A recombinant protein formulation to optimize human pluripotent stem cell-derived kidney organoids

Abstract

The application discloses a kind of recombinant protein preparations of optimizing human pluripotent stem cell-derived kidney organoids, the recombinant protein preparation includes ANXA2, the present application first discovers that ANXA2 can significantly improve early differentiation cell to the fate specialization of kidney lineage, significantly improve the stability of kidney organoid product obtained between different experimental batches, significantly improve the overall vascularization level and maturity of differentiation end-stage kidney organoid product, provide new ideas and strategies for the construction of human pluripotent stem cell-derived kidney organoids, with wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and in particular, relates to a recombinant protein preparation for optimizing human pluripotent stem cell-derived kidney organoids. BACKGROUND

[0002] Currently, there is no effective treatment for chronic kidney disease (CKD) except dialysis and kidney transplantation. However, dialysis can only delay disease progression, and the only cure, kidney transplantation, is faced with the current situation of organ donor shortage, and it is urgent to find new effective treatment options. Studying the pathogenesis of CKD helps to find new potential therapeutic targets, and the first requirement 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 in vivo kidneys in terms of gene expression profiles, physiological functions, or physiological structures. Animal models also cannot truly simulate the human body due to interspecies differences, so there is an urgent need for an in vitro model that can more realistically simulate the kidney organ in the human body.

[0003] Kidney organoids are obtained by simulating in vivo kidney organ development using human pluripotent stem cells (hPSCs) with multiple potentials and then differentiating them in vitro. They not only have a human genetic background, which can avoid interspecies differences in animal models, but also can produce complex three-dimensional structures and functions similar to corresponding organs in vivo, which single-type cell lines or primary cells cannot achieve. Therefore, kidney organoids are an ideal in vitro model for simulating kidney injury disease phenotypes, screening therapeutic drugs, and predicting potential drug responses in humans. The current bottleneck problems in the field of kidney organoids include: lack of overall maturity, insufficient vascularization level, and insufficient batch-to-batch stability, which may be largely due to the lack of key microenvironments and signaling pathways during in vitro differentiation. Therefore, exploring and supplementing the missing key factors may solve the above bottleneck problems. The work reported in the field has attempted to solve the above problems from different angles:

[0004] (1) There are reports that the maturation and vascularization of kidney organoids can be improved by transplanting kidney organoids into healthy living hosts. For example, kidney organoids were transplanted under the kidney capsule of immunodeficient mice, and the grafts showed podocyte maturation and connection with 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, multiple blood vessels, and blood circulation. However, further testing of these transplanted kidney organoids found that most of the vascular endothelial cells in the glomeruli came from the host animals, not from the grafts. This phenomenon suggests a potential problem with the animal in vivo transplantation method: the development of cells in kidney organoids depends on cell-cell communication, such as the interaction between podocytes and vascular endothelial cells to promote further maturation of each other. If the endothelial cells in the graft mainly come from the host animal, considering the differences between species, the cell-cell communication does not truly reflect kidney development in the human body, so the further development of the graft may be biased under the wrong signal. In addition, this technical method relies on animal in vivo transplantation, which is complex, costly, and not suitable for high-throughput applications.

[0005] (2) There are reports that microfluidic technology can significantly improve the maturation and vascularization of kidney organoids cultured in vitro. Microfluidics is a microfluidic manipulation technology. Because 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 in a physiological state. Microfluidics can simulate the in vivo microenvironment to some extent, such as providing fluid shear force, which is an important factor for kidney organoid differentiation that is lacking in static culture conditions. A study demonstrated that culturing kidney organoids under flow conditions in a microfluidic device induced the formation of perfusable vascular networks within the organoids, supporting the role of fluid flow in inducing vascularization, and the level of vascularization was positively correlated with fluid velocity. Kidney unit cells, including podocytes and renal tubular epithelial cells, cultured in this system expressed higher levels of lineage markers than kidney unit cells cultured under conventional static conditions, which may be due to the communication and interaction between them and the horizontally elevated vascular endothelial cells to further mature. This study suggests that microfluidics has a significant effect on the in vitro maturation of kidney organoids. The disadvantage of this technical method is that the reliance on microfluidic devices makes it difficult to repeat and promote due to high technical threshold and cost.

[0006] (3) There is a study using a technology based on "extrusion three-dimensional cell bioprinting" to manufacture kidney organoids with high throughput and high repeatability. When manufacturing organoids for drug research and development in vitro, reliability and repeatability are crucial. To achieve this, methods for stably obtaining organoid products with small individual differences between different hPSC cell lines and different experimental batches need to be explored. A study has shown that by using the "extrusion three-dimensional cell bioprinting" technology, the repeatability of kidney organoid products can be improved, significantly increasing the throughput while reducing the differences in cell number, diameter and cell activity between products. The disadvantage of this technology is that the dependence on 3D printing devices increases the technical threshold and manufacturing cost of kidney organoids. SUMMARY

[0007] In view of the technical problems existing in the prior art, the purpose of the present application is to provide a recombinant protein preparation for optimizing human pluripotent stem cell-derived kidney organoids, which is a human ANXA2 recombinant protein, and can significantly improve the fate specialization of early differentiated cells to the kidney lineage, significantly improve the stability of obtaining kidney organoid products between different experimental batches, and significantly improve the overall vascularization level and maturity of the differentiated end-stage kidney organoid product.

