Method for preparing renal progenitor cells and use thereof

Abstract

Disclosed in the present invention are a method for preparing renal progenitor cells and use thereof. The method specifically comprises: inducing differentiation of pluripotent stem cells into an intermediate mesodermal cell stage by means of a GSK-3α / β inhibitor, and then inducing differentiation of cells in the intermediate mesodermal cell stage into a cap mesenchyme stage by using a combination of FGF-2 and an RAR / RXR nuclear receptor agonist, so as to obtain renal progenitor cells having distinct characteristics and potential specific to the cap mesenchyme. According to the method, high-purity and high-quality renal progenitor cells can be obtained without the need for sorting and purification, and the purity can reach 99.9%; the obtained renal progenitor cells have the differentiation potential of kidney-like organs, are basically free of residual PSCs, have the differentiation potential of kidney-like organs, and thus are suitable for clinical cell therapy. Moreover, the method features simple, stable, and controllable operations, facilitating the large-scale preparation of safe, effective, high-purity, and homogeneous renal progenitor cells.

Description

Preparation method and application of renal progenitor cells TECHNICAL FIELD

[0001] The present application relates to the field of cell culture technology, in particular to a preparation method and application of renal progenitor cells. BACKGROUND

[0002] Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), collectively known as human pluripotent stem cells (hPSCs), both have the potential of self-renewal and multi-directional differentiation, and thus can be ideal seed cells for differentiating into renal lineage cells. Currently, the commonly used method for inducing differentiation of pluripotent stem cells (PSCs) into renal progenitor cells (NPCs) is to make the cells undergo three stages of mesendoderm (ME) cells, intermediate mesoderm (IM) cells, and metanephric mesenchyme (MM) cells during the induction and differentiation process. First, GSK-3β inhibitors / Wnt agonists / BMP4 and other small molecules are used to induce PSCs to the ME stage, then FGF2, Activin A, BMP4, CHIR, Retinoic Acid and other small molecule combinations are used to induce to the IM stage, and then FGF9 and Activin A are used to induce to the MM stage. The MM cells contain a group of pluripotent NPCs. The cell population obtained through the above three stages of differentiation contains only a part of NPCs, and these NPCs mainly have the characteristics of ITGA8+, OSR1+ and WT1+.

[0003] The NPCs obtained by the method reported in the current literature for inducing differentiation of PSCs to NPCs have the following problems: first, a considerable proportion of PSCs remains, which has a high risk of tumorigenicity and is not conducive to clinical application; second, the NPCs are not pure in lineage, the differentiation efficiency is low, the cell morphology is not uniform, and it is difficult to obtain high-purity target cells, which need to be further sorted and purified; third, the differentiation steps are complex and involve too many small molecules / cytokines, which is not conducive to large-scale production and commercialization. Human nephron truly originates from cap mesenchyme (CM), which is the main precursor cell of glomerulus. In the early embryo, cap mesenchyme with self-renewal ability not only maintains the number and function of precursor cells in the process of nephron development, but also participates in the formation of renal tubular epithelial cells. The current reported method for inducing differentiation of PSCs to NPCs, and then obtaining kidney organoids through various in vitro induction culture systems, basically ignores that glomerulus is derived from cap mesenchyme, and more attention is paid to whether metanephric mesenchyme (MM) cells have the potential to differentiate into kidney organoids through various induction culture methods. SUMMARY

[0004] To solve the above problems, the present application develops a preparation method of safe and effective, high-purity, and homogeneous renal progenitor cells suitable for large-scale preparation. In the induction and differentiation process, the cells mainly undergo intermediate mesoderm (IM) cell stage and cap mesenchyme (CM) stage, i.e., PSC→IM→CM.

[0005] The technical scheme adopted by the present application is:

[0006] In a first aspect of the present application, a culture medium kit is provided, which comprises a first culture medium and a second culture medium; the first culture medium comprises a GSK-3α / β inhibitor; and the second culture medium comprises FGF-2 and a RAR / RXR nuclear receptor agonist.

[0007] In some preferred embodiments, the working concentration of the GSK-3α / β inhibitor in the first culture medium is 1-16 μM; specifically, 1-3 μM, or 2-5 μM, or 5-8 μM, or 7-12 μM, or 12-16 μM.

[0008] In some preferred embodiments, the GSK-3a / b inhibitor of the first medium is selected from Elraglusib (9-ING-41), GSK-3 Inhibitor IX, AZD2858, CHIR-99021, CHIR 98014, or a combination thereof.

[0009] In some preferred embodiments, the working concentration of the FGF-2 is 10-300 ng / mL; specifically, 10-20 ng / mL; or 20-40 / mL; or 40-80 ng / mL; or 80-150 ng / mL; or 150-300 ng / mL.

[0010] In some preferred embodiments, the working concentration of the RAR / RXR nuclear receptor agonist is 0.1-20 mM, specifically, 0.1-1 mM; or 1-2 mM; or 2-5 mM; or 5-10 mM; or 10-15 mM; or 15-20 mM.

[0011] In some preferred embodiments, the RAR / RXR nuclear receptor agonist is selected from Retinoic Acid, BMS493, Bexarotene, Adapalene, or a combination thereof.

