A high-throughput human kidney organoid differentiation method

Abstract

The present invention discloses a high-throughput human kidney organoid induction differentiation method, including: adding ROCK pathway inhibitor to human induced pluripotent stem cells for 18 to 24 hours; replacing with GSK-3 inhibitor for 1 to 4 days; replacing with culture medium B containing GSK-3 inhibitor, FGF-9 and Heparin for 1 to 3 days; carrying out high-throughput 3D culture, efficiently obtaining a large number of organoid spheres; after treatment with GSK-3 inhibitor, culture medium B containing FGF-9 and Heparin for 1 to 6 days; replacing with culture medium B without addition for 1 to 10 days to obtain mature kidney organoids. The method is simple to operate, highly targeted, improves the efficiency and differentiation stability of induced differentiation, reduces the time required for differentiation and maturation, increases the proliferation efficiency of kidney organoids, and solves the problem that the differentiation efficiency in conventional methods is uneven and the operation is cumbersome.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a high-throughput kidney organoid induction and differentiation method. Background Art

[0002] Kidney disease is a global health threat, affecting approximately 750 million people worldwide. The onset of kidney disease often places heavy physical, psychological, and economic burdens on patients. Due to the complex structure of the kidney, the development of in vitro kidney disease research models has been slow, hindering the study of kidney disease-related pathological mechanisms and drugs.

[0003] In related technologies, kidney organoids based on stem cell differentiation have gradually become an important model for studying kidney physiology and diseases. Since scientists first reported the method for establishing kidney organoids in 2014, they have now been successfully applied in the study of glomerular and tubular diseases. However, there are still many problems and defects in the current differentiation methods. The main problems include two aspects: the first aspect is that the differentiation quality of kidney organoids is poor, and the existing differentiation methods have problems such as incomplete differentiation and low differentiation efficiency. On the other hand, the cost of differentiation is too high. Most of the inducers used in the existing differentiation methods are expensive growth factors, which leads to high production costs and makes it difficult to achieve universal use in a real sense. These problems combined have greatly limited the large-scale production of key tissues or organs such as endoderm derived from stem cells. Therefore, there is an urgent need for a differentiation method with high efficiency and low cost. Summary of the Invention

[0004] The present invention aims to address at least one of the technical problems existing in the aforementioned prior art. To this end, the present invention proposes a high-throughput method for inducing the differentiation of human kidney organoids. This method can address the problems of existing differentiation methods, such as high differentiation costs, low differentiation quality, incomplete differentiation, and low differentiation efficiency. It is simple and easy to operate, produces stable results, and enables high-throughput, large-scale culture, thereby obtaining more mature organoids with the main structures and functional cells of the kidney, thus possessing extremely high practical value.

[0005] The first aspect of the present invention provides a method for inducing differentiation of human kidney organoids, comprising the following steps:

[0006] (1) Human induced pluripotent stem cells were treated with medium A containing a ROCK pathway inhibitor for 18 to 24 hours;

[0007] (2) Change to medium B containing GSK-3 inhibitor for 1 to 4 days;

[0008] (3) Change to medium B containing GSK-3 inhibitor, FGF-9, and Heparin for 1 to 3 days;

[0009] (4) Digest the cells to obtain a cell suspension, perform 3D culture, and obtain organoid spheroids;

[0010] (5) Treat with medium B containing GSK-3 inhibitor for 50-70 min, transfer to a culture vessel, and culture with medium B containing FGF-9 and Heparin for 1-6 days;

[0011] (6) Change to medium B without additives and culture for 1 to 10 days to obtain mature kidney organoids.

[0012] In some embodiments of the present invention, the ROCK pathway inhibitor includes but is not limited to Y-27632.

[0013] In some embodiments of the present invention, the final concentration of the ROCK pathway inhibitor in (1) is 9 to 11 μM.

[0014] In some embodiments of the present invention, the final concentration of the ROCK pathway inhibitor in (1) is 10 μM.

[0015] In some embodiments of the present invention, the GSK-3 inhibitor includes but is not limited to at least one of LY2090314, CHIR-98014, BIO-acetoxime, AZD2858, SAR502250, CHIR99021, and CHIR-99021trihydrochloride.