[0008] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] The first aspect of the present application provides the use of ANXA2 in promoting the induction and differentiation of hPSCs to kidney organoids.

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

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

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

[0013] In the present application, the ANXA2 is annexin A2 (ANXA2), a calcium ion-mediated phospholipid-binding protein that 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 cell exocytosis, endocytosis, migration and proliferation, etc. As a receptor for plasminogen and tissue plasminogen activator, ANXA2 can promote the production of plasmin and make the homeostasis of blood coagulation, fibrinolysis and matrix degradation; as an antigen expressed on the cell membrane, ANXA2 can induce local inflammation and injury by binding to autoantibodies.

[0014] In some embodiments, the ANXA2 described in the present application can be obtained from any commercial source or prepared by biosynthesis methods well known to those skilled in the art, without being limited to the human recombinant protein ANXA2 from Wuhan Yunkelone Technology Co., Ltd. described in the specific embodiments of the present application. In preferred embodiments, the ANXA2 described in the present application is a human recombinant protein ANXA2.

[0015] In some embodiments, the use concentration of the ANXA2 described in the present application is not particularly limited, and any use concentration of ANXA2 that can promote the induction and differentiation of hPSCs into kidney organoids, improve the maturation 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 to the kidney lineage is within the scope of the present application. In preferred embodiments, the use concentration of the ANXA2 described in the present application is 100-300 ng / mL, and in more preferred embodiments, the use concentration of the ANXA2 described in the present application is 200 ng / mL.

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

[0017] (1) the use of ANXA2 in improving the maturation and vascularization level of kidney organoids;

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

[0019] (3) the use of ANXA2 in improving the fate specialization of early differentiated cells to the kidney lineage.

[0020] Further, the kidney organoid is a kidney organoid derived from hPSCs;

[0021] Preferably, the early differentiated cells are early differentiated cells derived from hPSCs;

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

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

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

[0025] In specific embodiments, the present application is demonstrated by experiments that the addition of 200 ng / mL human recombinant protein ANXA2 in the time period of day 6 to day 16 can significantly improve the fate specification of early differentiated cells (day 9) to kidney lineage, significantly improve the stability of kidney organoid products obtained between different experimental batches, and significantly improve the overall vascularization level and maturity of kidney organoid products at the end of differentiation (day 24). Therefore, the use of the ANXA2 in the above-mentioned second aspect will also fall within the protection scope of the present application.

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

[0027] Further, the recombinant protein preparation comprises ANXA2.

[0028] Further, the use concentration of the ANXA2 is 100-300 ng / mL.

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

[0030] In some embodiments, the recombinant protein preparation comprises ANXA2, and in other embodiments, the recombinant protein preparation can further comprise other reagents that can be used for optimizing hPSC-derived kidney organoids or reagents that assist ANXA2 in optimizing hPSC-derived kidney organoids.

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

[0032] Further, the method comprises the following step: adding ANXA2 to the hPSC culture system;

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

[0034] More preferably, the use concentration of the ANXA2 is 200 ng / mL.

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

[0036] Further, the method comprises 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 small molecule CHIR99021 in a culture medium for 4 days to differentiate them to primitive streak cells;

[0039] (3) Day 5-6: the primary streak cells obtained in step (2) are contacted with FGF9, heparin in the culture medium for 2 days to differentiate into mesendoderm cells;

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

[0041] (5) Day 7: the cells obtained in step (4) are digested into single cells, and the cells are prepared into cell clusters by centrifugation, contacted with CHIR99021 in the culture medium for 2h, and then the cells are contacted with FGF9, heparin, ANXA2 in the culture medium for 5 days to sequentially differentiate into metanephric mesenchymal cells, nephron precursor cells, and nephron units;

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

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

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

[0045] Preferably, the culture condition in step (1) is to change the medium every day until the cell confluence reaches 50%;

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

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

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

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

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

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

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

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

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

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

[0056] Preferably, the 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 pg / mL.

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

[0061] 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. Further, the number of cells in each cell cluster in step (5) is about (1-10) x 10 5 cells.

[0062] Preferably, the number of cells in each cell cluster in step (5) is about 5 x 10 5 cells.

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

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

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

[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 pg / mL.

[0069] More preferably, the concentration of heparin in step (5) is 1 pg / 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 medium every two days.

[0073] Preferably, the 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 medium every two days.

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

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

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

[0080] (1) In combination with the fields of developmental biology, gene editing, etc., a kidney disease research model is established by using the induction and differentiation of the kidney organoid.

[0081] (2) In combination with the fields of precision medicine and pharmacy, the kidney organoid is used in vitro for the testing of drug nephrotoxicity and drug effectiveness.

