provided sufficient biomechanical support and precise biochemical signal guidance to promote the differentiation of hiPSCs into the pancreatic endocrine lineage.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention is to provide a polypeptide hydrogel, which is an AG73-GelMA / AlgMA hydrogel, which is prepared by cross-linking an AG73 polypeptide with a GelMA / AlgMA hydrogel; wherein, in the GelMA / AlgMA hydrogel, the mass concentration of GelMA is 5%, and the mass concentration of AlgMA is 2%; the amino acid sequence of the AG73 polypeptide is CGGGRKRLQVQLSIRTC.

[0009] Furthermore, the preparation steps of the polypeptide hydrogel include:

[0010] Step S1, preparation of GelMA / AlgMA hydrogel: dissolving GelMA and AlgMA in PBS containing lithium phenyl-2,4,6-trimethylbenzoylphosphonate, heating the mixture to 40-45° C. to ensure complete dissolution, and obtaining a GelMA / AlgMA hydrogel mixture; filtering and sterilizing the prepared GelMA / AlgMA hydrogel mixture to obtain a GelMA / AlgMA hydrogel solution;

[0011] Step S2, cross-linking of AG73 polypeptide and GelMA / AlgMA hydrogel: dissolve AG73 polypeptide at 0.05-2 mg / mL (preferably 1 mg / mL) in the GelMA / AlgMA hydrogel solution obtained in step S1, and shake for 18-30 hours to obtain AG73-GelMA / AlgMA hydrogel.

[0012] Further, in step S1, PBS contains 0.25% (w / v) lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), and the mixture is heated to 42°C.

[0013] Furthermore, in step S1, a 0.22 μm filter is used for filtration sterilization.

[0014] Furthermore, in step S2, the oscillation time is 24 hours.

[0015] Furthermore, the pore size of the AG73-GelMA / AlgMA hydrogel is 120-150 μm. In a specific embodiment, the pore size is 136.4±9.3 μm.

[0016] The second aspect of the present invention is to provide a pancreatic organoid constructed by any of the polypeptide hydrogels described in the first aspect, and the construction method thereof comprises: 3D printing an AG73-GelMA / AlgMA hydrogel system loaded with hiPSCs, transferring the printed construct to a culture medium, incubating for 48 to 72 hours, and then inducing differentiation in four stages to obtain the pancreatic organoid.

[0017] Furthermore, the 3D printing step includes: placing a load of 1×10 6 ~1×10 7 (Preferably 5×10 6 cells / mL) cells / mL hiPSCs were encapsulated in an AG73-GelMA / AlgMA hydrogel system and 3D printed using a bioprinter (preferably a DLP printer); before printing, a structure with a side length of 5 mm and a chain distance of 0.4 μm was designed; at 10 mW / cm 2 The printing was performed for 60 seconds under a UV light intensity of 100 μm, and a total of 10 layers were printed, each with a thickness of 100 μm. It is understandable that the above printing parameters can be adaptively adjusted according to the volume size.

[0018] Further, the printed constructs were transferred to culture plates and cultured in complete mTeSR TM After incubation in Plus medium for 48 hours, four stages of differentiation were performed, and the pancreatic organoids were obtained 5 days after the fourth stage.

[0019] Furthermore, the differentiation is a four-stage differentiation, specifically:

[0020] Phase 1 (DE, 3 days): basal medium 1: 500 mL MCDB131 + 0.8 g glucose + 0.587 g sodium bicarbonate + 0.5 g BSA + 5 mL GlutaMAX + 22 mg vitamin C; induction medium (day 1): basal medium 1 + 100 ng / mL Activin A + 3 μM CHIR99021; induction medium (days 2 and 3): basal medium 1 + 100 ng / mL Activin A;

[0021] Phase II (PG, 3 days): basal medium 2: 500 mL MCDB131 + 0.4 g glucose + 0.587 g sodium bicarbonate + 5 mL GlutaMAX + 22 mg vitamin C; induction medium: basal medium 2 + 50 ng / mL KGF;

[0022] Phase III (PFG, 3 days): basal medium 3: 500 mL MCDB131 + 0.22 g glucose + 0.877 g sodium bicarbonate + 10 g BSA + 2.5 mL ITS-X + 5 mL GlutaMAX + 22 mg vitamin C; induction medium: basal medium 3 + 50 ng / mL KGF + 200 nM LDN193189 + 500 nM TPPB + 2 μM retinoic acid + 0.25 μM SANT1;

[0023] Stage 4 (PP, 5 days): Induction medium: basal medium 3+50 ng / mL KGF+200 nM LDN193189+500 nM TPPB+0.1 μM retinoic acid+0.25 μM SANT1.

[0024] Furthermore, the pancreatic organoids include PDX1 + / NKX6.1 + Pancreatic endocrine organoids, SOX9 + / PDX1 + Dual-potent pancreatic organoids.

[0025] The third aspect of the present invention is to provide an application of any polypeptide hydrogel described in the first aspect, which is selected from at least one of the following applications: application in constructing pancreatic organoids, application in preparing SC-β cells, application in preparing drugs for treating diabetes, and application in determining the regulatory effects and molecular mechanisms of ECM components and mechanical stress on pancreatic lineage development.

[0026] The fourth aspect of the present invention is to provide an application of the pancreatic organoid described in any one of the second aspects, which is selected from at least one of the following applications: application in the preparation of SC-β cells, and application in the preparation of drugs for treating diabetes.

[0027] Furthermore, the collected pancreatic organoids were further cultured and differentiated (specifically, differentiated in AG73-GelMA / AlgMA hydrogel-coated 6-well ultra-low adsorption culture plates) to form endocrine progenitor cells (EPs) and stem cell-derived β cells (SC-β cells).

[0028] Further, differentiation into endocrine progenitor cells (EPs) and stem cell-derived β cells (SC-β cells) is specifically as follows:

[0029] Phase 5 (EP, 7 days): Induction medium: DMEM + 1% GlutaMAX + 1% B27 + 10μM ALK5 inhibitor II + 0.3μM LDN193189 + 1μM T3 + 10μM ISX9, 10μg / mL heparin + 0.1μM γ-secretase inhibitor Xxi + 100nM Wnt-C59 + 10μM Y27632 + 0.25mM vitamin C;

[0030] Stage 6 (SC-β, 21 days): Induction medium: DMEM+10μM ALK5i II+20ng / mL β-cell membrane +0.1μM retinoic acid+0.25μM SANT1+1μM T3+1μM XXI.

[0031] Furthermore, in the application, the diabetes includes type 1 diabetes and severe type 2 diabetes, etc.

[0032] Furthermore, in the application, the GelMA / AlgMA hydrogel simulates the physiological stiffness of the pancreas and provides optimal mechanical stress and spatial structure for pancreatic organoid differentiation; the AG73-GelMA / AlgMA hydrogel promotes PDX1 + / NKX6.1 + The generation of pancreatic endocrine organoids improves the differentiation efficiency and function of SC-β cells; AG73-SDC4-mediated mechanical signal transduction promotes SOX9 by regulating the SDC4-ITGAV-AKT signaling axis + / PDX1 + Formation of pancreatic bipotent organoids.