[0016] In some embodiments of the present invention, the GSK-3 inhibitor is CHIR99021.

[0017] In some embodiments of the present invention, the final concentration of the GSK-3 inhibitor in (2) and (3) is 7 to 9 μM.

[0018] In some embodiments of the present invention, the final concentration of the GSK-3 inhibitor in (2) and (3) is 8 μM.

[0019] In some embodiments of the present invention, the final concentration of FGF-9 in (3) and (5) is 180-220 ng / mL.

[0020] In some embodiments of the present invention, the final concentration of FGF-9 in (3) and (5) is 200 ng / mL.

[0021] In some embodiments of the present invention, the final concentration of Heparin in (3) and (5) is 0.8-1.2 μg / mL.

[0022] In some embodiments of the present invention, the final concentration of Heparin in (3) and (5) is 1.0 μg / mL.

[0023] In some embodiments of the present invention, the inoculation amount of cells in (1) is 0.5×10 5 ~1.0×10 5 Cells / 9-10cm 2 .

[0024] In the present invention, the cell culture container is mainly a 6-well plate, and the bottom area of ​​the 6-well plate is about 9.6 cm 2 , the volume of liquid added is about 2 to 2.5 mL. In the present invention, the inventors have verified through experiments that the stem cell plating density has a substantial impact on the induced differentiation effect of human kidney organoids. The main reason is that in the differentiation process of kidney organoids, since the digestion and passage operation is not performed after the start of the first stage of the induced differentiation process (i.e., day 0 to 7), the cells are in a continuous proliferation process during this process, which can easily cause excessive cell proliferation and accumulation in the later stage of this stage, resulting in problems such as insufficient cell nutrition, poor state, and even extrusion and death. Therefore, the initial stem cell seeding density is particularly important for the effect of induced differentiation. The inventors found that based on a 6-well plate, 0.5×10 5 ~1.0×10 5 The relative density of cells / well has a better effect on inducing differentiation.

[0025] Of course, based on the change of culture container, 0.5×10 5 ~1.0×10 5 Cells / 9-10cm 2 It can be converted to 0.5×10 5 ~1.0×10 5 cells / 2 to 2.5 mL or other expressions.

[0026] In some embodiments of the present invention, the inoculation amount of cells in (1) is 0.6×10 5 Cells / 9-10cm 2 .

[0027] When the culture container is a 6-well plate, the inoculation amount of cells in (1) is 0.6×10 5 cells / well.

[0028] In the present invention, when the seeding density is 0.6×10 5 cells / well, the cell density on the 7th day was just close to covering the entire culture plate, about 95%, with clear cell outlines and observable cell tentacles. Organoids were formed on the 17th day of differentiation, with obvious tubule structures, abundant numbers, and compact structures.

[0029] In some embodiments of the present invention, the human induced pluripotent stem cells described in (1) include human urine-derived induced pluripotent stem cells.

[0030] In the present invention, the inventors used human urine-derived induced pluripotent stem cells, human skin fibroblast pluripotent stem cells and human umbilical cord blood mesenchymal pluripotent stem cells as test subjects for comparison. It was found that under the same differentiation conditions, human urine-derived induced pluripotent stem cells differentiated into kidney organoids with uniform structure, compact and regular tubule aggregation, and abundant tubules. Mesenchymal pluripotent stem cells can also differentiate into kidney organoids with some characteristics. Although there are tubule-like structures, the number of tubules is significantly less than that of urine-derived stem cell differentiation, and its structure is also looser than the former. The marginal cells of the kidney organoids differentiated from skin fibroblasts did not show obvious tubular structures, formed compact clumps, and no other obvious differentiated cells were seen. Further immunofluorescence identification results showed that the human kidney organoids differentiated from human urine-derived stem cells specifically and highly expressed the renal tubular cell marker protein LTL, the collecting duct cell marker protein GATA3, and the podocyte marker protein synaptopodin. The stem cells from the other two sources can only express the tubular cell marker protein LTL, while the expression levels of GATA3 and synaptopodin are very low, indicating that their differentiation is incomplete. This shows that the method of the present invention is mainly aimed at differentiating human induced pluripotent stem cells, especially human urine-derived induced pluripotent stem cells into human kidney organoids.