[0082] (3) In combination with the fields of regenerative medicine and precision medicine, the kidney organoid is used as a graft to replace the diseased kidney tissue.

[0083] Further, the kidney disease research model can be used in the research of key pathogenic genes of kidney diseases, such as the research of key pathogenic genes of polycystic kidney disease, etc.

[0084] Compared with the prior art, the application has the following advantages and beneficial effects:

[0085] Currently, there are two methods in the art that can improve the maturity and vascularization level of kidney organoids, one is to transplant the kidney organoids under the kidney capsule of mice or into chicken embryos, and to promote the further maturity of the kidney organoids by the in vivo vascular network of the host; the other is to use a microfluidic control system in vitro to promote the generation of microvessels inside the kidney organoids by using liquid shear force, and thereby improve the overall maturity. The above two methods are complex in operation, high in cost, and have certain technical threshold, especially not suitable for high-throughput preparation of functionally mature kidney organoids. The recombinant protein preparation provided by the present application has the greatest advantage that it is a single peptide segment with a determined composition, and there are commercialized recombinant proteins available. Only a fixed concentration of the preparation needs to be added to the existing in vitro differentiation scheme of the kidney organoids at a fixed time period, and incubated with the cells in the culture medium, without the need for additional complex instruments or operation methods to significantly improve the maturity and vascularization level. The operation is simple, the cost is reduced, and it is suitable for future large-scale obtaining of kidney organoids and is suitable for high-throughput application scenarios such as drug screening. BRIEF DESCRIPTION OF DRAWINGS

[0086] Figure 1 A schematic diagram of using human ANXA2 recombinant protein treatment in hPSC differentiation into kidney organoids;

[0087] Figure 2 The effect of human ANXA2 recombinant protein treatment on kidney organoid products, wherein the upper panel: immunofluorescence staining detects kidney vesicles (PAX8 + ), proximal tubular epithelial cells (LTL + ), podocyte precursor cells (WT1 + ), vascular endothelial cells (CD34 + ), scale, 50 pm. ANX-Orgs, kidney organoids treated with 200 ng / mL human ANXA2 recombinant protein during differentiation. The lower panel: Quantitative statistics of the number of pronephric aggregates, kidney vesicles, podocyte precursor cells and the length of vascular endothelial cells in kidney organoid products based on day 9 immunofluorescence results. Data from 1 independent experiment mean values ± SEM (replicates = 5, one randomly selected photo represents one replicate), statistical method is unpaired t-test, ns, P>0.05; *, P<0.05; ***, P<0.001. PTAs, pronephric aggregates; RVs, kidney vesicles; Ctrl-Orgs, kidney organoid products without ANXA2 treatment; ANX-Orgs, kidney organoid products treated with 200 ng / mL human ANXA2 recombinant protein during differentiation;

[0088] Figure 3 To label and quantify tubular structures in day 12 kidney organoids, Ctrl-Orgs, kidney organoid products without ANXA2 treatment; ANX-Orgs, kidney organoid products treated with 200 ng / mL human ANXA2 recombinant protein during differentiation, where, upper panel: labeling tubular structures in day 12 kidney organoids. Dark red represents tubular structures, green represents non-tubular structures. Scale bar, 30 pm. Lower panel: quantification of the proportion of tubular structure area in day 12 kidney organoids. Data from 3 independent experiments, statistical method 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 treated with human ANXA2 recombinant protein (day 24), scale bar, 200 pm;

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

[0091] Figure 6 For transmission electron microscopy to detect proximal tubular microvilli (blue arrow), peritubular capillary with perforated structure (orange arrow, fc), podocytes with primary foot process (green arrow, pp) and secondary foot process (red arrow, sp), and basement membrane structure (yellow arrow, bm) in kidney organoid products treated with human ANXA2 recombinant protein (day 24);

[0092] Figure 7 For GGT assay to detect GGT activity of kidney organoids, Ctrl-Orgs, kidney organoid products without ANXA2 treatment; ANX-Orgs, kidney organoid products treated with 200 ng / mL human ANXA2 recombinant protein during differentiation. Human proximal tubular cell line HK2 as positive control. Data from 1 independent experiment, mean values ± SD (replicates = 4), statistical method unpaired t-test, **, P < 0.01;

[0093] Figure 8 Figure 21. Western blot and quantification of NICDs (N1 ICD, N2 ICD, N4 ICD) protein levels in kidney organoids products at different time points (day 7, day 9, day 12). Ctrl-Orgs, kidney organoids products without ANXA2 treatment; ANX-Orgs, kidney organoids products treated with 200 ng / mL human ANXA2 recombinant protein during differentiation. Upper panel: Western blot detection of NICDs protein levels in kidney organoids products (replicates = 3). Lower panel: Quantification of NICDs protein levels in kidney organoids products. Data are from mean values ± SD of at least 2 independent experiments, statistical method unpaired t-test, ns, P > 0.05; *, P < 0.05; **, P < 0.01;