[0033] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0034] The development and functional maintenance of pancreatic endocrine and exocrine cells depend on the complex structure of the pancreatic-specific ECM, which is not only a physical and immune barrier to maintain pancreatic cell homeostasis, but also contributes to intercellular communication and matrix signal transduction. The present invention discovered through proteomic detection that SDC4 is a potential key signal transduction protein related to pancreatic organogenesis, differentiation and maturation. In order to simulate the spatiotemporal characteristics of pancreatic β cell development in vivo, the present invention combines tissue engineering with proteomics to reconstruct the pancreatic endocrine cell development microenvironment, thereby obtaining high-efficiency pancreatic organoids in vitro, and then obtaining a stable source of SC-β cells for diabetes transplantation treatment. The present invention uses AG73 polypeptide and GelMA / AlgMA hydrogel to construct a cross-linked hydrogel with stable chemical properties, adjustable physical structure and simple production process as a mimic of pancreatic cell differentiation; the above-mentioned AG73-GelMA / AlgMA hydrogel culture can stably and efficiently form relatively uniform PDX1 + / NKX6.1 + Pancreatic endocrine lineage organoids enhance biosafety, reduce off-target effects, and lower production costs; they can also use iPSCs from the patient's own source to differentiate into pancreatic organoids, further solving the problem of seed cell sources in diabetic stem cell therapy.

[0035] The AG73-GelMA / AlgMA hydrogel prepared by the present invention was used to generate pancreatic progenitor cell (PP) organoids and optimize the SC-β cell differentiation method. The laminin-derived homolog AG73 polypeptide can mimic certain cell-matrix interactions, promote the activation of the AKT signaling pathway, and promote PDX1 + / NKX6.1 +PP organoids form and effectively regulate the subsequent epithelial-mesenchymal transition (EMT) in the endocrine lineage. GelMA / AlgMA hydrogels mimic the physiological stiffness of the pancreas and provide optimal mechanical stress and spatial structure for PP organoid differentiation. Syndecan-4 (SDC4)-ITGAV complex plays a key role in the early stages of pancreatic development, promoting SOX9 + / PDX1 + Formation of dual-energy PPs. AG73-GelMA / AlgMA hydrogel-derived SC-β cells exhibited enhanced insulin secretion and accelerated hyperglycemia reversal in vivo. The present invention provides a cost-effective, stable and efficient alternative for comprehensive 3D culture of SC-β cells in vitro by reducing the uncertainties associated with traditional culture methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0037] Figure 1 Schematic diagram of the results of verifying the role of SDC4 in pancreatic β cell differentiation in one embodiment of the present invention; wherein, part a: DIA analysis schematic diagram; part b: SDC4 is highly expressed in the pancreas of 8-week-old adult mice and human pancreas, which is related to the expression location of insulin; part c: SDC4 expression is downregulated in the PG and PFG stages, and continues to differentiate into PPs and SC-β cells; part d: FCM results show that NKX6.1 in PPs of the SDC4-shR group + / PDX1 + The positive rate was significantly reduced.

[0038] Figure 2Schematic diagram of the preparation and characterization of AG73-GelMA / AlgMA hydrogel in one embodiment of the present invention; wherein, part a: schematic diagram of AG73-GelMA / AlgMA hydrogel; part b: hydrogen bond formed between GLN-8 site of AG73 and HE-13 site of SDC4; part c: GelMA / AlgMA hydrogel and AG73 hydrogel with final concentrations of 0.1 mg / mL and 1 mg / mL, respectively; part d: maleimide characteristic peak of AG73 hydrogel determined by nuclear magnetic resonance analysis; part e: rheological properties of three groups of hydrogels (G'=storage modulus, G"=loss modulus Part f: swelling ratio of the three groups of hydrogels; Part g: stress (kPa)-strain (%) change curves of the three groups of hydrogels on the 1st day and the 14th day; Part h: compression modulus of the three groups of hydrogels on the 1st day and the 14th day; Part i: scanning electron microscopy of the three groups of hydrogels; Part j: Calcein-AM / PI staining showed that no large number of dead cells were found in the three groups of hydrogels; Part k: pore sizes of the three groups of hydrogels; Part l: CCK-8 results showed that there was no statistical difference in cell proliferation among the three groups of hydrogels; Part m: compared with single hydrogel, high AG73 gel significantly improved the differentiation efficiency of PPs.

[0039] Figure 3 It is a schematic diagram of the results of hiPSCs differentiation into PP organoids in one embodiment of the present invention; wherein, part a: a schematic diagram of hiPSCs differentiation into PP organoid cells and SC-β cells in hydrogel; part b: hiPSCs form cell clusters in the early stage of hydrogel and continue to proliferate until the PP stage; part c: IF analysis found that the main TFs were positively expressed in different stages of cell differentiation, including DE stage (FOXA2 / SOX17), PFG stage (FOXA2 / PDX1), and PP stage (PDX1 / NKX6.1), and the hydrogel-derived PP organoids showed a clear luminal structure; part d: FCM results showed that compared with the Matrigel group and the hydrogel group, the positive expression of PDX1 / NKX6.1 in the PP organoids of the AG73 hydrogel group was significantly higher; part e: EPCAM of the PP organoids in the AG73 hydrogel was significantly upregulated.

[0040] Figure 4Schematic diagram of the results of PPs differentiation into EPs and SC-β cells in one embodiment of the present invention; wherein, part a: PP organoids differentiated into EPs and SC-β cells; part b: expression of NGN3 and YAP1 in EPs of Matrigel group and AG73 hydrogel group; part c: EPs and SC-β cells of Matrigel group and AG73 hydrogel group; part d: both groups of SC-β cells expressed NKX6.1, insulin and MAFA; part e: FCM results showed that the NKX6.1 / INS positivity rate in AG73 hydrogel group was higher than that in Matrigel group; part f: glucose stimulation experiment showed that AG73-derived SC-β cells had a higher insulin secretion level.

[0041] Figure 5 This is a schematic diagram of the results of SC-β cell transplantation in diabetic mice in one embodiment of the present invention; wherein, part a: schematic diagram of SC-β cells implanted into the renal capsule of T1DM mice; part b: SC-β cells implanted into the renal capsule of T1DM mice; part c: the transplanted SC-β cells differentiated in the Matrigel group and the AG73 hydrogel group continued to express insulin and NKX6.1 after in vivo transplantation; part d: changes in blood glucose levels within 120 minutes after intraperitoneal injection of glucose; part e: changes in fasting blood glucose in diabetic mice 12 weeks after SC-β cell transplantation in different groups.