[0012] In some preferred embodiments, the medium kit further comprises a third medium, wherein the third medium comprises a ROCK inhibitor.

[0013] In some preferred embodiments, the working concentration of the ROCK inhibitor is 0.5-20 mM; specifically, 0.5-2 mM; or 2-5 mM; or 5-8 mM; or 7-12 mM.

[0014] In some preferred embodiments, the ROCK inhibitor comprises Thiazovivin, azaindole1, RKI-1447, Y27632, or a combination thereof.

[0015] In some preferred embodiments, the first medium, the second medium further comprises one or more of a serum replacement, NEAA, GluMax.

[0016] In some preferred embodiments, the working concentration of the serum replacement is 5-30% (v / v).

[0017] In some preferred embodiments, the serum replacement is KOSR.

[0018] In some preferred embodiments, the working concentration of the NEAA is 0.5-1.5X.

[0019] In some preferred embodiments, the working concentration of the GluMax is 0.5-1.5X.

[0020] In some preferred embodiments, the base medium of the first medium, the second medium or the third medium can be selected from pluripotent stem cell culture media, such as Gibco E8, Gibco DMEM F12, TeSR-AOF, RPMI1640, Advanced RPMI 1640 medium or other similar media, without special limitation.

[0021] In some preferred embodiments, the second medium further comprises Activin A and a GSK-3α / β inhibitor.

[0022] In some preferred embodiments, the working concentration of the Activin A is 1-100 ng / mL, specifically 1-5 ng / mL; or 5-15 ng / mL; or 15-50 ng / mL; or 50-100 ng / mL.

[0023] In some preferred embodiments, the GSK-3α / β inhibitor of the second medium is selected from lraglusib, GSK-3 Inhibitor IX, AZD2858, CHIR-99021, CHIR 98014 or a combination thereof, wherein CHIR99021 is preferred.

[0024] In some preferred embodiments, the working concentration of the GSK-3α / β inhibitor of the second medium is 0.5-3 μM; specifically 0.5-2 μM, or 1-3 μM.

[0025] In a second aspect of the present application, the use of the medium kit of the first aspect of the present application in the preparation of kidney progenitor cells, glomeruli or kidney-like organs is provided.

[0026] In a third aspect of the present application, a method for preparing kidney progenitor cells is provided, comprising the following steps

[0027] S1: culturing pluripotent stem cells using the first medium of the first aspect of the present application for >96 hours; differentiating to the intermediate mesoderm (IM) stage;

[0028] S2: replacing the medium with the second medium of the first aspect of the present application to culture the cells for ≥24 hours; differentiating to the cap mesenchyme stage; obtaining kidney progenitor cells.

[0029] In some preferred embodiments, the cell density of the pluripotent stem cells in step S1 is 2 x 10^3 Cells / cm2 ~5 x 10^4 Cells / cm 2 , preferably 3.5 x 10^3 Cells / cm 2 ~1.0 x 10^4 Cells / cm 2 .

[0030] In some preferred embodiments, the culturing time of step S1 is ≤168 hours, preferably 120-144 hours.

[0031] In some preferred embodiments, the culturing time of step S2 is 24-60 hours; preferably 36-48 hours; or 42-56 hours, or 48 hours-60 hours, which is enough to culture the pluripotent stem cells to cap mesenchyme (CM) / metanephric mesenchyme (MM).

[0032] In some preferred embodiments, the pluripotent stem cells in step S1 are undifferentiated pluripotent stem cells.

[0033] In some preferred embodiments, the pluripotent stem cells are cultured in the third medium of the first aspect of the present application before step S1 to maintain the undifferentiated stage.

[0034] In some preferred embodiments, the pluripotent stem cells are seeded on feeder cells such as inactivated mouse embryonic fibroblasts, or in Matrigel such as Matrigel, Vitronectin XF Matrigel or Laminin 521 Matrigel, etc. during the maintenance of the undifferentiated stage.

[0035] In some preferred embodiments, the medium is replaced every 16-48 hours during the preparation of the renal progenitor cells.

[0036] In some preferred embodiments, the pluripotent stem cells are selected from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs); preferably, the pluripotent stem cells are human pluripotent stem cells.

[0037] In the fourth aspect of the present application, a method for obtaining a glomerular cell model by further differentiating the renal progenitor cells is provided, comprising the following steps:

[0038] The renal progenitor cells are cultured in a medium containing FGF-9 to obtain a glomerular cell model.

[0039] In some preferred embodiments, the working concentration of FGF-9 is 10-300 ng / mL, specifically 10-20 ng / mL; or 20-40 / mL; or 40-80 ng / mL; or 80-150 ng / mL; or 150-300 ng / mL; or 20-100 ng / mL, or 50-150 ng / mL, or 75-125 ng / mL, or 50-150 ng / mL, or 100-300 ng / mL.

[0040] In a fifth aspect, the present application provides a method for preparing renal progenitor cells, which takes mesodermal cells (which can be differentiated from human induced pluripotent stem cells or embryonic stem cells) as starting point, and comprises the following steps: treating the mesodermal cells with FGF-2 and RAR / RXR nuclear receptor agonist to obtain renal progenitor cells.