[0094] Figure 9 Figure 22. Western blot and quantification of NOTCH receptors (NOTCH1-4) protein levels in kidney organoids products at different time points (day 7, day 9, day 12). Ctrl-Orgs, kidney organoids products without ANXA2 treatment; ANX-Orgs, kidney organoids products treated with 200 ng / mL human ANXA2 recombinant protein during differentiation. Upper panel: Western blot detection of NOTCH receptors protein levels in kidney organoids products (replicates = 3). Lower panel: Quantification of NOTCH receptors protein levels in kidney organoids products. Data are from mean values ± SD of at least 2 independent experiments, statistical method unpaired t-test, ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Figure 10Figure 6. qPCR detection of NOTCH2 or NOTCH4 expression levels in 293 cells or HUVEC cells under different conditions. Left panel: qPCR detection of NOTCH2 expression levels in 293 cells under different conditions. Data are mean values ± SD (replicates = 4) from 2 independent experiments, unpaired t-test, **, P < 0.01. Control, 293 cells without ANXA2 treatment; ANXA2, 293 cells treated with 200 ng / mL human ANXA2 recombinant protein for 72 h. Right panel: qPCR detection of NOTCH4 expression levels in HUVEC cells under different conditions. Data are mean values ± SD (replicates = 4) from 2 independent experiments, unpaired t-test, *, P < 0.05. Control, HUVEC cells without ANXA2 treatment; ANXA2, HUVEC cells treated with 200 ng / mL human ANXA2 recombinant protein for 72 h.

[0095] Figure 11 Figure 7. Western blot detection and quantification of NOTCH2 and N2ICD, or NOTCH4 and N4ICD protein levels in 293 cells or HUVEC cells under different conditions. Left panel: Western blot detection and quantification of NOTCH2 and N2ICD protein levels in 293 cells under different conditions. Data are mean values ± SD (replicates = 3) from 5 independent experiments, unpaired t-test, *, P < 0.05. Control, 293 cells without ANXA2 treatment; ANXA2, 293 cells treated with 200 ng / mL human ANXA2 recombinant protein for 72 h. Right panel: Western blot detection and quantification of NOTCH4 and N4ICD protein levels in HUVEC cells under different conditions. Data are mean values ± SD (replicates = 3) from 4 independent experiments, unpaired t-test, **, P < 0.01. Control, HUVEC cells without ANXA2 treatment; ANXA2, HUVEC cells treated with 200 ng / mL human ANXA2 recombinant protein for 72 h.

[0096] Figure 12Figure 6 shows the binding of NOTCH2 and NOTCH4 to ANXA2 in 293 cells and HUVEC cells treated with human ANXA2 recombinant protein for 72 h. Left panel: Co-IP analysis of the binding of NOTCH2 to ANXA2 in 293 cells treated with human ANXA2 recombinant protein for 72 h. Right panel: Co-IP analysis of the binding of NOTCH4 to ANXA2 in HUVEC cells treated with human ANXA2 recombinant protein for 72 h;

[0097] Figure 13 Figure 8 shows the changes in the solvent accessible surface area of the LNR region of NOTCH2 and NOTCH4 after binding to ANXA2, and the position changes of the LNR region of NOTCH receptors at 0 ns and 100 ns after ANXA2 binds to NOTCH2 or NOTCH4. Upper panel: Changes in the solvent accessible surface area of the LNR region of NOTCH2 and NOTCH4 after binding to ANXA2. SASA, solvent accessible surface area; Control, changes in the solvent accessible surface area of the LNR region of NOTCH2 or NOTCH4 protein; NOTCH2-ANXA2, changes in the solvent accessible surface area of the LNR region of NOTCH2 protein in the complex of NOTCH2 and ANXA2; NOTCH4-ANXA2, changes in the solvent accessible surface area of the LNR region of NOTCH4 protein in the complex of NOTCH4 and ANXA2. Lower panel: Position changes of the LNR region of NOTCH receptors at 0 ns and 100 ns after ANXA2 binds to NOTCH2 or NOTCH4;

[0098] Figure 14Figure 2 shows the changes in the solvent accessible surface area of the LNR region of NOTCH1 and NOTCH3 after binding to ANXA2, as well as the changes in the position of the LNR region of the NOTCH receptor at 0 ns and 100 ns after ANXA2 binds to NOTCH1 or NOTCH3. The upper panel shows the changes in the solvent accessible surface area of the LNR region of NOTCH1 and NOTCH3 after binding to ANXA2. SASA, solvent accessible surface area; Control, changes in the solvent accessible surface area of the LNR region of NOTCH1 or NOTCH3 protein; NOTCH1-ANXA2, changes in the solvent accessible surface area of the LNR region of NOTCH1 protein in the complex of NOTCH1 and ANXA2; NOTCH3-ANXA2, changes in the solvent accessible surface area of the LNR region of NOTCH3 protein in the complex of NOTCH3 and ANXA2. The lower panel shows the changes in the position of the LNR region of the NOTCH receptor at 0 ns and 100 ns after ANXA2 binds to NOTCH1 or NOTCH3. DETAILED DESCRIPTION

[0099] The present application will be further described below in conjunction with specific examples, which are intended to explain the present application, but should not be construed as limiting the present application. Those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these examples without departing from the principles and spirit of the present application, and the scope of the present application is defined by the claims and their equivalents. The experimental methods in the following examples, unless otherwise specified, are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer. The reagents, biological materials, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0100] Example 1: Application of human ANXA2 recombinant protein in promoting the induction and differentiation of hPSC into kidney organoids and related mechanism research

[0101] 1. Experimental materials

[0102] (1) The culture medium is as follows:

[0103] PGM1: Cellapy, CA1007500;

[0104] APEL2: STEMCELL Technologies, 05270.