[0031] In some embodiments of the present invention, the 3D culture step in (4) is: high-throughput cell culture using a V-bottom 96-well culture plate and assisted by mechanical stimulation.

[0032] In some embodiments of the present invention, the intensity of the mechanical stimulation is 50-150 rpm.

[0033] In some embodiments of the present invention, the intensity of the mechanical stimulation is 65-100 rpm.

[0034] In some embodiments of the present invention, the duration of mechanical stimulation is 20 to 50 hours.

[0035] In some embodiments of the present invention, the duration of mechanical stimulation is 24 to 48 hours.

[0036] In this study, the inventors discovered that adding a certain degree of mechanical stimulation during the 3D suspension culture stage can effectively promote the proliferation of organoid spheroids. This is primarily due to the fact that, as cells gradually form spheroids, a certain degree of mechanical stimulation increases the nutrient availability of the spheroids from the culture medium. Experimental verification revealed that, under identical cell inoculation conditions, spheroids in the 65 rpm mechanical stimulation group produced significantly larger volumes than those in static culture, with the major and minor diameters of the spheroids in the mechanical stimulation group being nearly twice as large as those in the static control group.

[0037] In this study, the inventors broke away from the conventional method of directly transferring the cell suspension to a transwell culture plate for direct culture. Instead, they seeded the cells in a V-bottom 96-well culture plate for high-throughput 3D suspension culture for 22 to 26 hours before transferring them to a transwell culture plate for culture. This step significantly improved the proliferation and efficiency of organoids during the differentiation phase, resulting in improved organoid uniformity. On day 16 of differentiation, the long and short diameters of kidney organoids grew to 3.56±0.69 and 2.57±0.76 times those on day 9, respectively, compared to the long and short diameters that did not increase significantly under conventional culture.

[0038] In some embodiments of the present invention, the culture vessel in (5) is coated with Matrigel in advance.

[0039] In some embodiments of the present invention, the coating time is 25 to 35 minutes.

[0040] In some embodiments of the present invention, the coating time is 30 min.

[0041] In some embodiments of the present invention, the coating temperature is 35-37°C.

[0042] In some embodiments of the invention, the coating temperature is 37°C.

[0043] In the present invention, the inventors found that under the experimental conditions of pre-coating with matrix gel, the kidney organoids were significantly larger than the uncoated organoids on the 15th day, and the morphology and richness of tubular differentiation under coated conditions were also significantly higher than those without coating. This may be because matrix gel coating improves the nutritional level of cells during the differentiation process, resulting in better cell growth and differentiation effects.

[0044] In some embodiments of the present invention, the culture vessel in (5) includes but is not limited to a transwell plate.

[0045] In some embodiments of the present invention, culture medium A is a pluripotent stem cell culture medium.

[0046] In some embodiments of the present invention, culture medium A includes but is not limited to mTeSR1 medium.

[0047] In some embodiments of the present invention, culture medium B is an adherent culture matrix or a cell differentiation medium.

[0048] In some embodiments of the present invention, culture medium B includes but is not limited to STEMdiff APEL2 medium.

[0049] In some embodiments of the present invention, the method for inducing differentiation of human kidney organoids is specifically as follows:

[0050] On day 0 of the experiment, human induced pluripotent stem cells were collected at a concentration of 0.5×10 5 ~1.0×10 5 Cells / 9-10cm 2 Inoculate into 2-2.5 mL of mTeSR1 medium containing the ROCK pathway inhibitor Y-27632 and culture for 18-24 hours.

[0051] On days 1 to 4 of the experiment, the culture medium was replaced with STEMdiff APEL2 medium supplemented with CHIR99021;

[0052] On days 5 to 7 of the experiment, the culture medium was replaced with STEMdiffAPEL2 medium supplemented with CHIR99021, FGF-9, and Heparin to allow further differentiation;

[0053] On day 8 of the experiment, the cells were digested and resuspended in STEMdiff APEL2 medium containing CHIR99021, FGF-9, and Heparin to obtain a cell suspension. The cell suspension was then transferred to a V-bottom 96-well culture plate for high-throughput 3D culture. The culture plate was placed on a horizontal shaker at 65 rpm for mechanical stimulation in a 37°C, 5% CO2 incubator for 18–24 hours to allow the cells to aggregate into spheres, thereby obtaining high-throughput organoid spheroids.