[0041] In some preferred embodiments, the mesodermal cells are treated by culturing the cells in a medium containing FGF-2 and RAR / RXR nuclear receptor agonist for ≥24 hours.

[0042] In some preferred embodiments, the working concentration of FGF-2 is 10-300 ng / mL, specifically 10-20 ng / mL; or 20-40 / mL; or 40-80 ng / mL; or 80-150 ng / mL; or 150-300 ng / mL.

[0043] In some preferred embodiments, the working concentration of RAR / RXR nuclear receptor agonist is 0.1-20 μM, specifically 0.1-1 μM; or 1-2 μM; or 2-5 μM; or 5-10 μM; or 10-15 μM; or 15-20 μM.

[0044] In some preferred embodiments, the RAR / RXR nuclear receptor agonist is selected from Retinoic Acid, BMS493, Bexarotene, Adapalene or a combination thereof.

[0045] In a sixth aspect, the present application provides the use of the renal progenitor cells obtained in the second aspect in the preparation of a medicament for preventing and treating kidney diseases.

[0046] Preferably, the kidney diseases include renal ischemia-reperfusion injury.

[0047] The present application has the following advantages:

[0048] The application develops a preparation method of kidney progenitor cells which is suitable for large-scale preparation, safe and effective, high-purity and homogeneous; specifically, the method comprises the following steps: inducing pluripotent stem cells to differentiate into intermediate mesoderm cell stage by using GSK-3 alpha / beta inhibitor, and then inducing the intermediate mesoderm cell stage cells to differentiate into cap mesenchyme stage by using FGF-2 and RAR / RXR nuclear receptor agonist, so as to obtain kidney progenitor cells which have obvious characteristics and potential of cap mesenchyme; the method can obtain kidney progenitor cells with high purity and high quality without sorting and purification, the purity can reach 99.9%, the obtained kidney progenitor cells have differentiation potential of kidney organoids, are basically free of PSCs residues, have differentiation potential of kidney organoids, are suitable for clinical cell therapy, and the method is simple, stable and controllable, and is beneficial to large-scale preparation of kidney progenitor cells. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 is the cell culture result in Example 1.

[0050] Figure 2 is the cell culture result in Example 2.

[0051] Figure 3 is the cell culture result in Example 3.

[0052] Figure 4 is the cell culture result in Example 4.

[0053] Figure 5 is a microscope image of the kidney glomerulus cell model differentiated from the NPCs obtained in Example 2.

[0054] Figure 6 is a microscope image of the kidney glomerulus cell model in Example 8.

[0055] Figure 7 is the cell culture result in Comparative Example 1.

[0056] Figure 8 is the cell culture result in Comparative Example 2.

[0057] Figure 9 is the relative expression amount of marker genes of the NPCs obtained in Example 1.

[0058] Figure 10 is the relative expression amount of marker genes of the NPCs obtained in Example 2.

[0059] Figure 11 is the relative expression amount of marker genes of the NPCs obtained in Example 3.

[0060] Figure 12 is the relative expression amount of marker genes of the NPCs obtained in Example 4.

[0061] Figure 13 is the relative expression amount of marker genes of the cells obtained in Comparative Example 1.

[0062] Figure 14 is the relative expression amount of marker genes of the cells obtained in Comparative Example 2.

[0063] Figure 15 is the flow cytometry detection result of the NPCs obtained in Example 2.

[0064] Figure 16 is the RNA-seq results of NPCs obtained in Example 2.

[0065] Figure 17 is the differentiation of NPCs obtained in Example 4 into kidney organoid model.

[0066] Figure 18 is the results of NPCs obtained in Examples 1-4 in reducing the serum creatinine and blood urea nitrogen levels of IRI.

[0067] Figure 19 is the morphology of cells (mesodermal cells) after step 2 of Example 6 under a microscope (magnification: 20x).

[0068] Figure 20 is the morphology of hiPSCs before differentiation, kidney progenitor cells of Example 5 and Example 6 under a microscope, wherein Figure 20A is the microscope image of hiPSCs before differentiation (magnification: 10x), Figure 20B is the microscope image of kidney progenitor cells of Example 5 (magnification: 4x), and Figure 20C is the microscope image of kidney progenitor cells of Example 6 (magnification: 4x).

[0069] Figure 21 is the results of qPCR detection of cells after step 2 of Example 6 of Example 6, relative expression of IM markers.

[0070] Figure 22 is the results of qPCR detection of cell markers of hiPSCs before differentiation, kidney progenitor cells of Example 5 and Example 6 of Example 6.

[0071] Figure 23 is the results of immunofluorescence detection of cell markers of kidney progenitor cells of Example 7.

[0072] Figure 24 is the results of immunofluorescence staining of WT-1 and NPHS1 proteins of the model of Example 8.

[0073] Figure 25 is the results of immunofluorescence staining of Claudin-1 protein of the glomerular model of Example 8.

[0074] Figure 26 is the results of immunofluorescence staining of CD133 and CD24 proteins of the glomerular model of Example 8.