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

[0106] The attribute is a human recombinant protein, which is purchased from Wuhan Yunclone Technology Co., Ltd.

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

[0108] 2. Experimental method

[0109] (1) In vitro induction method of hPSC into kidney organoids

[0110] ① Day 0: hPSCs were cultured in cell culture dishes using human pluripotent stem cell (hPSC)-specific medium PGM1, with daily medium replacement until the cell confluence reached 50%;

[0111] ② Day 1-4: hPSCs were contacted with small molecule CHIR99021 (10 μM) in APEL2 for 4 days to differentiate them into primitive streak cells, with medium replacement every two days;

[0112] ③ Day 5-6: primitive streak cells were contacted with FGF9 (200 ng / mL) and heparin (1 μg / mL) in APEL2 for 2 days to differentiate them into mesendoderm cells, with medium replacement every two days;

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

[0114] ⑤ Day 7: cells were digested into single cells, prepared into cell clusters (about 5 x 10 5 cells per cell cluster) by centrifugation, and transferred to the upper filter membrane of a Transwell. Cells were contacted with CHIR99021 (10 μM) in APEL2 for 2 hours. After 2 hours, cells were re-contacted with FGF9 (200 ng / mL), heparin (1 μg / mL), and annexin A2 (200 ng / mL) in APEL2 for 5 days to sequentially differentiate them into metanephric mesenchymal cells, nephron precursor cells, and nephron units, with medium replacement every two days;

[0115] ⑥ Day 12-16: the above cells were continued to be contacted with annexin A2 (200 ng / mL) in APEL2 for 4 days to generate more nephron unit structures inside the cell clusters, with medium replacement every two days;

[0116] ⑦ Day 17-24: the above cells were continued to be cultured in APEL2, with medium replacement every two days. The entire kidney organoid differentiation cycle was 24 days.

[0117] (2) Detection method

[0118] 1. qRT-PCR experiment

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

[0120] 2. Immunofluorescence staining

[0121] Fresh samples were incubated in 4% paraformaldehyde in PBS for 30 min, embedded in Tissue-Tek O.C.T. (ThermoFisher) and flash-frozen in liquid nitrogen. Cryosections were made using a cryostat (Leica). Other steps were similar to paraffin sections, except that no antigen retrieval was performed. Primary antibodies included LTL-biotin conjugated (1:300, Vector Laboratories, B-1325), PECAM1 (1:200, Abeam, ab9498), WT1 (1:200, Abeam, ab89901), CD34 (1:200, Abeam, ab81289), PAX8 (1:200, CST, 59019S), incubated at 4°C overnight, washed with PBS for 3 times, and then incubated with the corresponding secondary antibodies at 37°C for 2 hours. After washing with PBS for 3 times, DAPI was used for staining at 1:10000 for 15 min. After washing with PBS for 3 times, immunofluorescence imaging was performed using Zeiss (LSM 780) and Leica confocal microscopes.

[0122] 3. Western blot

[0123] The kidney tissue and other cell samples were transferred to 1.5 mL tubes, washed twice with PBS, and then placed in lysis buffer (RIPA, P0013B) containing protease and phosphatase inhibitors (1:100, NCM Biotech, P002) for four times for 30 minutes, with vibration every 5 minutes. The lysate was centrifuged at 13000g for 30 minutes at 4°C, and the supernatant was transferred to a new 1.5 mL tube. The total protein concentration was determined using the BCA protein assay kit (Beyotime, P0010) according to the instructions. The protein lysate was diluted 1:5 with Beyotime, P0015L buffer and boiled at 100°C for 10 minutes. A total of 30 μg of denatured protein per sample was subjected to 4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a 0.45 μm PVDF membrane. The cell membrane was blocked with 5% skim milk solution at room temperature for 1 hour, and the immunoblot was incubated with primary antibodies against NOTCH1 (1:2,000, Proteintch, 20687-1-AP), NOTCH2 (1:2,000, Proteintch, 28580-1-AP), NOTCH3 (1:2,000, Abeam, 23426), NOTCH4 (1:2,000, Abeam, AB184742), N1ICD (1:200, R&D, AF3647), N2ICD (1:200, R&D, AF3735), N4ICD (1:200, R&D, AF3847). Incubate overnight at 4°C, and incubate with HRP-conjugated secondary antibodies (1:5,000, CST, 5127S; CST, 7074; AbClone, AS014) for 1 hour. Specific bands were detected by enhanced chemiluminescence (ECL) method, and measured by Image J software (NIH, Bethesda, MD, United States). The level of β-actin was used as an internal standard.

