Recombinant protein preparation for optimizing human pluripotent stem cell-derived kidney organoid

Abstract

The invention discloses a recombinant protein preparation for optimizing a human pluripotent stem cell-derived kidney organoid, which comprises ANXA2, a human pluripotent stem cell-derived kidney organoid and a human pluripotent stem cell-derived kidney organoid, it is found for the first time that ANXA2 can significantly improve fate specialization of early differentiated cells to kidney lineages, significantly improve the stability of kidney organoid products obtained in different experiment batches, and significantly improve the overall vascularization level and maturity degree of kidney organoid products in the final differentiated stage; a new thought and strategy are provided for construction of the human pluripotent stem cell-derived kidney organoid, and the human pluripotent stem cell-derived kidney organoid has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a recombinant protein preparation for optimizing kidney organoids derived from human pluripotent stem cells. Background Art

[0002] The prevalence of chronic kidney disease (CKD) is as high as 10%, bringing a huge burden to individuals and society. At present, except for dialysis and kidney transplantation, there is still no effective treatment method. However, dialysis can only delay the progression of the disease, and the only curative kidney transplantation is faced with the serious shortage of organ donors. There is an urgent need to find new effective treatment options. Studying the pathogenesis of CKD helps to find new potential therapeutic targets, and the primary condition is to establish an effective disease model. The kidney is composed of multiple cell types and has a complex physiological structure. Traditional in vitro cell models are often single immortalized cell lines, which cannot be compared with the in vivo kidney in terms of gene expression profiles, physiological functions, or physiological structures; while animal models have species differences with humans and cannot truly simulate the human body. Therefore, there is an urgent need for an in vitro model that can more truly simulate the kidney organ in the human body.

[0003] Kidney organoids are obtained by simulating the development of the kidney organ in vivo using pluripotent human pluripotent stem cells (hPSCs) and differentiating them in vitro. It not only has a human genetic background, can avoid species differences brought by animal models, but also can produce complex three-dimensional structures and functions similar to the corresponding organs in vivo, with advantages not possessed by single-type cell lines or primary cells. Therefore, kidney organoids are ideal in vitro models for simulating kidney injury disease phenotypes, screening therapeutic drugs, and predicting the human body's response to potential drugs. Currently, the bottleneck problems in the field of kidney organoids include: lack of overall maturity, vascularization level, and insufficient batch-to-batch stability. The main reason may be the lack of key microenvironments and signaling pathways for kidney development during in vitro differentiation. Therefore, exploring and supplementing the missing key factors may solve the above bottleneck problems. The work reported in this field has tried to solve the above problems from different angles:

[0004] (1) Some studies have reported that transplanting kidney organoids into healthy living hosts can improve the maturity and vascularization levels of kidney organoids. For example, when kidney organoids are transplanted under the renal capsule of immunodeficient mice, the grafts show phenomena such as podocyte maturation and connection to the host's vascular network; another study transplanted kidney organoids into the chorioallantoic membrane (CAM) of chicken embryos, and the grafts showed successful implantation, vascularization, multi-vascularization, and blood circulation. However, further examination of these transplanted kidney organoids found that most of the vascular endothelial cells in their glomeruli were from the host animals, rather than from the grafts. This phenomenon indicates the potential problems existing in the in-vivo transplantation method: the development of cells in kidney organoids requires cell-cell communication. For example, the interaction between podocytes and vascular endothelial cells promotes their further maturation. If the endothelial cells in the graft are mainly from the host animal, considering differences between species, for example, cell-cell communication at this time cannot truly reflect kidney development in the human body, so the further development of the graft may deviate under the action of incorrect signals. In addition, this technical method relies on in-vivo transplantation in animals, resulting in complex operations, high costs, and the disadvantage of being unsuitable for high-throughput applications.

[0005] (2) Some studies have reported using microfluidics technology to significantly improve the maturity and vascularization levels of kidney organoids cultured in vitro. Microfluidics technology is a microfluidic manipulation technology. Since the operating environment of traditional cell culture methods is very different from the in-vivo environment, it is difficult to objectively and truly reflect the biological characteristics of cells under physiological conditions. Microfluidics can simulate the in-vivo microenvironment to a certain extent, for example, providing factors such as fluid shear stress, which is important for the differentiation of kidney organoids, that are lacking under static culture conditions. A study demonstrated that culturing kidney organoids under flowing conditions on a microfluidic device induces the formation of a perfusable vascular network within the organoids, supporting the role of fluid flow in inducing vascularization, and the level of vascularization is positively correlated with the fluid velocity. The renal unit cells cultured in this system, including podocytes and renal tubular epithelial cells, express higher levels of lineage markers than renal unit cells cultured under conventional static conditions, which may be due to their communication and interaction with the increased level of vascular endothelial cells, resulting in further maturation. This study suggests that microfluidics has a significant effect on the in-vitro maturation of kidney organoids. The drawback of this technical method is that its dependence on microfluidic devices makes the technical threshold and cost high, and it is difficult to repeat and popularize.

[0006] (3) Some studies have used a technique based on "extrusion-based three-dimensional cell bioprinting" to fabricate kidney organoids with high throughput and high reproducibility. When manufacturing organoids for drug development in vitro, reliability and reproducibility are crucial. To achieve this, methods for stably obtaining organoid products with small individual differences from different hPSC cell lines and different experimental batches need to be explored. One study demonstrated that by using the "cellular extrusion bioprinting" technique, the reproducibility of kidney organoid products can be improved. While significantly increasing the throughput, it also reduces the differences in cell number, diameter, and cell viability among products. The drawback of this technical method is that the dependence on 3D printing devices increases the technical threshold and manufacturing cost of kidney organoids. Summary of the Invention

[0007] In view of the above technical problems existing in the prior art, the object of the present invention is to provide a recombinant protein preparation for optimizing kidney organoids derived from human pluripotent stem cells. The recombinant protein preparation is human ANXA2 recombinant protein, which can significantly enhance the fate specialization of early differentiated cells into the kidney lineage, significantly improve the stability of obtaining kidney organoid products among different experimental batches, and significantly enhance the overall vascularization level and maturity of kidney organoid products at the end of differentiation.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides the application of ANXA2 in promoting the induced differentiation of hPSC into kidney organoids.