[0075] Figure 27 is the results of immunofluorescence staining of PAX2 and WT-1 proteins of the glomerular model of Example 8. Embodiments of the present application

[0076] The concept and technical effects of the present application will be described clearly and completely in combination with the embodiments, so as to fully understand the purposes, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0077] Embodiment 1

[0078] The preparation method of the renal progenitor cells is specifically as follows:

[0079] Step S1: human pluripotent stem cells (iPSCs) are inoculated on inactivated mouse embryonic fibroblasts at a seeding density of 3.5×10 3 / cm 2 Normal culture in the iPSC maintenance medium, and Essential 8 medium is used in the present embodiment (other suitable pluripotent stem cell culture medium can also be selected). This step is to allow the cells to grow adherently, and the subsequent operation is performed after the cells adhere;

[0080] Step S2: replace the Advance RPMI 1640 medium containing AZD2858 (working concentration 2 μM) and GluMax (working concentration 1X) to induce and culture the cells for 168 hours;

[0081] Step S3: replace the Advance RPMI 1640 medium containing FGF-2 (working concentration 20 ng / mL) and BMS493 (working concentration 1 μM) and GluMax (working concentration 1X) to continue to culture the cells for 60 hours;

[0082] During the culture process, the fresh medium is replaced every 24-48 hours.

[0083] Results: as shown in FIG. 1, the cells are dense and uniform in morphology, and the confluence reaches 100%

[0084] Embodiment 2

[0085] The preparation method of the renal progenitor cells is specifically as follows:

[0086] Step S1: iPSCs are inoculated on Laminin 521 matrix glue at a seeding density of 1×10 4 / cm 2 The cells are cultured in TeSR-AOF medium containing Y27632 (working concentration 10 μM);

[0087] Step S2: replace the Advance RPMI 1640 medium containing CHIR 98014 (working concentration 8 μM), CTS TM KnockOutTM SR (KOSR, 10% v / v), NEAA (1x) RPMI1640 medium for 144 hours;

[0088] Step S3: Replace with KOSR (10% v / v), NEAA (1x) RPMI1640 medium containing FGF-2 (working concentration 100 ng / mL), Retinoic Acid (working concentration 1 mM) and CTS TM KnockOut TM SR (KOSR, 10% v / v), NEAA (1x) RPMI1640 medium for 48 hours;

[0089] During the culture process, fresh medium was replaced every 24-48 hours.

[0090] Results: As shown in Figure 2, the cells were dense, uniform in morphology, and the confluence reached 100%.

[0091] Example 3

[0092] The preparation method of renal progenitor cells is as follows:

[0093] Step S1: iPSCs were seeded at a density of 6.5 x 10 3 / cm 2 The cells were seeded in Vitronectin XF Matrigel at a seeding density and cultured in TeSR -AOF maintenance medium containing Y27632 (working concentration 20 mM);

[0094] Step S2: Replace with KOSR (10% v / v), NEAA (1x) RPMI1640 medium containing CHIR-99021 (working concentration 16 mM), KOSR (10% v / v), and NEAA (1x) for 108 hours;

[0095] Step S3: Replace with KOSR (10% v / v), NEAA (1x) RPMI1640 medium containing FGF-2 (working concentration 300 ng / mL) and Retinoic Acid (working concentration 10 mM) for 48 hours;

[0096] During the culture process, fresh medium was replaced every 24-48 hours.

[0097] Results: As shown in Figure 3, the cells were dense, uniform in morphology, and the confluence reached 100%.

[0098] Example 4

[0099] Step S1: Embryonic stem cells (ES) were seeded at a density of 1.2 x 10 4 / cm 2Seeding density: Cells were seeded on gelatin matrix and cultured in mTeSR™1 medium containing Thiazovivin (working concentration: 1 μΜ);

[0100] Step S2: Replace the old medium with Advance RPMI 1640 medium containing GSK-3 Inhibitor IX (working concentration: 5 μΜ) and GluMax (working concentration: 1X) to induce the cells for 120 hours;

[0101] Step S3: Replace the old medium with Advance RPMI 1640 medium containing FGF-2 (working concentration: 150 ng / mL) and Adapalene (working concentration: 5 μΜ) and GluMax (working concentration: 1X) to culture the cells for 48 hours;

[0102] During the culture process, the fresh medium was replaced every 24-48h.

[0103] Results: As shown in Figure 4, the cells were dense, uniform in morphology, and the confluence reached 100%.

[0104] Example 5

[0105] The preparation method of renal progenitor cells is as follows:

[0106] Step S1: Human induced pluripotent stem cells (hiPSCs) were added to a 6-well plate coated with Matrigel matrix at a density of 5 x 105cells / well, and the culture plate was shaken before and after to disperse the cells evenly, then mTeSR plus medium containing Y27632 (10 μΜ) was added to culture the cells for 1 day. 4 Step S1: Human induced pluripotent stem cells (hiPSCs) were added to a 6-well plate coated with Matrigel matrix at a density of 5 x 105cells / well, and the culture plate was shaken before and after to disperse the cells evenly, then mTeSR plus medium containing Y27632 (10 μΜ) was added to culture the cells for 1 day.

[0107] Step S2: The old medium was aspirated, and Advanced RPMI 1640 medium containing CHIR-99021 (5 μΜ), GlutaMax (1x), and penicillin / streptomycin double-antibiotic solution (1x) was added to each well, and the cells were cultured for 6 days.