[0054] On days 9–14 of the experiment, the culture medium was replaced with STEMdiff APEL2 medium containing CHIR99021 for 50–70 min, and the organoid spheroids were transferred to an incubator and cultured in medium B containing FGF-9 and Heparin for 1–6 days.

[0055] On day 15 of the experiment, fresh STEMdiff APEL2 medium was replaced and cultured for 1 to 10 days to obtain mature kidney organoids;

[0056] Among them, the final concentration of CHIR99021 was 7-9 μM, the final concentration of FGF-9 was 180-220 ng / mL, and the final concentration of Heparin was 0.8-1.2 μg / mL.

[0057] In some embodiments of the invention, the mature kidney organoids are obtained on day 24 of the experiment.

[0058] In the present invention, the method of the present invention obtains a high-throughput method for inducing kidney organoid differentiation based on optimal culture conditions (stem cell type, optimal seeding density, coating method, mechanical force stimulation, culture days, etc.), which can stably obtain high-quality kidney organoids in a short time.

[0059] The second aspect of the present invention provides the application of the human kidney organoid induction differentiation method described in the first aspect of the present invention in drug screening, toxicology testing, natural immunity research or viral infection.

[0060] The method of the present invention can efficiently obtain mature kidney organoids for routine kidney organoid-related research and applications, including drug screening, toxicology testing, innate immunity research or viral infection.

[0061] The beneficial effects of the present invention are:

[0062] 1. The high-throughput human kidney organoid induction differentiation method of the present invention is simple to operate and highly targeted. Through clear operations such as stem cell type selection, plating density, differentiation time, mechanical stimulation, and matrix coating, the efficiency of induced differentiation is effectively improved, the differentiation stability of kidney organoids is significantly improved, the time required for differentiation and maturation is reduced, and the proliferation efficiency of kidney organoids is increased, thus solving the problems of uneven differentiation efficiency and complicated operations in conventional methods.

[0063] 2. Based on the method of the present invention, kidney organoids are efficiently constructed with good differentiation effect. The obtained kidney organoids are of high quality, and relevant markers such as the renal tubular marker protein LTL, the collecting duct marker protein GATA3, and the podocyte marker protein synaptopodin can all be detected, which is helpful for research such as drug screening, toxicology testing, innate immunity research, and viral infection testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Flowchart of the stem cell differentiation process in an embodiment of the present invention.

[0065] Figure 2 Schematic diagram of inducers and cell morphology at various stages in the examples of the present invention.

[0066] Figure 3 This is the immunofluorescence detection result of mature kidney organoid markers obtained by differentiating pluripotent stem cells of the present invention.

[0067] Figure 4 Comparison of the structural morphology of kidney organoids differentiated from pluripotent stem cells from different sources.

[0068] Figure 5 To compare the expression of markers of renal organoids differentiated from pluripotent stem cells from different sources.

[0069] Figure 6 Comparison of the morphology of kidney organoids differentiated into different stem cell seeding densities on differentiation day 0.

[0070] Figure 7To compare the proliferation effects of different stem cell inoculation methods during the differentiation of kidney organoids on day 8 of differentiation.

[0071] Figure 8 Comparison of the effects of mechanical stimulation on the proliferation of renal organoids on the 8th day of differentiation. (a) is a physical picture, and (b) is a bar chart. Compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001, and ns indicates no statistical difference.

[0072] Figure 9 Comparison of the effect of matrix gel coating on the proliferation of renal organoids after 9 days of differentiation. a is a physical picture, b is a bar chart. Compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001, ns represents no statistical difference.

[0073] Figure 10 Comparison of the effects of different differentiation times on the maturity of kidney organoids. DETAILED DESCRIPTION

[0074] In order to make the invention purpose, technical solution and technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the present invention and are not intended to limit the present invention.

[0075] Unless otherwise specified, the experimental materials and reagents used are consumables and reagents that can be obtained from conventional commercial channels.