[0042] Figure 6 Schematic diagram of the results of single-cell transcriptome analysis of PP organoids in one embodiment of the present invention; wherein, part a: PPs were divided into 15 categories by t-SNE analysis; part b: PDX1, SOX9, NKX6.2, GATA4, GATA6 and HES1 were significantly upregulated in clusters 1, 2 and 7; part c: expression patterns of SOX9, GATA4, PDX1 and SDC4 and their distribution in different clusters; part d: cells expressing PDX1, SOX9 and YAP1 were identified as bipotential pancreatic precursor cells; part e: TFs analysis of the entire subgroup showed that cluster 7 exhibited high levels of PDX1, SOX9 and YAP1 expression; part f: expression of PDX1 and SOX9 in two groups of PP organoids.

[0043] Figure 7The figure is a schematic diagram of the molecular mechanism of AG73 hydrogel promoting PP organoid formation in one embodiment of the present invention; wherein, part a: AG73 hydrogel-derived PP organoids showed higher abundance of ligand receptor pairs among different clusters; part b: a large number of receptors such as FN and integrin family were observed to be activated in the AG73 hydrogel group; part c: HSPG signaling family was upregulated in AG73 hydrogel-derived PP organoids; part d: String database suggested that there were extensive connections between SDC4, FN and integrin; part e: t-SNE analysis showed the expression pattern of ITGAV in the two groups; part f: SDC4 was expressed in both groups, and ITGAV expression was significantly stronger in the AG73 hydrogel group; part g: IF analysis showed that along with the decrease in ITGAV expression, key TFs in PP organoids The levels of PDX1 and SOX9 were decreased; Part h: qRT-PCR analysis confirmed that the expression levels of GATA4, GATA6, NKX6.1, PDX1 and SOX9 in PP organoids were downregulated as ITGAV was downregulated; Part i: IF analysis showed that inhibition of AKT signaling pathway reduced the expression of SOX9 and GATA4 in PP organoids; Part j: qRT-PCR analysis confirmed that after inhibition of AKT signaling pathway, the expression of GATA4, SOX9, PDX1 and NKX6.1 was reduced; Part k: N-CAD expression was upregulated in the AG73 hydrogel group; Part i: Molecular mechanism by which AG73 hydrogel promotes the formation of PP organoids. DETAILED DESCRIPTION

[0044] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0045] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0046] In the following embodiments, the detection methods used include:

[0047] (1) mRNA-seq: Total RNA was extracted from PPs using a total RNA kit (Servicebio, China). Complementary DNA (cDNA) libraries were constructed by GeneDenovo Biotechnology Co., Ltd. (Guangzhou, China), and sequencing and transcriptome data analysis were performed. DESeq2 software was used for differential expression analysis between the two groups. All differentially expressed genes (DEGs) were analyzed for biological processes using gene ontology (GO) biological processes and Kyoto Gene and Genome Database (KEGG) pathway enrichment analysis.

[0048] (2) scRNA-seq and data analysis: Cells were separated into single cells using accutase (07920, STEMCELL Technologies) and filtered through a 40-μm cell filter (352340, Corning). Single cells were then isolated from the emulsion, and each cell was labeled with a unique set of oligonucleotides. The cDNA library was amplified by PCR. The cDNA libraries of all groups were paired-end sequenced on the Illumina 10×Genomics Chromium platform (10×Genomics, Illumina). scRNA-seq analysis was performed by Gene Denovo (Gene Denovo, China), see the mRNA-seq section. UMI counts and cell barcoding are matrices generated by cell genes and imported into Seurat version 3.1.1 for cell cluster identification. Cells that meet specific criteria, such as having more than 8000 UMIs, fewer than 500 or more than 4000 genes, and a mitochondrial gene percentage greater than 10%, will be excluded from further analysis. Dimensionality reduction was performed using principal component analysis (PCA); cells were embedded into a shared nearest neighbor (SNN) graph based on similar gene expression patterns using a graph-based clustering method implemented using Seurat. Construction of the SNN graph involved refining edge weights between cells based on common overlap and Jaccard distance calculations. Clusters were generated using the Louvain method and visualized using t-distributed stochastic neighbor embedding (t-SNE), using the same principal components as used for PCA visualization.

[0049] (3) FCM: Cells were fixed / permeabilized for 20 minutes, and the antibodies were incubated for 30 minutes at 4°C. After washing with PBS and resuspending, the cells were detected with BDLSRFortessa and analyzed with FlowJo v10. All antibodies used were conventional antibodies in the field.

[0050] (4) IF: Cells or tissues were fixed with formaldehyde, embedded in OCT complex, and sliced, and antigens were extracted using sodium citrate buffer (Beyotime, China). The slices were then treated with 0.5% Triton X-100 and 5% bovine serum albumin (BSA); then incubated with primary antibodies overnight at 4°C. The slices were thoroughly washed the next day and then exposed to secondary antibodies and DAPI (Solarbio, China) for 1 hour and 10 minutes, respectively. Finally, the slices were visualized and imaged under a Nikon Ti2-E microscope. The primary and secondary antibodies used were conventional antibodies in the field.

[0051] (5) qRT-PCR: When extracting total RNA, a total RNA kit (Vazyme, China) was used, and then cDNA synthesis was performed using a first-strand cDNA synthesis kit (Vazyme, China). qRT-PCR analysis was performed using SYBR Green Master Mix (Vazyme, China), and detection was performed using the QuantStudio 5PCR system (Thermo Fisher Scientific, USA). -ΔΔCt The method quantifies the gene expression level. The gene primers used are all conventional primers in the art.

[0052] (6) Statistical analysis: SPSS22 software was used for statistical analysis. Data are presented as mean ± standard deviation and were obtained from at least three independent experiments. All data sets were evaluated for normal distribution before performing Student's t-test or one-way ANOVA for inter-group comparison. P < 0.05 was considered statistically significant.

[0053] In the following examples, 5% GelMA / 2% AlgMA hydrogel, 1 mg / mL AG73-5% GelMA / 2% AlgMA hydrogel, and Matrigel matrix gel (used after diluting the stock solution 1:10 according to the instructions) were used. The present invention is exemplified below.

[0054] Example 1 - Preparation of AG73-GelMA / AlgMA hydrogel, pancreatic organoids, and SC-β cells

[0055] This embodiment is a method for preparing a preferred AG73-GelMA / AlgMA hydrogel, and using the hydrogel to construct pancreatic organoids, which are further differentiated to prepare SC-β cells, which specifically includes the following steps:

[0056] (1) Preparation of AG73-GelMA / AlgMA hydrogel;

[0057] Referring to the previous study (Zhu S, Xu Y, Li Y, Wang L, Huang Y, Wan J. Biomimetic Hydrogels Promote Pseudoislet Formation to Improve Glycemic Control in Diabetic Mice. ACS Biomater Sci Eng 2024, 10(4): 2486-2497.), 5% GelMA / 2% GelMA hydrogel was prepared, as briefly described as follows: GelMA (EFL, China) and AlgMA (EFL, China) were dissolved in PBS containing 0.25% (w / v) lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), and the mixture was heated to 42°C to ensure complete dissolution, ultimately reaching a GelMA mass concentration of 5% and an AlgMA mass concentration of 2%; the prepared hydrogel mixture was sterilized using a 0.22 μm filter and then stored at -20°C until use. AG73 peptide (CGGGRKRLQVQLSIRTC) was biosynthesized by Sangon Biotech Co., Ltd. During peptide grafting, AG73 peptide was dissolved in GelMA / AlgMA hydrogel solution at 1 mg / mL and shaken for 24 hours to obtain AG73-GelMA / AlgMA hydrogel.