[0108] Step S3: The old medium was removed, and Advanced RPMI 1640 medium containing FGF-2 (50 ng / mL), Retinoic Acid (3 μΜ), GlutaMAX (1x), penicillin / streptomycin double-antibiotic solution (1x) was added to culture the cells for 48 hours to obtain renal progenitor cells;

[0109] During the culture process, the fresh medium was replaced every 24-48h.

[0110] Example 6

[0111] The preparation method of renal progenitor cells is as follows:

[0112] Step S1: hiPSCs were seeded at 5 x 10 4 cells / well in 6-well plates coated with Matrigel, and the plates were shaken to distribute the cells evenly, then the cells were cultured in mTeSR plus medium containing Y27632 (10 μM) for 1 day.

[0113] Step S2: The old medium was removed, and Advanced RPMI 1640 medium containing CHIR-99021 (12 μM), GlutaMax (1x), and penicillin / streptomycin solution (1x) was added to each well, and the cells were cultured for 4 days.

[0114] Step S3: Advanced RPMI 1640 medium containing FGF-2 (200 ng / mL), Retinoic Acid (0.5 μM), GlutaMax (1x), CHIR99021 (1 μM), Activin A (10 ng / mL), and penicillin / streptomycin solution (1x) was added, and the cells were cultured for 48 hours to obtain kidney progenitor cells.

[0115] During the culture process, the fresh medium was replaced every 24-48 hours.

[0116] Example 7

[0117] The method for differentiating the NPCs of Example 2 into a glomerular cell model is as follows:

[0118] The cells obtained in Step S3 of Example 2 were subcultured, and the seeding density was adjusted to 1.2 x 10 6 cells / cm 2 The cells were seeded on Laminin 521 gel and cultured in RPMI1640 medium containing FGF-9 (working concentration 200 ng / mL), KOSR (10% by volume), and NEAA (1x), and the medium was replaced every 36 hours.

[0119] Results: The cells began to differentiate to form glomerulus-like structures 36 hours after FGF-9 induction, and obvious glomerulus-like structures were observed at 72 hours (Figure 5). The NPCs differentiated to form glomerulus-like structures spontaneously under 2D culture conditions without changing the culture system, which proves that the NPCs obtained by the method of Example 2 have obvious characteristics and potential of capillary mesenchyme.

[0120] Example 8

[0121] The method for preparing a glomerular cell model from iPSCs is as follows:

[0122] Step S1: iPSCs at 6.5 × 10 3 / cm 2 The cells were seeded in Vitronectin XF substrate and cultured in TeSR maintenance medium (this step is to allow the cells to adhere to the culture medium before proceeding with subsequent operations).

[0123] Step S2: Replace with a solution containing CHIR-99021 (working concentration 8 μM) and KOSR (volume fraction 10%).

[0124] Cells were cultured in 0.8× NEAA RPMI 1640 medium for 120 hours, with the medium changed every 24 hours.

[0125] Step S3: Replace with a solution containing FGF-2 (working concentration 100 ng / mL) and Retinoic Acid (working concentration...

[0126] Cells were cultured in RPMI 1640 medium containing 10 μM, KOSR (10% by volume) and NEAA (0.8×) for 48 hours, with the medium changed every 24 hours.

[0127] Step S4: Adjust the seeding density to 3.2 × 10⁻⁶ cells for cell passage culture. 5 pcs / cm 2 Cells were seeded in Vitronectin XF matrix gel and cultured in RPMI 1640 medium containing FGF-9 (working concentration 100 ng / mL), KOSR (volume fraction 5%), and NEAA (0.8×), with the medium changed every 24 hours.

[0128] Results: As shown in Figure 6, after 96 hours of FGF-9-induced differentiation, the cell model clearly exhibited structures similar to glomeruli (glomeruli), renal capsules, and renal capsule walls.

[0129] Comparative Example 1

[0130] The method for preparing kidney progenitor cells in Comparative Example 1 is the same as in Example 2, except for step S2, in which the cells are cultured for 96 hours; during the culture process, the culture medium is replaced with fresh medium every 24 to 48 hours.

[0131] Results: As shown in Figure 7, the cells were not dense, had uneven morphology, and had a confluence of 60-70%.

[0132] Comparative Example 2

[0133] The preparation method of the renal progenitor cells of Comparative Example 2 is the same as that of Example 3, except that in step S3, the RPMI1640 culture medium containing FGF-9 (working concentration 100 ng / mL), Retinoic Acid (working concentration 10 μM), KOSR (10% by volume), and NEAA (1x) is replaced to culture the cells for 48 hours.

[0134] Results: As shown in Figure 8, the cells are dense, morphologically heterogeneous, and highly heterogeneous. Small tubular structures can be observed.

[0135] Test Example 1 QPCR Detection

[0136] The mRNA of the cells prepared in Examples 1-4 and Comparative Examples 1-2 was extracted, and qPCR detection was performed to detect the relative expression (internal reference GAPDH) of the genes OCT4, ESRG, OSR1, WT1, ITGA8, and PAX2, according to the SYBR Green qPCR Mix kit instructions.