[0010] Furthermore, the usage concentration of the ANXA2 is 100 - 300 ng / mL;

[0011] Preferably, the usage concentration of the ANXA2 is 200 ng / mL;

[0012] Preferably, the addition time of the ANXA2 is Day 6 - 16.

[0013] In the present invention, the ANXA2 is annexin A2, which is a calcium ion-mediated phospholipid-binding protein and can be widely expressed in various types of cells and tissues. As a multifunctional molecule, ANXA2 can participate in the regulation of various cellular activities, such as: processes of cell exocytosis, endocytosis, migration, and proliferation. As a receptor for plasminogen and tissue plasminogen activator, ANXA2 can promote the production of plasmin and maintain the homeostasis of blood coagulation, fibrinolysis, and matrix degradation; as an antigen expressed on the cell membrane, ANXA2 can trigger local inflammation and damage by binding to autoantibodies.

[0014] In some embodiments, ANXA2 described in the present invention can be obtained from any commercial source or prepared by biosynthetic methods well-known to those skilled in the art, and is not limited to the human recombinant protein ANXA2 from Wuhan Yunke Long Biotechnology Co., Ltd. as described in the specific embodiments of the present invention. In a preferred embodiment, ANXA2 described in the present invention is a human recombinant protein ANXA2.

[0015] In some embodiments, there is no particular limitation on the use concentration of ANXA2 described in the present invention. Any use concentration of ANXA2 that can promote the induction and differentiation of hPSCs into kidney organoids, improve the maturity and vascularization level of kidney organoids, improve the stability of kidney organoid products obtained between different experimental batches, and / or improve the fate specialization of early differentiated cells into the kidney lineage is within the protection scope of the present invention. In a preferred embodiment, the use concentration of ANXA2 described in the present invention is 100 - 300 ng / mL. In a more preferred embodiment, the use concentration of ANXA2 described in the present invention is 200 ng / mL.

[0016] The second aspect of the present invention provides the application of ANXA2 in any of the following aspects:

[0017] (1) The application of ANXA2 in improving the maturity and vascularization level of kidney organoids;

[0018] (2) The application of ANXA2 in improving the stability of kidney organoid products obtained between different experimental batches;

[0019] (3) The application of ANXA2 in improving the fate specialization of early differentiated cells into the kidney lineage.

[0020] Furthermore, the kidney organoids are hPSC-derived kidney organoids;

[0021] Preferably, the early differentiated cells are hPSC-derived early differentiated cells;

[0022] Preferably, the use concentration of ANXA2 is 100 - 300 ng / mL;

[0023] More preferably, the use concentration of ANXA2 is 200 ng / mL;

[0024] Preferably, the addition time of ANXA2 is Day 6 - 16.

[0025] In a specific embodiment, the present invention has experimentally verified that adding 200 ng / mL of human recombinant protein ANXA2 during the time period from day 6 to day 16 can significantly enhance the fate specialization of early differentiated cells (day 9) into the kidney lineage, significantly improve the stability of obtaining kidney organoid products among different experimental batches, and significantly enhance the overall vascularization level and maturity of kidney organoid products at the end of differentiation (day 24). Therefore, the applications of the ANXA2 in the above second aspect will all fall within the protection scope of the present invention.

[0026] The third aspect of the present invention provides a recombinant protein preparation for optimizing hPSC-derived kidney organoids.

[0027] Furthermore, the recombinant protein preparation contains ANXA2.

[0028] Furthermore, the usage concentration of the ANXA2 is 100 - 300 ng / mL;

[0029] Preferably, the usage concentration of the ANXA2 is 200 ng / mL.

[0030] In some embodiments, the recombinant protein preparation contains ANXA2. In other embodiments, the recombinant protein preparation may further contain other reagents that can be used to optimize hPSC-derived kidney organoids or reagents that assist ANXA2 in optimizing hPSC-derived kidney organoids.

[0031] The fourth aspect of the present invention provides a method for inducing the differentiation of hPSCs into kidney organoids.

[0032] Furthermore, the method includes the following steps: adding ANXA2 to the hPSC culture system;

[0033] Preferably, the usage concentration of the ANXA2 is 100 - 300 ng / mL;

[0034] More preferably, the usage concentration of the ANXA2 is 200 ng / mL;

[0035] Preferably, the addition time of the ANXA2 is Day 6 - 16.

[0036] Furthermore, the method includes the following steps:

[0037] (1) Day 0: Culturing hPSCs in a culture medium;

[0038] (2) Day 1 - 4: Contacting the hPSCs obtained in step (1) with the small molecule CHIR99021 in the culture medium for 4 days to differentiate them into primitive streak cells;

[0039] (3) Day 5 - 6: Contact the primitive streak cells obtained in step (2) with FGF9 and heparin in the culture medium for 2 days to induce their differentiation into intermediate mesoderm cells;

[0040] (4) Day 6: Contact the cells obtained in step (3) with FGF9, heparin, and ANXA2 in the culture medium for 1 day;

[0041] (5) Day 7: Digest the cells obtained in step (4) into single cells, prepare cell aggregates by centrifugation, contact them with CHIR99021 in the culture medium for 2 h, and then re - contact the cells with FGF9, heparin, and ANXA2 in the culture medium for 5 days to sequentially induce their differentiation into metanephric mesenchyme cells, nephron progenitor cells, and nephrons;

[0042] (6) Day 12 - 16: Contact the cells obtained in step (5) with ANXA2 in the culture medium for 4 days to generate more nephron structures inside the cell aggregates;

[0043] (7) Day 17 - 24: Continuously culture the cells obtained in step (6) in the culture medium to obtain kidney organoids.