[0058] (2) encapsulation and differentiation of hiPSCs to obtain PPs, EPs, and SC-β cells;

[0059] hiPSCs were purchased from the cell bank of the Chinese Academy of Sciences. The above hiPSCs can also directly use patient-derived hiPSCs, which are briefly described as follows: adipose tissue is obtained from the greater omentum of patients undergoing partial pancreatectomy and separated by collagenase digestion. P3-P5 ADSCs are used to generate hiPSCs in approximately 20 to 28 days using ReproRNATM-OKSGM (STEMCELL Technologies, 05930). It is understandable that hiPSCs can also be obtained by other means, such as direct purchase.

[0060] hiPSCs (5 × 10 ) in the G73-GelMA / AlgMA hydrogel system were prepared using a DLP printer (EFL-BP8601 series) according to the methods described in previous studies (Zhu S, Xu Y, Li Y, Wang L, Huang Y, Wan J. Biomimetic Hydrogels Promote Pseudoislet Formation to Improve Glycemic Control in Diabetic Mice. ACS Biomater Sci Eng 2024, 10(4): 2486-2497). 6 cells / mL) for 3D printing. Before printing, a structure with a side length of 5 mm and a chain distance of 0.4 μm was designed. 2 The printed constructs were then transferred to a culture plate and printed under UV light intensity of 100 μm for 60 s. A total of 10 layers were printed, each 100 μm thick. TM Plus medium (100-0276, STEMCELL Technologies) for 48 hours before differentiation, using a four-stage approach to obtain the desired PPs. On day 5 of the fourth stage, the hydrogels were treated with collagenase to collect PPs, cultured, and further differentiated in AG73-GelMA / AlgMA hydrogel-coated 6-well ultra-low attachment culture plates to obtain EPs and SC-β cells.

[0061] The above differentiation method (see Figure 3 Part a) is as follows:

[0062] Phase 1 (DE, 3 days): basal medium 1: 500 mL MCDB131 + 0.8 g glucose + 0.587 g sodium bicarbonate + 0.5 g BSA + 5 mL GlutaMAX + 22 mg vitamin C; induction medium (day 1): basal medium 1 + 100 ng / mL Activin A + 3 μM CHIR99021; induction medium (days 2 and 3): basal medium 1 + 100 ng / mL Activin A;

[0063] Phase II (PG, 3 days): basal medium 2: 500 mL MCDB131 + 0.4 g glucose + 0.587 g sodium bicarbonate + 5 mL GlutaMAX + 22 mg vitamin C; induction medium: basal medium 2 + 50 ng / mL KGF;

[0064] Phase III (PFG, 3 days): basal medium 3: 500 mL MCDB131 + 0.22 g glucose + 0.877 g sodium bicarbonate + 10 g BSA + 2.5 mL ITS-X + 5 mL GlutaMAX + 22 mg vitamin C; induction medium: basal medium 3 + 50 ng / mL KGF + 200 nM LDN193189 + 500 nM TPPB + 2 μM retinoic acid + 0.25 μM SANT1;

[0065] Stage 4 (PP, 5 days): Induction medium: basal medium 3+50 ng / mL KGF+200 nM LDN193189+500 nM TPPB+0.1 μM retinoic acid+0.25 μM SANT1.

[0066] Phase 5 (EP, 7 days): Induction medium: DMEM + 1% GlutaMAX + 1% B27 + 10μM ALK5 inhibitor II + 0.3μM LDN193189 + 1μM T3 + 10μM ISX9, 10μg / mL heparin + 0.1μM γ-secretase inhibitor Xxi + 100nM Wnt-C59 + 10μM Y27632 + 0.25mM vitamin C;

[0067] Stage 6 (SC-β, 21 days): Induction medium: DMEM+10μM ALK5i II+20ng / mL β-cell membrane +0.1μM retinoic acid+0.25μM SANT1+1μM T3+1μM XXI.

[0068] Example 2 - SDC4 affects pancreatic development and differentiation of PPs

[0069] This example verifies the effect of SDC4 on pancreatic development and PPs differentiation, which specifically includes the steps of:

[0070] (1) To investigate the involvement of DEPs in different stages of pancreatic development in mice, hierarchical cluster analysis was used in this example to detect a set of 1753 DEPs that were consistently differentially expressed in three developmental stages, briefly described as follows: DIA-based proteomic analysis and validation Pancreatic tissues were obtained from C57 / BL6 mice (Jiangsu Jicui Pharmaceutical Kang Biological Co., Ltd.) at the first week (Fw, n = 3), third week (Tw, n = 3), and eighth week (Ew, n = 3) after birth and subjected to DIA sequencing to create a comprehensive map of differentially expressed proteins (DEPs) during mouse pancreatic development. Protein extraction, high pH reverse phase separation, high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) analysis and data analysis methods are described in Huang Y, Xu Y, Zhu J, Wan J, Xiong Y, Jiang Z, et al. An artificial LAMA2-GelMA hydrogel microenvironment for the development of pancreatic endocrine progenitors. Biomaterials 2022, 291: 121882. To verify the results of DIA analysis, pancreatic tissues of C57 / BL6 mice and humans (obtained from patients with pancreatic diseases requiring partial pancreatectomy) were fixed, permeabilized and immunofluorescence (IF) stained for analysis.

[0071] By cluster sequence testing, eight different protein expression profiles were identified, among which, e.g. Figure 1 As shown in part a of Figure 3, the expression of SDC4 was stable and consistent, especially in the pancreas of TW and EW mice. Figure 1 As shown in part b, IF analysis confirmed that the expression of SDC4 in the pancreatic islet tissue of EW mice was significantly greater than that in the pancreatic islet tissue of FW and TW mice, which is related to the location of insulin expression; in addition, the same high level of SDC4 expression was observed in the pancreatic islets of human pancreatic tissue.

[0072] (2) Using differentiation of SC-β cells induced by hiPSCs to verify the relationship between SDC4 and pancreatic lineage development;

[0073] hiPSCs cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences. Figure 1 As shown in part c, the SC-β cell differentiation strategy is briefly described as follows: For SC-β cell differentiation, 3×10 5 cells / cm 2hiPSCs were seeded onto Matrigel-coated 6-well plates and differentiated in 2D culture using a 6-stage method. Stage 1 (3 days): definitive endoderm (DE); Stage 2 (3 days): primitive gut (PG); Stage 3 (3 days): posterior foregut (PFG); Stage 4 (5 days): pancreatic progenitors (PPs); Stage 5 (7 days): endocrine progenitors (EPs); Stage 6 (21 days): stem cell-derived β cells (SC-β cells).