[0137] Results:

[0138] As shown in Figure 9, the iPSCs of Example 1 have a high degree of differentiation, and the expression levels of the iPSCs stemness genes OCT4 and ESRG after cell differentiation are only 0.53% and 0.16% relative to the undifferentiated iPSCs (100%). The qPCR results in Figure 9 show that when the expression levels of the NPCs stemness genes after cell differentiation are normalized to 1 relative to the undifferentiated iPSCs, WT, ITGA8, and PAX2 have very high expression, and OSR1 has low expression.

[0139] Figure 10 qPCR results suggest that the iPSCs of Example 2 have a very high degree of differentiation, and the expression levels of the iPSCs stemness genes OCT4 and ESRG after cell differentiation are only 0.07% and 0.01% relative to the undifferentiated iPSCs (100%). The qPCR results in Figure 10 show that when the expression levels of the NPCs stemness genes after cell differentiation are normalized to 1 relative to the undifferentiated iPSCs, WT, ITGA8, PAX2, and OSR1 have very high expression.

[0140] Figure 11 qPCR results suggest that the iPSCs have a very high degree of differentiation, and the expression levels of the iPSCs stemness genes OCT4 and ESRG after cell differentiation are only 0.08% and 0.01% relative to the undifferentiated iPSCs (100%). The qPCR results in Figure 11 show that when the expression levels of the NPCs stemness genes after cell differentiation are normalized to 1 relative to the undifferentiated iPSCs, WT, ITGA8, PAX2, and OSR1 have very high expression.

[0141] The qPCR results of Example 4 shown in Figure 12 suggest that the iPSCs differentiation is very high, the iPSCs stemness genes OCT4 and ESRG expression levels after cell differentiation are only 0.01% and 0.02% relative to the undifferentiated iPSCs (100%). The qPCR results of Figure 12 show that the WT, ITGA8, PAX2, and OSR1 have very high expression levels when the NPCs stemness gene expression levels are normalized to 1 relative to the undifferentiated iPSCs.

[0142] As shown in Figure 13, the iPSCs differentiation of Comparative Example 1 is low, the iPSCs stemness genes OCT4 and ESRG expression levels are 5.11% and 1.05% relative to the undifferentiated iPSCs (100%), indicating that a considerable portion of iPSCs remains (iPSCs remain undifferentiated). The qPCR results of Figure 13 show that the WT has higher expression and the ITGA8, PAX2, and OSR1 have lower expression when the NPCs stemness gene expression levels are normalized to 1 relative to the undifferentiated iPSCs.

[0143] The qPCR results of Figure 14 suggest that the iPSCs differentiation of Comparative Example 2 is low, the iPSCs stemness genes OCT4 and ESRG expression levels are 0.16% and 0.01% relative to the undifferentiated iPSCs (100%). The qPCR results of Figure 14 show that the WT1, ITGA8, PAX2 have higher expression and the OSR1 has lower expression when the NPCs stemness gene expression levels are normalized to 1 relative to the undifferentiated iPSCs.

[0144] Test Example 2 Flow cytometry detection of NPCs related marker gene expression

[0145] Cells from Example 2 were dissociated with Accutase for 10 minutes and cell clumps were removed using a 40 pm cell strainer (Corning, #352340). Cells were fixed with 2% paraformaldehyde on ice for 15 minutes and permeabilized with 0.1% Triton on ice for 15 minutes. Cells were blocked with PBS + 5% donkey serum for 15 minutes and incubated with primary antibodies (PAX8 1:2500, ITGA8 1:100, WT1 1:100) for 30 minutes. After washing with PBS + 1% BSA for 3 times, cells were incubated with secondary antibodies (Alexa Fluor 594 conjugated donkey anti-rabbit 1:5000 [Life Technologies], Cy5 conjugated donkey anti-mouse 1:2500 [Jackson Nutrition], or Alexa Fluor 647 conjugated donkey anti-mouse 1:5000 [Life Technologies]) for 20 minutes on ice. Cells were then washed with PBS + 1% BSA for 3 times. Flow cytometry was performed.

[0146] Results: As shown in Figure 15, 99.96% of cells from Example 2 expressed WT-1 and 99.99% of cells expressed PAX-2, which reflected that the NPCs obtained by the differentiation protocol of Example 2 were very pure and did not need further sorting and purification. The NPCs obtained by the methods in the prior art (Takasato et al. Nat Cell Biol. 2014 (DOI: 10.1038 / ncb2894)) were basically difficult to reach 80% purity, and the purity of the target cell markers was very uneven and varied greatly, indicating that the NPCs obtained by the prior methods were very heterogeneous.

[0147] Example 3 RNA-seq proved that the NPCs obtained by the method were different from conventional NPCs

[0148] The NPCs obtained in Example 2 were entrusted to Beijing Nuowu Ziyuan Technology Co., Ltd. for RNA-seq detection.

[0149] Results: Significantly up-regulated genes: logFC > 1 and P.Val < 0.05;

[0150] Results: As shown in Figure 16, the RNAseq results showed that the NPCs obtained by the protocol of Example 2 highly expressed cap mesenchyme characteristic genes. The NPCs obtained by the methods reported in the prior art were basically defined as metanephric mesenchyme (MM), and were highly heterogeneous and low purity.