[0044] Furthermore, the culture medium described in step (1) is the hPSC - specific culture medium PGM1;

[0045] Preferably, the culture conditions in step (1) are to change the culture medium daily until the cell confluence reaches 50%;

[0046] Preferably, the culture medium described in step (2) is APEL2 medium;

[0047] Preferably, the concentration of CHIR99021 in step (2) is 1 - 20 μM;

[0048] More preferably, the concentration of CHIR99021 in step (2) is 10 μM;

[0049] Preferably, the culture conditions in step (2) are to change the culture medium every two days;

[0050] Preferably, the culture medium described in step (3) is APEL2 medium;

[0051] Preferably, the concentration of FGF9 in step (3) is 100 - 300 ng / mL;

[0052] More preferably, the concentration of FGF9 in step (3) is 200 ng / mL;

[0053] Preferably, the concentration of heparin in step (3) is 0.1 - 5 μg / mL;

[0054] More preferably, the concentration of heparin in step (3) is 1 μg / mL;

[0055] Preferably, the culture condition in step (3) is to change the culture medium every two days;

[0056] Preferably, the culture medium in step (4) is APEL2 medium;

[0057] Preferably, the concentration of FGF9 in step (4) is 100 - 300 ng / mL;

[0058] More preferably, the concentration of FGF9 in step (4) is 200 ng / mL;

[0059] Preferably, the concentration of heparin in step (4) is 0.1 - 5 μg / mL;

[0060] More preferably, the concentration of heparin in step (4) is 1 μg / mL;

[0061] Preferably, the usage concentration of ANXA2 in step (4) is 100 - 300 ng / mL; more preferably, the usage concentration of ANXA2 in step (4) is 200 ng / mL. Further, in step (5), each cell mass contains approximately (1 - 10) × 10 5 cells;

[0062] Preferably, each cell mass in step (5) contains approximately 5 × 10 5 cells;

[0063] Preferably, the culture medium in step (5) is APEL2 medium;

[0064] Preferably, the concentration of CHIR99021 in step (5) is 1 - 20 μM;

[0065] More preferably, the concentration of CHIR99021 in step (5) is 10 μM;

[0066] Preferably, the concentration of FGF9 in step (5) is 100 - 300 ng / mL;

[0067] More preferably, the concentration of FGF9 in step (5) is 200 ng / mL;

[0068] Preferably, the concentration of heparin in step (5) is 0.1 - 5 μg / mL;

[0069] More preferably, the concentration of heparin in step (5) is 1 μg / mL;

[0070] Preferably, the concentration of ANXA2 used in step (5) is 100 - 300 ng / mL;

[0071] More preferably, the concentration of ANXA2 used in step (5) is 200 ng / mL;

[0072] Preferably, the culture condition in step (5) is to change the culture medium every two days;

[0073] Preferably, the culture medium in step (6) is APEL2 medium;

[0074] Preferably, the concentration of ANXA2 used in step (6) is 100 - 300 ng / mL;

[0075] More preferably, the concentration of ANXA2 used in step (6) is 200 ng / mL;

[0076] Preferably, the culture condition in step (6) is to change the culture medium every two days;

[0077] Preferably, the culture medium in step (7) is APEL2 medium;

[0078] Preferably, the culture condition in step (7) is to change the culture medium every two days.

[0079] In addition, the present invention also provides the application of the kidney organoids obtained by the method as described above in any of the following aspects:

[0080] (1) In combination with fields such as developmental biology and gene editing, using the induction and differentiation of kidney organoids to establish a research model for kidney diseases;

[0081] (2) In combination with precision medicine and pharmacy fields, for in vitro testing of the renal toxicity and efficacy of drugs;

[0082] (3) In combination with regenerative medicine and precision medicine fields, for the application of using the kidney organoids as grafts to replace diseased kidney tissues.

[0083] Furthermore, the research model for kidney diseases can be used in the research of key pathogenic genes of kidney diseases, for example: for the research of key pathogenic genes of polycystic kidney disease, etc.

[0084] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0085] Currently, there are mainly two methods in this field that can improve the maturity and vascularization level of kidney organoids. One is to transplant kidney organoids under the renal capsule of mice or into chicken embryos, and promote the further maturation of kidney organoids by means of the host's in vivo vascular network; the other is to use a microfluidic control system in vitro to promote the generation of microvessels inside kidney organoids by means of liquid shear force, and thereby improve their overall maturity. The above two methods are complex in operation, high in cost, and have a certain technical threshold, especially not suitable for high-throughput preparation of functionally mature kidney organoids. The greatest advantage of the recombinant protein preparation provided by the present invention is that it is a single peptide segment with a determined composition, and there are already commercially available recombinant proteins. It only needs to add a fixed concentration of this preparation within a fixed time period in the existing in vitro differentiation protocol of kidney organoids, add it to the culture medium and co-incubate with cells. Without the need to additionally rely on complex instruments or operation means, a significant improvement in the maturity and vascularization level can be achieved. The operation is simple, the cost is reduced, and it is suitable for obtaining a large number of kidney organoids in the future, and is applicable to high-throughput application scenarios such as drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 Schematic diagram of the treatment with human ANXA2 recombinant protein in the differentiation of hPSCs into kidney organoids;

[0087] Figure 2 Effect of the treatment with human ANXA2 recombinant protein on the products of kidney organoids. Among them, the upper figure: Immunofluorescence staining was used to detect renal vesicles (PAX8 + ) in the products of kidney organoids (day 9) treated with human ANXA2 recombinant protein, proximal tubular epithelial cells (LTL + ), podocyte precursor cells (WT1 + ), vascular endothelial cells (CD34 + ), scale bar, 50 μm. ANX-Orgs, kidney organoids treated with 200 ng / mL human ANXA2 recombinant protein during the differentiation process. The lower figure: Quantitative statistics of the number of pronephric aggregates, renal vesicles, podocyte precursor cells and the length of vascular endothelial cells in the products of kidney organoids based on the immunofluorescence results on day 9. The data are mean values ± SEM from 1 independent experiment (replicates = 5, a randomly selected photo represents a replicate), and the statistical method is unpaired t-test, ns, P>0.05; *, P<0.05; ***, P<0.001. PTAs, pronephric aggregates; RVs, renal vesicles; Ctrl-Orgs, products of kidney organoids untreated with ANXA2; ANX-Orgs, products of kidney organoids treated with 200 ng / mL human ANXA2 recombinant protein during the differentiation process;

[0088] Figure 3 To label and quantitatively analyze the renal tubular structures in kidney organoids on day 12, Ctrl-Orgs, kidney organoid products untreated with ANXA2; ANX-Orgs, kidney organoid products treated with 200 ng / mL human ANXA2 recombinant protein during differentiation. Among them, the upper panel: labeling of renal tubular structures in kidney organoids on day 12. Dark red represents renal tubular structures, and green represents non-renal tubular structures. Scale bar, 30 μm. The lower panel: quantitative analysis of the area ratio of renal tubular structures in kidney organoids on day 12. Data are from 3 independent experiments, and the statistical method is unpaired t-test, **, P < 0.01;