[0074] Down-regulation of SDC4 expression in cells at the PG and PFG stages, and induction of differentiation into PPs; PFGs and PPs at stages 3 and 4 were transfected with shRNA SDC4; shRNA targeting SDC4 (SDC4-shRNA) was synthesized by Suzhou Gene Biotechnology Co., Ltd. shRNA transfection was performed using jetOPTIMUS (Polyplus, France) according to the manufacturer's instructions. Transfection efficiency was detected by flow cytometry (FCM).

[0075] like Figure 1 As shown in part d, the FCM results showed that NKX6.1 in the SDC4-shR group + / PDX1 + The proportion of PPs (15.35±2.659%) was significantly lower than that of the NC group (53.26±2.738%) (P<0.05), which indicated that the differentiation efficiency of PPs decreased significantly after downregulating cell SDC4.

[0076] The above experimental results show that SDC4 plays a key role in regulating the formation of PPs in the early stage of pancreatic lineage development. Matrigel-2D culture confirmed that SDC4 deficiency reduced PDX1 + / NKX6.1 + Differentiation efficiency of PPs.

[0077] Example 3 - Characterization of AG73-GelMA / AlgMA hydrogel

[0078] In previous studies, the inventors developed a biomimetic hydrogel composed of 5% GelMA and 2% AlgMA, whose physiological hardness is similar to that of pancreatic tissue. In order to reconstruct the microenvironment during pancreatic development and promote the response of cells to ECM mechanical signals, this embodiment designed AG73 hydrogel as a platform (such as Figure 2 to evaluate the performance of AG73-GelMA / AlgMA hydrogels and how AG73-SDC4-mediated signaling affects pancreatic differentiation.

[0079] In this example, the performance of the AG73-GelMA / AlgMA hydrogel prepared in Example 1 was characterized. Figure 2 As shown in part c of the figure, the hydrogels involved in the following verification process are: GelMA / AlgMA hydrogel (hereinafter referred to as hydrogel), AG73-GelMA / AlgMA hydrogel with a final concentration of 0.1 mg / mL (hereinafter referred to as low AG73 hydrogel); AG73-GelMA / AlgMA hydrogel with a final concentration of 1 mg / mL (hereinafter referred to as high AG73 hydrogel), and the specific steps include:

[0080] (1) The protein structure of SDC4 was retrieved from the PDB database, and the polypeptide structure of AG73 was predicted using the SWISS-MODEL software. The PDB templates of SDC4 and AG73 were uploaded to the HDOCK server for molecular docking prediction. The results are shown in Figure 2. Figure 2 As shown in part b, through molecular docking simulation, it was found that the GLN-8 residue of AG73 formed a hydrogen bond with the HE-13 of SDC4, indicating that there was a binding interaction between them.

[0081] (2) To confirm that AG73 was successfully grafted onto the GelMA / AlgMA hydrogel, the AG73-GelMA / AlgMA hydrogel was freeze-dried and ground into powder. After being dissolved in deuterated water, the maleimide peak was detected using an AVANCE III instrument. The results are shown in part 2d. Nuclear magnetic resonance (NMR) analysis confirmed the presence of a characteristic maleimide peak in the AG73 hydrogel, indicating that AG73 was successfully bound to 5% GelMA / 2% AlgMA.

[0082] (3) To evaluate the rheological behavior, the storage modulus (G') and loss modulus (G") were measured using an Anton Paar MCR 302 rheometer (Anton Paar Instruments, Graz, Austria); these measurements were performed at 37 °C, 1% strain, and using a 25 mm diameter cone-plate setup. Figure 2 As shown in part e, the rheological properties of the hydrogels, including storage modulus (G', indicating elasticity) and loss modulus (G", indicating viscosity), were analyzed, and the results showed that under exposure to UV radiation, G' exceeded G", and both low AG73 hydrogels and high AG73 hydrogels exhibited better elastic behavior and excellent mechanical durability.

[0083] (4) The equilibrium swelling ratio of the hydrogel was determined by evaluating the change in wet weight after PBS immersion. The initial wet weight of the hydrogel of appropriate size was recorded as W0. The weight of the hydrogel was then measured at a specific time point (Wt) and the swelling ratio was calculated as: swelling ratio = (Wt-W0) / W0×100%. Figure 2As shown in part f, after immersion in PBS for 48 hours, the swelling rates of the hydrogel, low AG73 hydrogel and high AG73 hydrogel were 38.5±3.4%, 40.6±5.4% and 35.5±2.2%, respectively, and there was no statistically significant difference between the two (P>0.05).

[0084] (5) The compression modulus of the hydrogel was determined by analyzing the stress-strain curves, which were obtained using an electronic universal material testing machine (CMT6103, MTS, USA). The curves were recorded at a deformation rate of 1 mm / min, and the modulus was calculated from the slope of the linear part of the curve. Figure 2 As shown in parts g and h of the figure, it can be seen from the stress-strain curves that the compression moduli of the hydrogel, low AG73 hydrogel, and high AG73 hydrogel were 10.9±1.9 kPa, 10.1±1.6 kPa, and 9.0±1.4 kPa on the first day, and 9.6±1.6 kPa, 9.1±0.9 kPa, and 8.5±1.2 kPa on the 14th day (P>0.05), respectively, and there was no significant difference in the compression modulus. Whether on the first day or the 14th day, in the three hydrogels, these results show that the addition of AG73 has no effect on the compression modulus of the hydrogel. In addition, no significant decrease in the compression modulus was observed on the 14th day, indicating that the prepared hydrogel has good stability.

[0085] (6) To investigate the microstructural characteristics of the hydrogels, the hydrogels were first freeze-dried for 48 h. The microstructure of the samples was analyzed by scanning electron microscopy (SEM; Gemini 300, Zeiss, Germany). The porosity of the hydrogels was quantified by analyzing the SEM images using ImageJ software. At least four samples in each experimental group were subjected to these tests to ensure consistent and reliable results. Figure 2 As shown in parts i and k of the figure, SEM results show that the pore sizes of the three groups of hydrogels are 180.0±15.8μm, 145.2±9.8μm, and 136.4±9.3μm, respectively (P<0.05). AG73 cross-linking leads to a decrease in pore size; however, the pore size of the resulting AG73 hydrogel remains within an appropriate range, providing sufficient space for cell growth.

[0086] (7) To investigate the cell compatibility of the hybrid hydrogels, CCK-8 analysis and calcein-AM / PI staining were performed at predetermined time points. The absorbance of each sample was measured at 450 nm using a microplate reader (MK3, Thermo Fisher Scientific, MA, USA), and the fluorescence images were observed and captured using a Nikon Ti2-E microscope. Figure 2As shown in part j of Figure 3, the calcein-AM / PI staining results showed that the survival rates of cells in the three hydrogel groups were satisfactory, and not many dead cells were observed. Figure 2 As shown in Part 1, there was no significant difference in the CCK-8 results of hiPSCs proliferation in the three groups of hydrogels for 1 day and 3 days (P>0.05).