[0151] Test Example 4 Kidney-like organ differentiation

[0152] The NPCs of Example 4 were differentiated into a kidney-like organ model. The induction differentiation was performed according to the method of Taguchi et al. Cell Stem Cell. 2014 (DOI: 10.1016 / j.stem.2013.11.010).

[0153] The results of Figure 17 suggest that the cells differentiate from aggregation, gradually forming structures similar to glomeruli, tubules, endothelial networks, etc., proving that the NPCs obtained by the method of Example 2 have differentiation potential into kidney-like organs.

[0154] Test Example 5 NPCs treatment of IRI

[0155] A mouse model of renal ischemia-reperfusion injury (IRI) was prepared. NOD-SCID mice (6-8 weeks old, male, 20-25 g) were randomly divided into 6 groups, 5 in each group, sham operation group (sham), control group (IRI+NS) and test group (IRI+NPCs). The operation was as follows: ① Anesthetize the mouse, depilate the abdomen and fix it prone; ② Iodophor disinfection of the skin area; ③ Incision on the midline of the abdomen, move the intestinal tissue, and expose the bilateral kidneys; ④ Release after clamping the bilateral renal pedicles for 30 min (the sham group only peeled off the bilateral renal pedicles without clamping); ⑤ According to the grouping, inject physiological saline (50 μL per mouse, control group) or inject renal progenitor cells of Examples 1-4 (1×10 5 / 50 μL per mouse, test group) into the renal parenchyma, respectively, and then replace the bilateral kidneys and intestinal tissue, and suture the abdominal muscle and skin layer by layer; ⑥ Postoperative rewarming until recovery; ⑦ On the third day after the operation, take the whole blood, separate the serum, and then use a biochemical instrument to detect the serum creatinine and urea nitrogen (BUN).

[0156] Results: As shown in Figure 18, the NPCs of Examples 1-4 can reduce the serum creatinine and urea nitrogen caused by IRI, and the NPCs have a certain therapeutic effect on IRI.

[0157] In addition, the safety of the NPCs was also detected by a tumorigenicity experiment of HELA cells and NPCs of Example 2 in nude mice:

[0158] Specifically, the HELA cells and NPCs were subcutaneously injected into the axillary of nude mice, respectively, and after 4 months of tracking, the results showed that no tumor was formed in the axillary of the mice injected with NPCs, indicating that the NPCs did not have tumorigenicity; while the nude mice injected with HELA cells developed obvious tumors after 1 month.

[0159] It is shown that the NPCs obtained by the preparation of the application are suitable for clinical use and have safety.

[0160] Test Example 6 IM cell detection

[0161] The cells after step S2 of Example 6 were taken under a microscope and showed a tight cell morphology, as shown in Figure 19. The RNA of the cells after step S2 of Example 6 was extracted, and the intermediate mesoderm marker genes PAX2, OSR1, and the endoderm and ectoderm marker genes SOX17 and PAX6 were detected according to the qPCR kit instructions.

[0162] The kidney progenitor cells after step S3 of Example 5 and the kidney progenitor cells after step S3 of Example 6, and the hiPSCs before differentiation were observed under a microscope, and the experimental results are shown in Figure 20. The edges of the hiPSCs before differentiation were relatively clear, and the growth state was good. The cell nuclei and cell membranes in the single clone were relatively clear, and the boundaries between the cells were relatively clear, and there were basically no differentiated cells (Figure 20A). A certain amount of cell aggregation structure was observed under a microscope in the cells of Example 5, but the aggregation structure was not tight and the amount was not particularly large (Figure 20B). Compared with Example 5, the cell aggregation structure of Example 6 was relatively dense, and there were many aggregation structures, and many aggregation structures were connected into larger sheet-like aggregation structures, and there were many protruding structures in the aggregation structure (Figure 20C). The RNA of the kidney progenitor cells after differentiation of Example 5 and Example 6, and the hiPSCs before differentiation was extracted, and the kidney progenitor cell characteristic marker genes SIX2, PAX2, HOXA10, HOXA11, EMX2 and LHX1 were detected according to the qPCR kit instructions.

[0163] qPCR result analysis:

[0164] The qPCR results of the cells after step S2 of Example 6 are shown in Figure 22 (with hiPSCs as a reference). After step S2, that is, after CHIR treatment, the cells significantly overexpressed the intermediate mesoderm marker genes PAX2 and OSR1, and basically did not express the endoderm and ectoderm marker genes SOX17 and PAX6. In short, after the hiPSCs were treated with GKS-3a / β inhibitors for 4 days, the IM cells entered the IM cell stage.

[0165] The qPCR results of the cells after step S3 of Example 5 and the cells after step S3 of Example 6 are shown in Figure 22, with undifferentiated human pluripotent stem cells (hiPSCs) as a reference, and the relative expression value of each gene was set to 1. The results showed that the cells of Example 5 and Example 6 both significantly expressed kidney progenitor cell characteristic marker genes, indicating that the terminal differentiated cells induced by the two examples were both kidney progenitor cells; compared with Example 5, the relative expression amounts of the kidney progenitor cell-related genes of Example 6 were significantly up-regulated. In short, the IM cells obtained NPCs after treatment with FGF-2 and RA.