[0089] Figure 4 For immunofluorescence staining to detect proximal tubular epithelial cells (LTL + )、podocytes (WT1 + ) and vascular endothelial cells (PECAM + ) in kidney organoid products (day 24) treated with human ANXA2 recombinant protein, scale bar, 200 μm;

[0090] Figure 5 For flow cytometry analysis to detect the proportions of proximal tubular epithelial cells (LTL + )、podocytes (WT1 + ) and vascular endothelial cells (PECAM + ) in kidney organoid products (day 24) treated with human ANXA2 recombinant protein;

[0091] Figure 6 For transmission electron microscopy to detect proximal renal tubular microvilli (blue arrows), peritubular capillaries with fenestrated structures (orange arrows, fc) (purple arrows, pc), podocytes with primary foot processes (green arrows, pp) and secondary foot processes (red arrows, sp), and basement membrane structures (yellow arrows, bm) in kidney organoid products (day 24) treated with human ANXA2 recombinant protein;

[0092] Figure 7 For GGT assay to detect the GGT activity of kidney organoids, Ctrl-Orgs, kidney organoid products untreated with ANXA2; ANX-Orgs, kidney organoid products treated with 200 ng / mL human ANXA2 recombinant protein during differentiation. Human proximal tubular cell line HK2 was used as a positive control. Data are mean values ± SD from 1 independent experiment (replicates = 4), and the statistical method is unpaired t-test, **, P < 0.01;

[0093] Figure 8 To detect the protein levels of NICDs (N1ICD, N2ICD, N4ICD) in kidney organoid products at different time points (day 7, day 9, day 12) by Western blot and quantitative statistics. Ctrl-Orgs: kidney organoid products untreated with ANXA2; ANX-Orgs: kidney organoid products treated with 200 ng / mL human ANXA2 recombinant protein during differentiation. Among them, the upper panel: Western blot to detect the protein levels of NICDs in kidney organoid products (replicates = 3). The lower panel: quantitative statistics of the protein levels of NICDs in kidney organoid products. Data are mean values ± SD from at least 2 independent experiments. Statistical method: unpaired t-test, ns, P > 0.05; *, P < 0.05; **, P < 0.01;

[0094] Figure 9 To detect the protein levels of NOTCH receptors (NOTCH1-4) in kidney organoid products at different time points (day 7, day 9, day 12) by Western blot and quantitative statistics. Ctrl-Orgs: kidney organoid products untreated with ANXA2; ANX-Orgs: kidney organoid products treated with 200 ng / mL human ANXA2 recombinant protein during differentiation. Among them, the upper panel: Western blot to detect the protein levels of NOTCH receptors in kidney organoid products (replicates = 3). The lower panel: quantitative statistics of the protein levels of NOTCH receptors in kidney organoid products. Data are mean values ± SD from at least 2 independent experiments. Statistical method: unpaired t-test, ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Figure 10To detect the expression levels of NOTCH2 or NOTCH4 in 293 cells or HUVEC cells under different treatment conditions by qPCR. Among them, the left figure: qPCR was used to detect the expression level of NOTCH2 in 293 cells under different treatment conditions. Data are mean values ± SD from 2 independent experiments (replicates = 4), and the statistical method was unpaired t-test, **, P < 0.01. Control, 293 cells untreated with ANXA2; ANXA2, 293 cells treated with 200 ng / mL human ANXA2 recombinant protein for 72 h. The right figure: qPCR was used to detect the expression level of NOTCH4 in HUVEC cells under different treatment conditions. Data are mean values ± SD from 2 independent experiments (replicates = 4), and the statistical method was unpaired t-test, *, P < 0.05. Control, HUVEC cells untreated with ANXA2; ANXA2, HUVEC cells treated with 200 ng / mL human ANXA2 recombinant protein for 72 h;

[0095] Figure 11 To detect and quantitatively analyze the protein levels of NOTCH2 and N2ICD, NOTCH4 and N4ICD in 293 cells or HUVEC cells under different treatment conditions by Western blot. Among them, the left figure: Western blot was used to detect and quantitatively analyze the protein levels of NOTCH2 and N2ICD in 293 cells under different treatment conditions. Data are mean values ± SD from 5 independent experiments (replicates = 3), and the statistical method was unpaired t-test, *, P < 0.05. Control, 293 cells untreated with ANXA2; ANXA2, 293 cells treated with 200 ng / mL human ANXA2 recombinant protein for 72 h. The right figure: Western blot was used to detect and quantitatively analyze the protein levels of NOTCH4 and N4ICD in HUVEC cells under different treatment conditions. Data are mean values ± SD from 4 independent experiments (replicates = 3), and the statistical method was unpaired t-test, **, P < 0.01. Control, HUVEC cells untreated with ANXA2; ANXA2, HUVEC cells treated with 200 ng / mL human ANXA2 recombinant protein for 72 h;

[0096] Figure 12To detect the binding of NOTCH2 to ANXA2 and NOTCH4 to ANXA2 in 293 cells or HUVEC cells treated with human ANXA2 recombinant protein for 72 h. Among them, the left figure: Detection of the binding of NOTCH2 to ANXA2 in 293 cells treated with human ANXA2 recombinant protein for 72 h by Co-IP. The right figure: Detection of the binding of NOTCH4 to ANXA2 in HUVEC cells treated with human ANXA2 recombinant protein for 72 h by Co-IP;

[0097] Figure 13 For molecular dynamics simulation analysis of the change in solvent accessible surface area of the LNR region after the binding of NOTCH2 and NOTCH4 to ANXA2 respectively, and the change in the position of the LNR region of the NOTCH receptor at two time points of 0 ns and 100 ns after the binding of ANXA2 to NOTCH2 or NOTCH4 in molecular dynamics simulation. Among them, the upper figure: Molecular dynamics simulation analysis of the change in solvent accessible surface area of the LNR region after the binding of NOTCH2 and NOTCH4 to ANXA2 respectively. SASA, solvent accessible surface area; Control, the change in solvent accessible surface area of the LNR region of NOTCH2 or NOTCH4 protein; NOTCH2-ANXA2, the change in solvent accessible surface area of the LNR region of NOTCH2 protein in the complex of NOTCH2 and ANXA2; NOTCH4-ANXA2, the change in solvent accessible surface area of the LNR region of NOTCH4 protein in the complex of NOTCH4 and ANXA2. The lower figure: The change in the position of the LNR region of the NOTCH receptor at two time points of 0 ns and 100 ns after the binding of ANXA2 to NOTCH2 or NOTCH4 in molecular dynamics simulation;