[0124] IV. Flow cytometry

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

[0126] (5) GGT activity experiment

[0127] GGT activity was detected with GGT Activity Assay Kit (Biovision, K784-100). The organoid samples were homogenized in 200 pL of GGT Assay Buffer and centrifuged (13000g, 10 minutes) and the insoluble material was removed. Then 10 pL / well of test sample was prepared in a 96-well plate with GGT Assay Buffer, and 90 pL of GGT Substrate Mixture was added to each well, which contained the test sample and positive control. For the PNA standard curve, 2 mM PNA standard solution was added to different wells of a 96-well plate in a volume of 0, 4, 8, 12, 16, 20 pL respectively, in duplicate, generating standard solutions of 0, 8, 16, 24, 32, 40 nM / well, and the final volume was adjusted to 100 pL with GGT Assay Buffer. For the measurement of the PNA standard curve, the OD was measured at 418 nm with a microplate reader. For the samples and positive control, the mixture was incubated at 37°C for 3 minutes, then the OD was measured at 418 nm with a microplate reader, and incubated at 37°C for 30 minutes to 2 hours, and the OD was measured again at 418 nm (A1); the incubation time will depend on the activity of GGT in the sample. When calculating, first draw the PNA standard curve, then calculate the GGT activity of the test sample: AOD = A1-A0, add AOD to the PNA standard curve to get the B nmol value of PNA produced by GGT in a given time. The formula used is as follows:

[0128]

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

[0130] 6. Scanning electron microscopy

[0131] The 1.0 mm 3 The kidney organoid samples were washed 3 times in 0.1 M acetate buffer overnight, then fixed in 1% cesium trioxide solution for 2 hours, and dehydrated in 30, 50, 70, 80, 90 and 100% acetone solutions. The samples were cut into 50 nm sections using an ultramicrotome (Leica EMUC7, Wetzlar, Germany), and observed under a transmission electron microscope (JEM-1400) after being treated with 2% uranyl acetate for 10 minutes and lead citrate for 5 minutes, respectively.

[0132] 7. Co-immunoprecipitation

[0133] The co-immunoprecipitation (Co-IP) kit (Thermo Science Piells, 26149) was used according to the manufacturer's instructions. 293 cells and human umbilical vein endothelial cells were cultured in 60 mm dishes, about 1 mg of cell lysate was incubated with 10 pg of AnxA2 antibody (Proteintech, 66035-1-Ig) fixed amino linkage resin at 4°C overnight, and mouse antibody (Beyotime, A7028) was used as a negative control. After washing, the isolated immune complexes were subjected to Western blot analysis as described above.

[0134] 8. Molecular dynamics simulation

[0135] Molecular dynamics simulation was 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 file for simulation. The solvated complex was immersed in a cubic periodic boundary box using a 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, standard ensemble (NVT) and constant pressure ensemble (NPT) simulations were performed to maintain constant temperature (298 K) and constant pressure (1 bar). The equilibrated system was subjected to 100 ns of molecular dynamics simulation.

[0136] 9. Solvent accessible surface area analysis

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

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

[0139] The (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 solvent accessible surface area (SASA).

[0140] 3. Experimental results

[0141] According to the existing literature reports, the in vitro induction of hPSC to kidney organoids needs 24 days, the inventors of the present application found that adding 200 ng / mL human ANXA2 recombinant protein (attached figure, Figure 1 ) in the time period of day 6 to day 16 has the following effects:

[0142] (1) Significantly improves the fate specialization of early differentiated cells (day 9) to kidney lineage. PAX8 + kidney vesicles, the number of which is 10 times higher than that of the control product without ANXA2 treatment (P<0.001), and which has initiated proximal tubule specialization (LTL + ); the number of podocyte precursor cells (WT1 + ) in the ANXA2 treatment product is 2.1 times higher than that of the control product without ANXA2 treatment (P<0.001); primary capillary network (CD34 + ) is generated in the ANXA2 treatment product, and the length of microvessels is 9.5 times higher than that of the control product without ANXA2 treatment (P<0.001) (attached figure, Figure 2 ).

[0143] (2) Significantly improves the stability of kidney organoid products obtained between different experimental batches. Using the proportion of tubular structure in organoids as the evaluation standard, 3 independent experiments were conducted, and the proportion of tubular structure in the ANXA2 treatment product was 50.0%, while that in the control product without ANXA2 treatment was 27.1% (P<0.01) (attached figure, Figure 3 ); the coefficients of variation of the ANXA2 treatment product and the control product without ANXA2 treatment were 2.1% and 29.6%, respectively.