[0087] (8) The hydrogels were degraded by collagenase, and the expression changes of transcription factors (TFs) of PPs cultured in different hydrogels were compared. PPs were collected for qRT-PCR analysis. To evaluate the potential of hydrogels and AG73 hydrogels to enhance the differentiation of hiPSCs into PPs, hiPSCs were encapsulated and subjected to a four-stage differentiation protocol to obtain PPs. Figure 2 As shown in part m, after evaluating the expression levels of key TFs associated with PPs (PDX1, NKX6.1, SOX9, GATA4, GATA6, and PTF1A), the results showed that high AG73 hydrogels significantly improved the differentiation efficiency of PPs compared with hydrogels alone.

[0088] From the above experimental results, it can be seen that AG73-GelMA / AlgMA hydrogel has good mechanical stability and cell compatibility, among which high AG73-GelMA / AlgMA can significantly promote the differentiation efficiency of PPs.

[0089] Example 4-Differentiation of hiPSCs into PP organoids

[0090] This example verifies the differentiation efficiency of hiPSCs into PP organoids, the differentiation potential of SC-β cells and their insulin secretion capacity under different conditions. The following experiments are grouped as follows: Matrigel 2D differentiation culture (referred to as Matrigel group), GelMA / AlgMA hydrogel 3D differentiation culture (referred to as hydrogel group), AG73-GelMA / AlgMA hydrogel 3D differentiation culture (referred to as AG73 hydrogel group). Specifically, it includes:

[0091] (1) The PPs, EPs and SC-β cells obtained from the Matrigel group, hydrogel group, and AG73 hydrogel group were encapsulated in 2% v / v agarose gel and subjected to IF staining analysis.

[0092] like Figure 3 As shown in part b of Figure 3, after hiPSCs were coated with hydrogel or AG73 hydrogel and differentiated into PPs, they formed early cell clusters and continued to proliferate until the PP stage. Figure 3As shown in part c, the differentiation efficiency of PP organoids was evaluated by IF analysis, and it was found that the main TFs were positively expressed in different stages of cell differentiation (DE stage (FOXA2 / SOX17), PFG stage (FOXA2 / PDX1), PP stage (PDX1 / NKX6.1)). The effect of AG73 hydrogel on the differentiation of PP organoids was evaluated, and the positive expression rate of TF in the simple hydrogel group was significantly reduced. Figure 3 As shown in part d of Figure 3, FCM results further showed that the positive expression of PDX1 / NKX6.1 in PP organoids in the AG73 hydrogel group was significantly higher than that in the Matrigel group and the hydrogel group. Figure 3 As shown in part e, IF analysis found that epithelial markers (such as EPCAM) were significantly upregulated in the AG73 hydrogel group, indicating that AG73 hydrogel promoted epithelial-mesenchymal transition (EMT) in PP organoids.

[0093] (3) In order to study the differentiation potential of SC-β cells, e.g. Figure 4 As shown in part a, this example uses PP organoids to directly generate EPs and SC-β cells.

[0094] like Figure 4 As shown in part c, in the Matrigel group, EPs were generated by polymerization in the AggreWell 400 plate, and in the AG73 hydrogel group, EPs were released from the hydrogel by collagenase treatment. The EPs in the Matrigel group seemed to be more compact than those in the AG73 hydrogel group. Figure 4 As shown in part b of Figure 1, IF detection was used to evaluate the expression of NGN3 and YAP1 in EPs of the Matrigel group and the AG73 hydrogel group. The expression level of YAP1 was higher in the Matrigel group. The inventors' previous studies have found that downregulating YAP1 expression in the late stage of PPs development can promote the differentiation of these cells into EPs, that is, the AG73 hydrogel group is more conducive to differentiation into EPs. Figure 4 As shown in part d, SC-β cells in both the Matrigel group and the AG73 hydrogel group expressed NKX6.1, insulin, and MAFA.

[0095] The differences in TF expression between the Matrigel group and the AG73 hydrogel group were further analyzed by FCM. Figure 4 As shown in part e, the positive rates of NKX6.1 / INS and INS / MAFA in the AG73 hydrogel group were 65.5±3.2% and 41.3±2.1%, respectively, which were significantly higher than those in the Matrigel group (52.8±3.0% and 31.6±2.9%, respectively) (P<0.05).

[0096] (4) Glucose stimulation experiment (GSIS) was applied to stimulate insulin secretion of SC-β cells. The SC-β cells in different groups were washed, incubated in Krebs-Ringer's solution (KRB) for 30 minutes, and then incubated in low glucose (2mM) or high glucose (20mM) KRB for 30 minutes. The insulin content in the supernatant was detected using an insulin enzyme-linked immunosorbent assay (ELISA) kit (Mercodia) and normalized to the DNA content. Single SC-β cells were dispersed with accutase (07920, STEMCELL Technologies) and counted using a cell counter (C100-SE, RWD). The results are shown in Figure 2. Figure 4 As shown in part f, the insulin secretion in the AG73 hydrogel group was significantly higher than that in the Matrigel group.

[0097] The above experimental results show that AG73 hydrogel can induce hiPSCs to differentiate into more mature SC-β cells.

[0098] Example 5 - Transplantation of SC-β cells in diabetic mice

[0099] This example verifies the therapeutic effect of SC-β cells obtained by 3D differentiation culture of hiPSCs using AG73-GelMA / AlgMA hydrogel on diabetes, and also conducts an experiment using SC-β cells obtained by 2D differentiation culture of Matrigel, specifically including the following steps:

[0100] In vivo transplantation: After fasting for 12 hours, 7-8 week old SCID-Beige mice were intraperitoneally injected with streptozotocin (STZ) 120 mg / kg on days 1 and 3. Fasting blood glucose levels exceeding 16.7 mM were considered a successful modeling indicator. Figure 5 As shown in parts a and b of the figure, different groups of SC-β cells (3×10 6 cells) were implanted under the renal capsule of type 1 diabetic (T1DM) mice (n=6), and diabetic mice without intervention were used as controls. Blood was collected from the tail vein of each group of mice at different time points to monitor fasting blood glucose levels. The mice were killed 12 weeks after transplantation, and the grafts were extracted for histological staining analysis. In addition, the intraperitoneal glucose tolerance test (IPGTT) was analyzed (methods refer to Xu Y, Mao S, Fan H, Wan J, Wang L, Zhang M, et al. LINC MIR503HG Controls SC-β Cell Differentiation and Insulin Production by Targeting CDH1 and HES1. Adv Sci (Weinh) 2024, 11 (13): e2305631.).