[0098] Figure 14To analyze the changes in the solvent accessible surface area of the LNR region after NOTCH1 and NOTCH3 bind to ANXA2 respectively by molecular dynamics simulation, and the position changes of the LNR region of the NOTCH receptor at two time points of 0 ns and 100 ns after ANXA2 binds to NOTCH1 or NOTCH3 by molecular dynamics simulation. Among them, the upper figure: the changes in the solvent accessible surface area of the LNR region after NOTCH1 and NOTCH3 bind to ANXA2 respectively by molecular dynamics simulation. SASA, solvent accessible surface area; Control, the changes in the solvent accessible surface area of the LNR region of NOTCH1 or NOTCH3 protein; NOTCH1-ANXA2, the changes in the solvent accessible surface area of the LNR region of NOTCH1 protein in the complex of NOTCH1 and ANXA2; NOTCH3-ANXA2, the changes in the solvent accessible surface area of the LNR region of NOTCH3 protein in the complex of NOTCH3 and ANXA2. The lower figure: the position changes of the LNR region of the NOTCH receptor at two time points of 0 ns and 100 ns after ANXA2 binds to NOTCH1 or NOTCH3 by molecular dynamics simulation. Detailed implementation mode

[0099] The present invention will be further described below in conjunction with specific embodiments. The specific embodiments are only used to explain the present invention and cannot be construed as a limitation to the present invention. Those of ordinary skill in the art can understand that: various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purposes of the present invention. The scope of the present invention is defined by the claims and their equivalents. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturers. The reagents, biological materials, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0100] Research on the application of human ANXA2 recombinant protein in promoting the induction and differentiation of hPSCs into kidney organoids and its related mechanism

[0101] 1. Experimental materials

[0102] (1) The culture media are as follows:

[0103] PGM1: Cellapy, CA1007500;

[0104] APEL2: STEMCELL Technologies, 05270.

[0105] (2) Annexin A2 (ANXA2) is as follows:

[0106] It is a human recombinant protein and is purchased from Wuhan Yunclon Technology Co., Ltd.

[0107] The concentration of ANXA2 used in the experimental system for the differentiation of hPSCs into kidney organoids is 200 ng / mL.

[0108] 2. Experimental methods

[0109] (1) In vitro induction method for differentiating hPSCs into kidney organoids

[0110] ① Day 0: Culture hPSCs in a cell culture dish using the hPSC-specific medium PGM1, and change the medium daily until the cell confluence reaches 50%;

[0111] ② Days 1 - 4: Contact hPSCs with the small molecule CHIR99021 (10 μM) in APEL2 for 4 days to induce their differentiation into primitive streak cells, and change the medium every two days;

[0112] ③ Days 5 - 6: Contact the primitive streak cells with FGF9 (200 ng / mL) and heparin (1 μg / mL) in APEL2 for 2 days to induce their differentiation into intermediate mesoderm cells, and change the medium every two days;

[0113] ④ Day 6: Contact the cells with FGF9 (200 ng / mL), heparin (1 μg / mL), and annexin A2 (200 ng / mL) in APEL2 for 1 day;

[0114] ⑤ Day 7: Digest the cells into single cells, prepare cell aggregates (about 5×10 5 cells per cell aggregate) by centrifugation, and transfer the cell aggregates to the upper filter membrane of a Transwell. Contact the cells with CHIR99021 (10 μM) in APEL2 for 2 hours. After 2 hours, re-contact the cells with FGF9 (200 ng / mL), heparin (1 μg / mL), and annexin A2 (200 ng / mL) in APEL2 for 5 days to sequentially induce their differentiation into metanephric mesenchyme cells, nephron progenitor cells, and nephrons, and change the medium every two days;

[0115] ⑥ Days 12 - 16: Continue to contact the above cells with annexin A2 (200 ng / mL) in APEL2 for 4 days to generate more nephron structures inside the cell aggregates, and change the medium every two days;

[0116] ⑦ Days 17 - 24: Continue to culture the above cells in APEL2, and change the medium every two days. The entire differentiation cycle of kidney organoids is 24 days.

[0117] (2) Detection method

[0118] ①qRT-PCR assay

[0119] Total ribonucleic acid was extracted from cells using Trizol (ThermoFisher, 15596026). 1000 ng of total RNA was reverse transcribed into cDNA using a one-step gDNA removal and cDNA synthesis SuperMix (Transgen Biotech, AU341-02). qRT-PCR assay was performed using TB PreMix Ex Taq TM II (Takara, RR820A). Its relative expression level was analyzed using the ΔΔCT method and normalized to GAPDH mRNA expression. Four technical replicates were set for all samples. The primer sequences used in this study were designed by conventional methods in the art.

[0120] ②Immunofluorescence staining

[0121] Fresh samples were incubated in PBS with 4% paraformaldehyde for 30 min, embedded in Tissue-Tek O.C.T. (ThermoFisher), and flash frozen in liquid nitrogen. Frozen sections were made using a cryostat (Leica). Other steps were similar to paraffin sections, except that antigen retrieval was not performed. The primary antibodies included LTL-biotin conjugate (1:300, Vector Laboratories, B-1325), PECAM1 (1:200, Abcam, ab9498), WT1 (1:200, Abcam, ab89901), CD34 (1:200, Abcam, ab81289), PAX8 (1:200, CST, 59019S), and were incubated overnight at 4°C. After washing 3 times with PBS, the corresponding secondary antibodies were incubated for 2 hours at 37°C. After washing 3 times with PBS, DAPI was used to stain at 1:10000 for 15 minutes. After washing 3 times with PBS, immunofluorescence imaging was performed using a Zeiss (LSM 780) and Leica confocal microscope.