[0076] Example 1 High-throughput kidney organoid differentiation induction method

[0077] In this example, the specific steps of the high-throughput kidney organoid differentiation method are as follows:

[0078] On day 0 of the experiment, normal human induced pluripotent stem cells were obtained and 0.6×10 5 Cells / well were seeded into 6-well plates, 2 mL of mTeSR1 medium containing the ROCK pathway inhibitor Y-27632 was added, and the plates were incubated at 37°C in a 5% CO2 incubator for 1 day. The final concentration of Y-27632 was 10 μM.

[0079] On days 1 to 4 of the experiment, the culture medium was replaced with 2 mL of STEMdiff APEL2 medium supplemented with CHIR99021 to initiate differentiation induction. The final concentration of CHIR99021 was 8 μM.

[0080] On days 5 to 7 of the experiment, the culture medium was replaced with 2 mL of STEMdiff APEL2 medium supplemented with CHIR99021, FGF-9, and Heparin for further differentiation. The final concentrations of CHIR99021, FGF-9, and Heparin were 8 μM, 200 ng / mL, and 1 μg / mL, respectively.

[0081] On day 8 of the experiment, cells were digested with 1 ml of Accutase, centrifuged at 1000 rpm for 5 minutes, and resuspended in 1 ml of STEMdiff APEL2 medium containing CHIR99021, FGF-9, and Heparin to obtain a cell suspension. Cells were counted and 9,000 cells were transferred to a 96-well V-bottom culture plate for high-throughput 3D culture. The plate was placed on a horizontal shaker at 65 rpm for mechanical stimulation and incubated in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to aggregate into spheroids, generating high-throughput organoid spheroids.

[0082] On days 9 to 14 of the experiment, the culture medium in the V-bottom 96-well culture plates was discarded, and 0.2 mL of STEMdiff APEL2 medium containing CHIR99021 (final concentration of CHIR99021 was 5 μM) was added to the organoid spheroids for 60 minutes. The spheroids were then gently aspirated and transferred to 6-well transwell chambers that had been pre-coated with Matrigel for 30 minutes at 37°C. After coating, the coating solution was discarded, and 2 mL of STEMdiff APEL2 medium containing FGF-9 (final concentration of FGF-9 was 200 ng / mL) and Heparin (final concentration of Heparin was 1 μg / mL) was directly added to continue differentiation culture.

[0083] During this process, the organoid spheres further differentiate. Microscopically, cells at the edge of the spheres can be observed to gradually differentiate, forming radiating antennae, and then gradually developing tubular structures.

[0084] On day 15 of the experiment, when numerous tubule-like structures were observed under the microscope, extending radially from the center to the edge of the sphere, it was determined that the kidney organoids had initially formed, kidney-specific genes began to be expressed, and tubules were formed. At this time, fresh STEMdiff APEL2 medium was replaced and cultured for another 9 days to allow the kidney organoids to further mature.

[0085] On the 24th day of the experiment, differentiation was completed and mature kidney organoids were obtained.

[0086] The steps in Example 1 are as follows Figure 1 shown.

[0087] The microscope images at the time points in Example 1 are as follows Figure 2 shown.

[0088] It can be found that before induced differentiation, the stem cells exhibit a typical stem cell morphology, with large nuclei, tightly aggregated cells, and distinct cell clusters with clear boundaries. Most cells are island-like, clustered, or elliptical. After directed differentiation and 3D culture according to the method of this example, the cell colonies form distinct spheres that gradually collapse to form hills. As the cells differentiate, they initially form at the edges of the spheres, then gradually form tubular structures toward the center of the spheres.

[0089] The obtained kidney organoids were further tested for characteristic markers. The specific steps are as follows:

[0090] Use a 200μL pipette tip to make a mark 1mm along the circular edge of the mature organoid obtained, and then use a pipette to gently blow a few times along the scratch to make the organoid fall off the culture plate. Carefully remove the organoid and place it in a 6-well plate containing 4% PFA (v / v, paraformaldehyde) at room temperature for 10 minutes, and wash it twice with PBS. Inject OCT embedding agent into the freezing embedding box in advance (be careful not to have bubbles), place the organoid washed with PBS with the largest plane facing up, and then place it flat in OCT, and then add another layer of OCT to completely cover the organoid to complete the embedding. Place the embedded freezing embedding box in a -80℃ refrigerator to allow the embedding material to fully solidify (about 1h), and use a microtome to make frozen sections with a thickness of 10μm. After the sectioning is completed, select appropriate sections for immunofluorescence experiments.