[0101] The ability of SC-β cells to regulate blood glucose levels in diabetic mice was confirmed after 12 weeks of transplantation in vivo. Figure 5 As shown in part c, IF staining showed that SC-β cells differentiated in the AG73 hydrogel group and Matrigel continued to express insulin and NKX6.1 after in vivo transplantation. Figure 5 As shown in part d, the changes in blood glucose levels within 120 minutes after intraperitoneal injection of glucose indicated that the glucose tolerance and clearance rate of mice treated with AG73 hydrogel-derived SC-β cells were significantly higher than those of diabetic mice treated with Matrigel group. Figure 5 As shown in part e, 6 weeks after transplantation, there was a significant difference in blood glucose levels between the AG73 hydrogel group and the Matrigel group (P<0.05); the blood glucose level in the AG73 hydrogel group was 6.5±1.1mmol / mL, and that in the Matrigel group was 11.3±1.5mmol / mL; 11 weeks after transplantation, the blood glucose levels of both groups improved, 6.2±0.4mmol / mL in the AG73 hydrogel group and 6.6±0.4mmol / mL in the Matrigel group, effectively restoring normal blood glucose levels; compared with the Matrigel group, the blood glucose levels of diabetic mice receiving AG73 hydrogel-derived SC-β cells decreased faster, and this ability to decrease appeared about 6 weeks after transplantation, while the Matrigel group required 9 weeks. Shortly after the graft was removed, both groups of mice experienced hyperglycemia, indicating that the transplanted SC-β cells altered glucose homeostasis.

[0102] The above experimental results show that AG73 hydrogel-induced hiPSCs differentiated SC-β cells can reverse the hyperglycemia state in diabetic mice more quickly.

[0103] Example 6 - Single-cell transcriptome analysis of PP organoids

[0104] This example studies the mechanism by which AG73 hydrogel promotes the differentiation of hiPSCs into PP organoids. The method used in this example includes: encapsulating and differentiating hiPSCs, collecting immature PPs from the GelMA / AlgMA group (abbreviated as the hydrogel group) and the AG73-GelMA / AlgMA group (abbreviated as the AG73 hydrogel group) for single-cell RNA sequencing (scRNA-seq) on the second day of PP differentiation; introducing targeted ITGAV siRNA or AKT pathway inhibitors into AG73-GelMA / AlgMA to explore its effects on the differentiation of hiPSCs into PP organoids and its potential mechanisms. The specific experiments include:

[0105] (1) To investigate how AG73 hydrogel affects the development of PP organoids at the cell cluster level, scRNA-seq was performed on day 2 after PFGs were converted into PPs. All cells were classified into 15 categories by t-SNE analysis. Figure 6 As shown in part a of FIG, the dominant clusters in the AG73 hydrogel group are clusters 1, 3, 7, 8, and 9, while clusters 0, 1, 4, and 6 are mainly observed in the hydrogel group.

[0106] Next, development-related genes expressed in PPs were identified from the hydrogel group and AG73 hydrogel group, such as Figure 6 As shown in part b of Figure 3, key TFs genes PDX1, SOX9, NKX6.2, GATA4, GATA6, and HES1 were significantly upregulated in clusters 1, 2, and 7. t-SNE analysis was then performed to study the expression patterns of SOX9, GATA4, PDX1, and SDC4 and their distribution in different clusters, as shown in Figure 3. Figure 6 As shown in part c, the expression of PDX1, SOX9, and GATA4 in the AG73 hydrogel group was upregulated compared with that in the hydrogel group, while the expression of SDC4 was not significantly different between the two groups. Figure 6 As shown in Figure e, analysis of the entire TF subsets revealed that cluster 7 had high levels of PDX1, SOX9, and YAP1 expression. Figure 6 As shown in part d, according to previous studies (Mamidi A, Prawiro C, Seymour PA, de Lichtenberg KH, Jackson A, Serup P, et al. Mechanosignalling via integrins directs fate decisions of pancreatic progenitors. Nature 2018, 564(7734): 114-118.), cells expressing PDX1, SOX9 and YAP1 were identified as bipotential PPs. Subsequently, as Figure 6 As shown in part f, IF analysis confirmed that PDX1 and SOX9 were expressed in PP organoids, which were detected in both experimental groups, but their levels were higher in the AG73 hydrogel group.

[0107] (2) The cell-cell and cell-ECM interactions in the hydrogel were further analyzed. Figure 7 As shown in part a of Figure 3, AG73 hydrogel-derived PP organoids presented more ligand-receptor pairs between different clusters, indicating enhanced intercellular communication. Figure 7As shown in part c, the HSPG signaling family, including SDC4, was upregulated in AG73 hydrogel-derived PP organoids; subsequent analysis showed that the abundance of SDC4-related receptors and ligands was significantly increased in AG73 hydrogel-derived PP organoids. Figure 7 As shown in part b of Figure 3, a large number of closely related receptors, such as those in the FN and integrin families, were observed to be activated. Figure 7 As shown in part d of Figure 3, STRING database analysis suggests that there are extensive connections between SDC4, FN, and integrins; the domains contained in fibronectin can simultaneously participate in SDC4 and integrins. Figure 7 As shown in part e of Figure , the expression patterns of ITGAV in the two groups were further verified by t-SNE analysis, and clusters 1 and 7 were found to be significantly enriched in the AG73 hydrogel group. IF experiments were performed to confirm the co-expression of SDC4 and ITGAV in PP organoids. Figure 7 As shown in part f, although SDC4 was expressed in both groups, the expression of ITGAV was significantly higher in the AG73 hydrogel group.

[0108] To further verify how ITGAV affects PP organoid development, siRNA targeting ITGAV was introduced into the AG73 hydrogel group at the PFG and PP stages and maintained in culture until the PP stage was completed. Figure 7 As shown in Figure 5g, IF analysis showed that the expression of ITGAV was reduced in PP organoids, along with the levels of its key TFs PDX1 and SOX9. Figure 7 As shown in part h, qRT-PCR analysis confirmed that along with the downregulation of ITGAV in PP organoids, the expression levels of TFs genes such as PDX1, NKX6.1, SOX9, GATA4, GATA6 and PAX4 in PP organoids also decreased. Subsequently, the role of AKT signaling in the differentiation of PP organoids was investigated by adding inhibitors of the AKT signaling pathway at the PG and PFG stages. Figure 7 As shown in part i of Figure 3, IF analysis showed that the expression of SOX9 and GATA4 was reduced in PP organoids. Figure 7 As shown in part j of Figure 2, qRT-PCR analysis confirmed that the expression levels of PDX1, NKX6.1, SOX9, and GATA4 decreased after inhibiting the AKT signaling pathway. Figure 7 As shown in part k of Figure 3, the changes in adhesion of PP organoids in hydrogels were studied, and it was demonstrated that N-CAD expression was upregulated in the AG73 hydrogel group, indicating that the hydrogel promoted the EMT of PP organoids.

[0109] The above experimental results show that AG73 hydrogel promotes SOX9 by regulating the SDC4-ITGAV-AKT signaling axis. + / PDX1 + Formation of pancreatic bipotent organoids.