[0122] ③Western blot

[0123] Transfer the renal tissue and other cell samples into 1.5 mL tubes, wash them twice with PBS, and then place them in lysis buffer (RIPA, P0013B) containing protease and phosphatase inhibitors (1:100, NCM Biotech, P002) at 4°C for 30 minutes, shaking every 5 minutes. The lysate is centrifuged at 13,000 g at 4°C for 30 minutes, and the supernatant is transferred to a new 1.5 mL tube. The total protein concentration is determined using a BCA protein assay kit (Beyotime, P0010) according to the instructions. The protein lysate is diluted 1:5 with Beyotime, P0015L buffer and boiled at 100°C for 10 minutes. 30 μg of the total denatured protein of each sample is transferred onto a 0.45 μm PVDF membrane after 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis chromatography. Block the cell membrane with 5% non-fat milk solution at room temperature for 1 hour. For immunoblotting, incubate with primary antibodies against NOTCH1 (1:2,000, Proteintch, 20687-1-AP), NOTCH2 (1:2,000, Proteintch, 28580-1-AP), NOTCH3 (1:2,000, Abcam, 23426), NOTCH4 (1:2,000, Abcam, AB184742), N1ICD (1:200, R&D, AF3647), N2ICD (1:200, R&D, AF3735), N4ICD (1:200, R&D, AF3847) overnight at 4°C. Incubate with HRP-conjugated secondary antibodies (1:5,000, CST, 5127S; CST, 7074; AbClone, AS014) for 1 hour. Detect specific bands using the enhanced chemiluminescence (ECL) method and measure them using Image J software (NIH, Bethesda, MD, United States). Use the β-actin level as an internal standard.

[0124] ④Flow cytometry

[0125] The samples were stained with the Cytofix / Cytoperm Fixation / Permeabilization Kit (BD, 554714). First, the organoids were dissociated with Acutase and completely resuspended into single cells, then 250 μL of fixation / permeabilization solution was added and allowed to act at 4°C for 20 minutes. The samples were washed 3 times with 1 mL of 1× buffer. Then the cells were blocked with 5% goat serum in PBS for 30 minutes. The samples were incubated overnight at 4°C with LTL-biotin conjugate (1:300, Vector Laboratories, B-1325), PECAM1 (1:200, Abcam, ab9498), and WT1 (1:200, Abcam, ab89901), and washed 3 times in 1 mL of 1× buffer. The cells were incubated with the secondary antibody at 37°C for 1 hour and washed 3 times in 1 mL of 1× buffer. Flow cytometry analysis was performed using a BD FACSVerse, and statistical analysis was performed using FlowJo software.

[0126] ⑤ GGT Activity Assay

[0127] The GGT activity was detected using a GGT activity kit (Biovision, K784-100). The organoid samples were homogenized in 200 μL of GGT assay buffer and centrifuged (13,000 g, 10 minutes) to remove insoluble materials. Then, 10 μL / well of the test sample was prepared in a 96-well plate with GGT assay buffer, and 90 μL of the GGT substrate mixture, which included the test sample and a positive control, was added to each well. For the PNA standard curve, 2 mM PNA standard solution was added to different wells of the 96-well plate in volumes of 0, 4, 8, 12, 16, and 20 μL in duplicate, generating standard solutions of 0, 8, 16, 24, 32, and 40 nM / well, and the final volume was adjusted to 100 μL with GGT assay buffer. For the measurement of the PNA standard curve, the OD was measured at 418 nm using a microplate reader. For the samples and the positive control, the mixture was incubated at 37°C for 3 minutes, then the OD was measured at 418 nm using a microplate reader, incubated at 37°C for 30 minutes to 2 hours, and the OD was measured again at 418 nm (A1); the incubation time would depend on the GGT activity in the sample. When calculating, first draw the PNA standard curve, and then calculate the GGT activity of the test sample: ΔOD = A1 - A0. Add ΔOD to the PNA standard curve to obtain the B nmol value of PNA produced by GGT within a given time. The formula used is as follows:

[0128]

[0129] Wherein, B is the total amount of pNA (nmol) in the standard curve, T is the incubation time (minutes), V is the volume of the sample added to the reaction well (mL), and the unit definition is that one unit of GGT produces 1.0 μmol of pNA per minute at 37°C. Note: One unit of pNA ≈ 1.5 IU.

[0130] ⑥ Scanning electron microscopy

[0131] Fix the kidney organoid samples with 2.5% glutaraldehyde at 4°C for 1.0 mm 3 overnight, wash 3 times in 0.1 M acetate buffer, then fix in 1% osmium tetroxide solution for 2 hours, and dehydrate in 30, 50, 70, 80, 90 and 100% acetone solutions. Cut the samples into 50-nm sections with an ultramicrotome (Leica EM UC7, Wetzlar, Germany), treat with 2% uranyl acetate for 10 minutes and lead citrate for 5 minutes respectively, and observe under a transmission electron microscope (JEM-1400).

[0132] ⑦ Immunoprecipitation

[0133] Immunoprecipitation (Co-IP) was performed using an immunoprecipitation kit (Thermo Science Piells, 26149) according to the manufacturer's instructions. 293 cells and human umbilical vein endothelial cells were cultured in 60-mm culture dishes. Approximately 1 mg of cell lysate was incubated overnight at 4°C with amino-link coupled resin immobilized with 10 μg of AnxA2 antibody (Proteintech, 66035-1-Ig), and mouse antibody (Beyotime, A7028) was used as a negative control. After washing, the isolated immune complexes were analyzed by Western blotting as described above.

[0134] ⑧ Molecular dynamics simulation

[0135] Molecular dynamics simulations were performed using the Gromacs 2022.2 program. The complex structures of NOTCH1, NOTCH2, NOTCH3, NOTCH4, and ANXA2 were obtained by HDOCK molecular docking and used as the original coordinate files for the simulations. The solvated complexes were immersed in a cubic periodic boundary box using the TIP3P water molecule model. Sodium chloride was added to neutralize the system charge to a concentration of 0.15 mol / L. Energy minimization was initially performed using the Particle-mesh Ewald (PME) method with a cutoff value of 1.2 nm to eliminate unfavorable contacts throughout the system. Subsequently, canonical ensemble (NVT) and isobaric-isothermal ensemble (NPT) simulations were performed, maintaining a constant temperature (298 K) and constant pressure (1 bar). The equilibrated system was subjected to 100 ns of molecular dynamics simulations.