[0144] (3) significantly improved the overall vascularization level and maturation degree of kidney organoid products at the end of differentiation (day 24). The number of proximal tubular epithelial cells (LTL + ), podocytes (WT1 + ) and vascular endothelial cells (PECAM + ) (Fig. Figure 4 ), were 1.8 times, 4.5 times and 2.0 times (Fig. Figure 5 ) of the control group products without ANXA2 treatment, respectively. In the ANXA2 treatment group products, microvilli similar to brush border structures were generated on the side of the proximal tubular lumen, peritubular capillary blood vessels with perforated structures were generated, mature podocytes with primary and secondary foot processes were developed, and glomerular filtration membrane structures were generated (Fig. Figure 6 ). At the physiological function level, the ANXA2 treatment group products had significantly higher amino acid transport activity (Gamma-glutamyl-transferase, GGT) specific to proximal tubular epithelial cells (Fig. Figure 7 ).

[0145] It was found through experiments that compared with the control group products without ANXA2 treatment, the NOTCH signal in the ANXA2 treatment group products was significantly activated and enhanced at the early stage of differentiation (day 7, day 9, day 12), which was manifested by the up-regulation of its activation marker NICD (Notch intracellular domain, NICD), which was produced by the cleavage of the NOTCH receptor and could enter the nucleus to regulate the expression of downstream genes. NOTCH signal subtype NOTCH2 is believed to play a key role in the development of renal units, and NOTCH4 is believed to be able to regulate vascular development. Their activation markers N2ICD and N4ICD were significantly up-regulated in the ANXA2 treatment group products (Fig. Figure 8 ). While the NOTCH2 and NOTCH4 receptors were significantly up-regulated starting at day 9, which was later than the change of NICD (Fig. Figure 9 ). To further verify whether ANXA2 can enhance the activation of NOTCH signal, human embryonic kidney cell line 293 cells (with NOTCH2 background activity) and human umbilical cord vein endothelial cells HUVEC cells (with NOTCH4 background activity) were treated with 200 ng / mL human ANXA2 recombinant protein for 72 h, respectively. qPCR detection showed that the expression amount of NOTCH2 gene in ANXA2-treated 293 cells was significantly up-regulated relative to the control group (Fig. Figure 10 , left); the expression amount of NOTCH4 gene in ANXA2-treated HUVEC cells was significantly up-regulated relative to the control group (Fig. Figure 10, right); Western blotting showed that the protein levels of NOTCH2 and N2ICD were significantly upregulated in ANXA2-treated 293 cells relative to the control (Figure 2, Figure 11 , left); the protein levels of NOTCH4 and N4ICD were significantly upregulated in ANXA2-treated HUVEC cells relative to the control (Figure 2, Figure 11 , right). These results indicated that ANXA2 could enhance the activation of NOTCH signaling in multiple cell models.

[0146] To elucidate the mechanism of ANXA2 activating NOTCH signaling, we found that ANXA2 could bind to NOTCH2 receptor in 293 cells (Figure 2, Figure 12 , left) and to NOTCH4 receptor in HUVEC cells (Figure 2, Figure 12 , right) by co-immunoprecipitation (Co-IP) experiments. It was thus speculated that ANXA2 might change the protein conformation of NOTCH receptors after binding to them, thereby facilitating the activation of signaling. It is known that the activation of NOTCH signaling depends on the change in the protein conformation of the Lin12-NOTCH repeat (LNR) region of the receptor, which exposes 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, we further used molecular dynamics (MD) simulation to analyze the change in the protein conformation of the LNR region of the receptor after the binding of ANXA2 to the NOTCH receptor. Analysis found that the solvent accessible surface area (SASA) of the LNR region of the NOTCH2-ANXA2 and NOTCH4-ANXA2 complex was significantly increased throughout the simulation time period (0-100 ns) compared with the protein conformation of the NOTCH receptor alone as a control, suggesting that the LNR region had undergone a conformational change (Figure 2, Figure 13 ). However, the LNR region of NOTCH1-ANXA2 and NOTCH3-ANXA2 did not undergo significant changes (Figure 2, Figure 14 ), which was consistent with the results that ANXA2 treatment failed to enhance the activation of NOTCH1 and NOTCH3 signaling in kidney organoids (Figure 2, Figure 8 , Figure 9 ).

Claims

1. Use of a human recombinant protein ANXA2 in promoting the induced differentiation of hPSCs into kidney organoids, characterized in that, The application comprises the following steps: (1) Day 0: culturing hPSCs in a culture medium; (2) Day 1-4: contacting the hPSCs obtained in step (1) with a small molecule CHIR99021 in a culture 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 culture medium for 2 days to differentiate them into mesendoderm cells; (4) Day 6: contacting the cells obtained in step (3) with FGF9, heparin and ANXA2 in a culture medium 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 cell clusters with CHIR99021 in a culture medium for 2 h, and then contacting the cell clusters with FGF9, heparin and ANXA2 in a culture medium for 5 days to sequentially differentiate them into metanephric mesenchymal cells, nephron precursor cells and nephrons; (6) Day 12-16: contacting the cells obtained in step (5) with ANXA2 in a culture medium 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 culture medium to obtain kidney organoids; The concentration of ANXA2 used in steps (4), (5) and (6) is 100-300 ng / mL.