[0110] As can be seen from the above examples, in the present invention, SDC4 is found to be a potential key signal transduction protein associated with pancreatic organogenesis, differentiation and maturation. The present invention aims to reconstruct the developmental cues present in the process of in vitro β-cell differentiation, and constructs AG73-GelMA / AlgMA hydrogel by utilizing the ligand-receptor interaction between SDC4 and laminin-derived AG73 to induce PDX1 + / NKX6.1 + Differentiation of PP organoids. AG73 hydrogel provides sufficient biomechanical support and precise biochemical signal guidance to promote the differentiation of hiPSCs into the pancreatic endocrine lineage.

[0111] The present invention further revealed that SDC4 plays a key role in regulating the formation of PPs in the early stage of pancreatic lineage development. 2D culture confirmed that SDC4 deficiency reduced PDX1 + / NKX6.1 + The addition of AG73 significantly improved the differentiation efficiency of PDX1 compared with GellMA / AlgMA hydrogels. + / NKX6.1 + Differentiation efficiency of PP organoids. In terms of mechanism, the addition of AG73 activated the SDC4 / ITGAV complex and promoted the expression of SOX9 in the early stage of differentiation through the AKT signaling pathway. + / PDX1 + The above results indicate that SDC4 has the ability to activate cell transcriptional function by accurately recognizing mechanical signals of ECM; after adding AG73, N-cadherin was upregulated in PP organoids, resulting in the attenuation of CDH1-CDH1 connection between cells but retaining CDH2-CDH2 connection, that is, AG73 can increase the adhesion, viability and N-cad expression of cells encapsulated in hydrogels.

[0112] The present invention integrates tissue engineering and proteomics to construct AG73 biomimetic hydrogels and reconstruct the pancreatic development microenvironment in order to obtain efficient in vitro PPs and stable SC-β cells for transplantation therapy of diabetes. In order to demonstrate the clinical applicability of the above technical solution, the present invention uses hiPSCs to generate PP organoids for subsequent differentiation into SC-β cells. The results show that AG73-GelMA / AlgMA hydrogel can promote PDX1 + / NKX6.1 +The generation of PP organoids improves the differentiation efficiency and function of SC-β cells. In addition, AG73-SDC4-mediated mechanical signal transduction promotes SOX9 by regulating the SDC4-ITGAV-AKT signaling axis. + / PDX1 + Formation of dual-energy PP organoids. The present invention helps to optimize the source of seed cells in stem cell replacement therapy for diabetes, and provides a new approach to combining tissue engineering with stem cell therapy for basic and clinical research on diabetes. In later studies, the method of the present invention can be used to screen additional effective peptides by simulating the complex microenvironment of natural pancreatic development to optimize the in vitro differentiation of SC-β cells; at the same time, the repeatable and adjustable components and structures of engineered hydrogels can also be used to study the key regulatory effects and molecular mechanisms of ECM components and mechanical stress on pancreatic lineage development.

[0113] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A polypeptide hydrogel, characterized in that: The polypeptide hydrogel is AG73-GelMA / AlgMA hydrogel, which is prepared by cross-linking AG73 polypeptide and GelMA / AlgMA hydrogel; wherein, in the GelMA / AlgMA hydrogel, the mass concentration of GelMA is 5%, and the mass concentration of AlgMA is 2%; the amino acid sequence of the AG73 polypeptide is CGGGRKRLQVQLSIRTC.

2. The polypeptide hydrogel according to claim 1, characterized in that The preparation steps of the polypeptide hydrogel include: Step S1, preparation of GelMA / AlgMA hydrogel: dissolving GelMA and AlgMA in PBS containing lithium phenyl-2,4,6-trimethylbenzoylphosphonate, heating the mixture to 40-45° C. to ensure complete dissolution, and obtaining a GelMA / AlgMA hydrogel mixture; filtering and sterilizing the prepared GelMA / AlgMA hydrogel mixture to obtain a GelMA / AlgMA hydrogel solution; Step S2, cross-linking of AG73 polypeptide and GelMA / AlgMA hydrogel: dissolve AG73 polypeptide at 0.05-2 mg / mL in the GelMA / AlgMA hydrogel solution obtained in step S1, and shake for 18-30 hours to obtain AG73-GelMA / AlgMA hydrogel.

3. A pancreatic organoid constructed by the polypeptide hydrogel according to claim 1 or 2, characterized in that: The method for constructing the pancreatic organoid comprises: 3D printing an AG73-GelMA / AlgMA hydrogel system loaded with hiPSCs, transferring the printed structure into a culture medium, incubating for 48 to 72 hours, and inducing differentiation through four stages to obtain the pancreatic organoid.

4. The pancreatic organoid according to claim 3, characterized in that The 3D printing steps include: placing a load of 1×10 6 ~1×10 7 cells / mL hiPSCs were encapsulated in the AG73-GelMA / AlgMA hydrogel system and 3D printed using a printer; before printing, a structure with a side length of 5 mm and a chain distance of 0.4 μm was designed; at 10 mW / cm 2 The film was printed under UV light intensity of 100 μm for 60 s, with a total of 10 layers, each with a thickness of 100 μm.

5. The pancreatic organoid according to claim 3, characterized in that Transfer the printed constructs to culture plates and place them in complete mTeSR TM After incubation in Plus medium for 48 hours, four stages of differentiation were performed, and the pancreatic organoids were obtained 5 days after the fourth stage.

6. The pancreatic organoid according to claim 3, characterized in that The pancreatic organoids include PDX1 + / NKX6.1 + Pancreatic endocrine organoids, SOX9 + / PDX1 + Dual-potent pancreatic organoids.

7. A use of the polypeptide hydrogel as claimed in any one of claims 1 to 2, characterized in that: The application is selected from at least one of the following applications: application in constructing pancreatic organoids, application in preparing SC-β cells, application in preparing drugs for treating diabetes, and application in determining the regulatory effects and molecular mechanisms of ECM components and mechanical stress on pancreatic lineage development.

8. A use of a pancreatic organoid as claimed in any one of claims 3 to 6, characterized in that: The application is selected from at least one of the following applications: application in preparing SC-β cells, and application in preparing drugs for treating diabetes.

9. The use according to claim 7 or 8, characterized in that: The collected pancreatic organoids were further cultured and differentiated to form endocrine progenitor cells and SC-β cells.

10. The use according to claim 7 or 8, characterized in that: The GelMA / AlgMA hydrogel simulates the physiological rigidity of the pancreas and provides optimal mechanical stress and spatial structure for pancreatic organoid differentiation; the AG73-GelMA / AlgMA hydrogel promotes PDX1 + / NKX6.1 + The generation of pancreatic endocrine organoids improves the differentiation efficiency and function of SC-β cells; AG73-SDC4-mediated mechanical signal transduction promotes SOX9 by regulating the SDC4-ITGAV-AKT signaling axis. + / PDX1 + Formation of bipotent pancreatic organoids.

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