[0136] ⑨ Solvent accessible surface area analysis

[0137] The calculation method of solvent accessible surface area (SASA) analysis includes regarding the peptide segment in the region of 1170-1435 as the LNR sequence, and regarding the entire protein of the complex and the sequence in the complex excluding LNR as "other".

[0138] The calculation formula is: SASA = LNR - (Other + LNR - Complex) / 2

[0139] (Other + LNR - Complex) / 2 in the formula represents the connection region between LNR and "other", especially the embedding region of LNR in the protein, which is called the solvent accessible surface area (SASA).

[0140] 3. Experimental Results

[0141] According to the reports in existing literature, the in vitro induction of hPSCs into kidney organoids requires a total of 24 days. The inventors of the present invention found that adding 200 ng / mL human ANXA2 recombinant protein in the time period from day 6 to day 16 (accompanying drawings, Figure 1 ) has the following effects:

[0142] (1) Significantly enhance the fate specialization of early differentiated cells (day 9) into the kidney lineage. The nephron precursor structure - PAX8 + renal vesicles are produced in the ANXA2 treatment group, and the number is 10 times higher than that of the control group without ANXA2 treatment (P < 0.001), and the proximal tubule specialization (LTL + ) has been initiated; the number of podocyte precursor cells (WT1 + ) in the ANXA2 treatment group is 2.1 times higher than that of the control group without ANXA2 treatment (P < 0.001); the primary capillary network (CD34 + ) is produced in the ANXA2 treatment group, and the microvascular length is 9.5 times higher than that of the control group without ANXA2 treatment (P < 0.001) (accompanying drawings, Figure 2 ).

[0143] (2) Significantly enhance the stability of the obtained kidney organoid products among different experimental batches. Using the proportion of the renal tubule structure in the organoids as the evaluation criterion, 3 independent experiments are carried out. The proportion of the renal tubule structure in the ANXA2 treatment group is 50.0%, and that of the control group without ANXA2 treatment is 27.1% (P < 0.01) (accompanying drawings, Figure 3 ); the coefficient of variation of the products in the ANXA2 treatment group and the control group without ANXA2 treatment are 2.1% and 29.6% respectively.

[0144] (3) Significantly improve the overall vascularization level and maturity of kidney organoid products at the end of differentiation (day 24). In the products of the ANXA2 treatment group, proximal tubular epithelial cells (LTL + ), podocytes (WT1 + ), and vascular endothelial cells (PECAM + ) (accompanying figures, Figure 4 ) were 1.8 times, 4.5 times, and 2.0 times, respectively, those of the products of the control group without ANXA2 treatment (accompanying figures, Figure 5 ). In the products of the ANXA2 treatment group, microvilli similar to the brush border structure were produced on the luminal side of the proximal renal tubule, peritubular capillaries with perforated structures were generated, mature podocytes with primary and secondary foot processes were developed, and the glomerular filtration membrane structure was produced (accompanying figures, Figure 6 ). At the physiological function level, the products of the ANXA2 treatment group had significantly higher specific amino acid transport activity of proximal tubular epithelial cells (Gamma-glutamyl-transferase, GGT) (accompanying figures, Figure 7 ).

[0145] Through experimental verification, it was found that compared with the products of the control group without ANXA2 treatment, the NOTCH signal in the products of the ANXA2 treatment group was significantly activated and enhanced at the early stage of differentiation (day 7, day 9, day 12), manifested as the upregulation of its activation marker NICD (Notch intracellular domain, NICD), which was generated by the cleavage of the NOTCH receptor and could enter the nucleus to regulate the expression of downstream genes. The NOTCH signal subtype NOTCH2 was considered to play a key role in the development of renal units, and NOTCH4 was considered to be able to regulate vascular development. Their activation markers N2ICD and N4ICD were significantly upregulated in the products of the ANXA2 treatment group (accompanying figures, Figure 8 ). The NOTCH2 and NOTCH4 receptors began to be significantly upregulated at day 9, later than the change of NICD (accompanying figures, Figure 9 ). To further verify whether ANXA2 could enhance the activation of the NOTCH signal, human embryonic kidney cell line 293 cells (with basal NOTCH2 activity) and human umbilical vein endothelial cells HUVEC cells (with basal NOTCH4 activity) were treated with 200 ng / mL human ANXA2 recombinant protein for 72 h respectively. qPCR detection showed that the expression level of the NOTCH2 gene in ANXA2-treated 293 cells was significantly upregulated compared with the control group (accompanying figures, Figure 10 , left); the expression level of the NOTCH4 gene in ANXA2-treated HUVEC cells was significantly upregulated compared with the control group (accompanying figures, Figure 10, right); Western blotting analysis showed that the protein levels of NOTCH2 and N2ICD in ANXA2-treated 293 cells were significantly upregulated compared with the control group (Figure, Figure 11 , left); the protein levels of NOTCH4 and N4ICD in ANXA2-treated HUVEC cells were significantly upregulated compared with the control group (Figure, Figure 11 , right). The above results indicate that ANXA2 can enhance the activation of NOTCH signaling in multiple cell models.

[0146] To clarify the mechanism by which ANXA2 activates NOTCH signaling, co-immunoprecipitation (Co-IP) experiments revealed that in 293 cells, ANXA2 could bind to the NOTCH2 receptor (Figure, Figure 12 , left), and in HUVEC cells, ANXA2 could bind to the NOTCH4 receptor (Figure, Figure 12 , right). Therefore, it is speculated that after ANXA2 binds to the NOTCH receptor, it may promote signal activation by changing its protein conformation. It is known that the activation of NOTCH signaling depends on the conformational change of the protein in the Lin12-NOTCH repeat (LNR) region on the receptor, exposing the originally hidden S2 site, and the exposure of this site is a prerequisite for the generation of NICD and the activation of NOTCH signaling. Therefore, molecular dynamics (MD) simulation was further used to analyze the conformational changes in the LNR region of the NOTCH receptor after ANXA2 binds to the NOTCH receptor. The analysis found that compared with the protein conformation of the NOTCH receptor alone as a control, the solvent accessible surface area (SASA) of the LNR region of the NOTCH2-ANXA2 and NOTCH4-ANXA2 complexes was significantly increased during the entire simulation time period (0-100 ns), indicating that the LNR region had a conformational change (Figure, Figure 13 ). However, there were no obvious changes in the LNR regions of NOTCH1-ANXA2 and NOTCH3-ANXA2 (Figure, Figure 14 ), which is consistent with the result that ANXA2 treatment failed to enhance the activation of NOTCH1 and NOTCH3 signaling in kidney organoids (Figure, Figure 8 , Figure 9 ).