2. Use according to claim 1, characterized in that, The concentration of ANXA2 used is 200 ng / mL.

3. The application according to claim 1, wherein the ANXA2 improves the maturation degree and vascularization level of the kidney organoids in the in vitro induced differentiation process of the hPSCs into the kidney organoids.

4. The application according to claim 1, wherein the ANXA2 improves the stability of the kidney organoid products obtained in different experimental batches in the in vitro induced differentiation process of the hPSCs into the kidney organoids.

5. The application according to claim 1, wherein the ANXA2 improves the fate specification of early differentiated cells to the kidney lineage in the in vitro induced differentiation process of the hPSCs into the kidney organoids. The early differentiated cells are early differentiated cells derived from the hPSCs.

6. A method of inducing differentiation of hPSCs into kidney organoids, characterized in that, The method comprises the following steps: (1) Day 0: culturing hPSCs in a culture medium; (2) Day 1-4: contacting the hPSCs obtained in step (1) with a small molecule CHIR99021 in a culture 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 culture medium for 2 days to differentiate them into mesendoderm cells; (4) Day 6: contacting the cells obtained in step (3) with FGF9, heparin and ANXA2 in a culture medium 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 cell clusters with CHIR99021 in a culture medium for 2 h, and then contacting the cell clusters with FGF9, heparin and ANXA2 in a culture medium for 5 days to sequentially differentiate them into metanephric mesenchymal cells, nephron precursor cells and nephrons; (5) Day 7: the cells obtained in step (4) are digested into single cells, the cells are prepared into cell clusters by centrifugation, and are contacted with CHIR99021 in a culture medium for 2 h, and then are contacted with FGF9, heparin and ANXA2 in a culture medium for 5 days, so as to sequentially differentiate into metanephric mesenchymal cells, nephron precursor cells and nephron units; (6) Day 12-16: the cells obtained in step (5) are contacted with ANXA2 in a culture medium for 4 days, so as to generate more nephron unit structures inside the cell clusters; (7) Day 17-24: the cells obtained in step (6) are continuously cultured in a culture medium, and kidney organoids are obtained; the concentration of CHIR99021 in step (2) is 1-20 μM; the concentration of FGF9 in step (3) is 100-300 ng / mL; the concentration of heparin in step (3) is 0.1-5 μg / mL; the concentration of FGF9 in step (4) is 100-300 ng / mL; the concentration of heparin in step (4) is 0.1-5 μg / mL; the concentration of ANXA2 used in steps (4), (5) and (6) is 100-300 ng / mL; the ANXA2 is a human recombinant protein ANXA2; the concentration of CHIR99021 in step (5) is 1-20 μM; the concentration of FGF9 in step (5) is 100-300 ng / mL; the concentration of heparin in step (5) is 0.1-5 μg / mL.

7. The method of claim 6, wherein, the concentration of ANXA2 used in steps (4), (5) and (6) is 200 ng / mL.

8. The method of claim 6, wherein, the culture medium in step (1) is a hPSC special culture medium PGM1.

9. The method of claim 6, wherein, the culture condition in step (1) is to change the medium every day until the cell confluence reaches 50%.

10. The method of claim 6, wherein, the culture medium in step (2) is APEL2 culture medium.

11. The method of claim 6, wherein, the concentration of CHIR99021 in step (2) is 10 μM.

12. The method of claim 6, wherein, the culture condition in step (2) is to change the medium once every two days.

13. The method of claim 6, wherein, the culture medium in step (3) is APEL2 culture medium.

14. The method of claim 6, wherein, the concentration of FGF9 in step (3) is 200 ng / mL.

15. The method of claim 6, wherein, the concentration of heparin in step (3) is 1 μg / mL.

16. The method of claim 6, wherein, the culture condition in step (3) is to change the medium once every two days.

17. The method of claim 6, wherein, the culture medium in step (4) is APEL2 culture medium.

18. The method of claim 6, wherein, the concentration of FGF9 in step (4) is 200 ng / mL.

19. The method of claim 6, wherein, the concentration of heparin in step (4) is 1 μg / mL.

20. The method of claim 6, wherein, Each cell cluster in step (5) is about (1-10) x 10 5 cells.

21. The method of claim 6, wherein, about 5 x 10 5 cells per cell cluster in step (5).

22. The method of claim 6, wherein, the culture medium in step (5) is APEL2 culture medium.

23. The method of claim 6, wherein, the concentration of CHIR99021 in step (5) is 10 μM.

24. The method of claim 6, wherein, the concentration of FGF9 in step (5) is 200 ng / mL.

25. The method of claim 6, wherein, the concentration of heparin in step (5) is 1 μg / mL.

26. The method of claim 6, wherein, the culture condition in step (5) is to change the medium once every two days.

27. The method of claim 6, wherein, the culture medium in step (6) is APEL2 culture medium.

28. The method of claim 6, wherein, The condition of the culture in step (6) is to change the liquid every two days.

29. The method of claim 6, wherein, The medium in step (7) is APEL2 medium.

30. The method of claim 6, wherein, The condition of the culture in step (7) is to change the liquid every two days.

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