[0166] Test Example 7 NPCs Immunofluorescence

[0167] The cells after differentiation of Example 5 were removed from the old culture medium, then fixed with 4% paraformaldehyde, permeabilized with Triton X-100 and blocked with BSA, incubated with primary and secondary antibodies, and then observed under a microscope after mounting.

[0168] Immunofluorescence result analysis: The immunofluorescence results are shown in Figure 23. The cells of Example 5 highly expressed NPC-related genes WT1, SIX2, PAX2, SALL1, etc. It is shown that the cells induced by Example 5 are NPCs.

[0169] Test Example 8 Glomerular Cell Model Immunofluorescence

[0170] The glomerular cell model of Example 8 was taken for immunofluorescence test, fixed with 4% paraformaldehyde, permeabilized with Triton X-100 and blocked with BSA, incubated with primary and secondary antibodies, and then observed under a microscope after mounting.

[0171] The results are shown in Figures 10-13. As can be seen from Figure 24, the core protein nephrin (NPHS1) of the slit diaphragm between the glomerular podocytes is widely distributed between the cytoplasm, while the Wilms tumor protein WT-1 is concentrated in the nucleus. In the position of the glomerulus, NPHS1 and WT-1 are more significantly expressed.

[0172] Figure 25 is an immunofluorescence image of the glomerular model single-stained Claudin-1 protein. Claudin-1 is a transmembrane protein commonly found in renal tubular epithelial cells and is a specific marker for Bowman's capsule. In this model, Claudin-1 is widely and localized to the glomerular structure.

[0173] Figure 26 is an immunofluorescence image of the glomerular model double-stained CD133 and CD24. The green fluorescent label in Figure 12 is

[0174] CD133 protein, and the red fluorescent label is CD24 protein. CD133 is a transmembrane glycoprotein commonly found in glomerular epithelial cells, while CD24 is a highly glycosylated glycosylphosphatidylinositol-anchored surface protein derived from glomerular epithelial cells. CD133 and CD24 are specific markers for glomerular parietal epithelial cells.

[0175] Figure 27 is an immunofluorescence image of the glomerular model double-stained PAX2 and WT-1. PAX2 and WT-1 are commonly found in glomerular capillary loop mesangial aggregates.

[0176] The above detailed description of the application has been made with reference to specific embodiments thereof, but the application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the application. Furthermore, the embodiments of the application and the features in the embodiments can be combined with each other in the case of no conflict.

Claims

1. A culture medium kit comprising a first culture medium and a second culture medium; the first culture medium containing a GSK-3α / β inhibitor; and the second culture medium containing FGF-2 and a RAR / RXR nuclear receptor agonist.

2. The culture medium kit according to claim 1, characterized in that, The GSK-3α / β inhibitor is selected from lraglusib, GSK-3 Inhibitor IX, AZD2858, CHIR-99021, CHIR 98014 or a combination thereof; preferably, the RAR / RXR nuclear receptor agonist is selected from Retinoic Acid, BMS493, Bexarotene, Adapalene or a combination thereof.

3. The culture medium kit according to claim 1, characterized in that, The working concentration of the GSK-3α / β inhibitor is 1–16 μM; preferably, the working concentration of the FGF-2 is 10–300 ng / mL; preferably, the working concentration of the RAR / RXR nuclear receptor agonist is 0.1–20 μM.

4. The culture medium kit according to any one of claims 1 to 3, characterized in that, The culture medium kit further includes a third culture medium, which includes a ROCK inhibitor; preferably, the ROCK inhibitor includes Thiazovivin, azaindole1, RKI-1447, Y27632 or a combination thereof; preferably, the working concentration of the ROCK inhibitor is 0.5–20 μM.

5. The culture medium kit according to any one of claims 1 to 3, characterized in that, The first culture medium and the second culture medium further include one or more of serum substitutes, NEAA, and GluMax; preferably, the working concentration of the serum substitute is 5-30% (v / v); preferably, the working concentration of the NEAA is 0.5-1.5X; preferably, the working concentration of the GluMax is 0.5-1.5X.

6. The use of the culture medium kit according to any one of claims 1 to 5 in the preparation of renal progenitor cells, glomeruli or kidney-like organs.

7. A method for preparing renal progenitor cells, comprising the following steps S1: Culture pluripotent stem cells in the first culture medium as described in any one of claims 1 to 5 for >96 hours; S2: Replace with the second culture medium as described in any one of claims 1 to 5 and culture the cells for ≥24 hours; obtain kidney progenitor cells.

8. The method according to claim 7, characterized in that, The culture time in step S1 is ≤168 hours; preferably, the pluripotent stem cells are selected from embryonic stem cells and induced pluripotent stem cells, and preferably, the pluripotent stem cells are human pluripotent stem cells.

9. The use of the renal progenitor cells prepared by the method according to any one of claims 7 to 8 in the preparation of drugs for the prevention and treatment of kidney disease.

10. A method for preparing renal progenitor cells, characterized in that: Kidney progenitor cells were obtained by treating intermediate mesodermal cells with FGF-2 and RAR / RXR nuclear receptor agonists.