Claims

1. Use of ANXA2 in promoting the induced differentiation of hPSCs into kidney organoids.

2. The application according to claim 1, characterized in that, The concentration of ANXA2 used is 100 - 300 ng / mL; Preferably, the concentration of ANXA2 used is 200 ng / mL; Preferably, the addition time of ANXA2 is Day 6 - 16.

3. Use of ANXA2 in any of the following aspects: (1) Use of ANXA2 in enhancing the maturity and vascularization level of kidney organoids; (2) Use of ANXA2 in enhancing the stability of kidney organoid products obtained among different experimental batches; (3) Use of ANXA2 in enhancing the fate specialization of early differentiated cells into the kidney lineage.

4. The application according to claim 3, wherein The kidney organoids are hPSC - derived kidney organoids; Preferably, the early differentiated cells are hPSC - derived early differentiated cells; Preferably, the concentration of ANXA2 used is 100 - 300 ng / mL; More preferably, the concentration of ANXA2 used is 200 ng / mL; Preferably, the addition time of ANXA2 is Day 6 - 16.

5. A recombinant protein preparation for optimizing kidney organoids derived from hPSCs, characterized in that, The recombinant protein preparation contains ANXA2.

6. The recombinant protein preparation according to claim 5, wherein The concentration of ANXA2 used is 100 - 300 ng / mL; Preferably, the concentration of ANXA2 used is 200 ng / mL.

7. A method for inducing the differentiation of hPSCs into kidney organoids, characterized in that, The method includes the following steps: adding ANXA2 to the hPSC culture system; Preferably, the concentration of ANXA2 used is 100 - 300 ng / mL; More preferably, the concentration of ANXA2 used is 200 ng / mL; Preferably, the addition time of ANXA2 is Day 6 - 16.

8. The method according to claim 7, wherein The method includes the following steps: (1) Day 0: Culturing hPSCs in a medium; (2) Day 1 - 4: Contacting the hPSCs obtained in step (1) with the small molecule CHIR99021 in a medium for 4 days to differentiate them into primitive streak cells; (3) Day 5 - 6: Contacting the primitive streak cells obtained in step (2) with FGF9 and heparin in a medium together for 2 days to differentiate them into intermediate mesoderm cells; (4) Day 6: Contacting the cells obtained in step (3) with FGF9, heparin, and ANXA2 in a medium together for 1 day; (5) Day 7: Digesting the cells obtained in step (4) into single cells, preparing the cells into cell clusters by centrifugation, contacting the cells with CHIR99021 in a medium for 2 h, and then contacting the cells with FGF9, heparin, and ANXA2 in a medium together for another 5 days to sequentially differentiate them into posterior kidney mesenchymal cells, nephron progenitor cells, and nephrons; (6) Day 12 - 16: Contacting the cells obtained in step (5) with ANXA2 in a medium together for 4 days to generate more nephron structures inside the cell clusters; (7) Day 17 - 24: Continuing to culture the cells obtained in step (6) in a medium to obtain kidney organoids.

9. The method according to claim 8, wherein The medium described in step (1) is the hPSC - specific medium PGM1; Preferably, the conditions for the culture in step (1) are changing the medium every day until the cell confluence reaches 50%; Preferably, the medium in step (2) is APEL2 medium; Preferably, the concentration of CHIR99021 in step (2) is 1 - 20 μM; More preferably, the concentration of CHIR99021 in step (2) is 10 μM; Preferably, the conditions for the culture in step (2) are changing the medium once every two days; Preferably, the medium in step (3) is APEL2 medium; Preferably, the concentration of FGF9 in step (3) is 100 - 300 ng / mL; More preferably, the concentration of FGF9 in step (3) is 200 ng / mL; Preferably, the concentration of heparin in step (3) is 0.1 - 5 μg / mL; More preferably, the concentration of heparin in step (3) is 1 μg / mL; Preferably, the conditions for the culture in step (3) are changing the medium once every two days; Preferably, the medium in step (4) is APEL2 medium; Preferably, the concentration of FGF9 in step (4) is 100 - 300 ng / mL; More preferably, the concentration of FGF9 in step (4) is 200 ng / mL; Preferably, the concentration of heparin in step (4) is 0.1 - 5 μg / mL; More preferably, the concentration of heparin in step (4) is 1 μg / mL; Preferably, the concentration of ANXA2 used in step (4) is 100 - 300 ng / mL; More preferably, the concentration of ANXA2 used in step (4) is 200 ng / mL.

10. The method according to claim 8, wherein In step (5), each cell mass contains approximately (1 - 10) × 10 5 cells; Preferably, each cell mass in step (5) contains approximately 5×10 5 cells; Preferably, the medium in step (5) is APEL2 medium; Preferably, the concentration of CHIR99021 in step (5) is 1 - 20 μM; More preferably, the concentration of CHIR99021 in step (5) is 10 μM; Preferably, the concentration of FGF9 in step (5) is 100 - 300 ng / mL; More preferably, the concentration of FGF9 in step (5) is 200 ng / mL; Preferably, the concentration of heparin in step (5) is 0.1 - 5 μg / mL; More preferably, the concentration of heparin in step (5) is 1 μg / mL; Preferably, the concentration of ANXA2 used in step (5) is 100 - 300 ng / mL; More preferably, the concentration of ANXA2 used in step (5) is 200 ng / mL; Preferably, the conditions for the culture in step (5) are changing the medium once every two days; Preferably, the medium in step (6) is APEL2 medium; Preferably, the concentration of ANXA2 used in step (6) is 100 - 300 ng / mL; More preferably, the concentration of ANXA2 used in step (6) is 200 ng / mL; Preferably, the conditions for the culture in step (6) are changing the medium once every two days; Preferably, the medium in step (7) is APEL2 medium; Preferably, the condition for the culture in step (7) is to change the culture medium every two days.

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