[0091] Before immunofluorescence analysis, frozen sections were fixed in 300 μL of 4% PFA solution (300 μL / section) at room temperature for 10 minutes. Residual PFA was washed off twice with PBS. Cells were then treated with 300 μL of 0.1% Triton X-100 for 10 minutes at room temperature to fully permeabilize the cells. Residual Triton X-100 was washed off twice with PBS. Blocking was performed with 5% BSA (300 μL / section) at room temperature for 1 hour. The blocking solution was discarded, and primary antibody dilutions (including anti-LTL at a 1:1000 dilution, anti-GATA3 at a 1:200 dilution, and anti-synaptopodin at a 1:200 dilution) diluted in 5% BSA were added and incubated at room temperature for 2 hours. Residual primary antibody was washed off three times with PBST (PBS containing 0.1% Tween 20). Add 300 μL of secondary antibody dilution (1:100 goat anti-rabbit Alexa-647 and 1:100 goat anti-mouse Alexa-594) diluted in 5% BSA and incubate at room temperature for 1 hour. Wash away any residual secondary antibody with PBST, repeating three times. Add one drop of DAPI-containing anti-fluorescence quenching mounting medium (Invitrogen) and incubate at room temperature for 1 hour in the dark.

[0092] Using confocal microscopy, the results were Figure 3 shown.

[0093] It can be found that the kidney organoids cultured in this example specifically express the renal tubular cell marker protein LTL, the collecting duct cell marker protein GATA3, and the podocyte marker protein synaptopodin, indicating that the kidney organoids differentiated into mature renal tubular cells, collecting duct cells, and podocytes, indicating that the organoids differentiated successfully.

[0094] Based on the morphology of the cell colonies and the results of marker protein detection, it can be seen that the differentiation method in the embodiment of the present invention can obtain kidney organoids with typical renal tubular cells.

[0095] Example 2 High-throughput kidney organoid differentiation induction method

[0096] In this example, the specific steps of the high-throughput kidney organoid differentiation method are as follows:

[0097] On day 0 of the experiment, normal human induced pluripotent stem cells were obtained and 0.6×10 5 Cells / well were seeded into 6-well plates, 2 mL of mTeSR1 medium containing the ROCK pathway inhibitor Y-27632 was added, and the plates were incubated at 37°C in a 5% CO2 incubator for 1 day. The final concentration of Y-27632 was 10 μM.

[0098] On days 1 to 4 of the experiment, the culture medium was replaced with 2 mL of STEMdiff APEL2 medium supplemented with CHIR99021 to initiate differentiation induction. The final concentration of CHIR99021 was 8 μM.

[0099] On days 5 to 7 of the experiment, the culture medium was replaced with 2 mL of STEMdiff APEL2 medium supplemented with CHIR99021, FGF-9, and Heparin for further differentiation. The final concentrations of CHIR99021, FGF-9, and Heparin were 8 μM, 200 ng / mL, and 1 μg / mL, respectively.

[0100] On day 8 of the experiment, cells were digested with 1 ml of Accutase, centrifuged at 1000 rpm for 5 minutes, and resuspended in 1 ml of STEMdiff APEL2 medium containing CHIR99021, FGF-9, and Heparin to obtain a cell suspension. Cells were counted and 18,000 cells were transferred to a 96-well V-bottom culture plate for 3D culture. The plate was placed on a horizontal shaker at 100 rpm for mechanical stimulation and incubated in a 37°C, 5% CO2 incubator for 48 hours to allow the cells to aggregate into spheroids, thus generating high-throughput organoid spheroids.

[0101] On days 9 to 14 of the experiment, the culture medium in the V-bottom 96-well culture plates was discarded, and 0.2 mL of STEMdiff APEL2 medium containing CHIR99021 (final concentration of CHIR99021 was 5 μM) was added to the organoid spheroids for 60 minutes. The spheroids were then gently aspirated and transferred to 6-well transwell chambers that had been pre-coated with Matrigel for 30 minutes at 37°C. After coating, the coating solution was discarded, and 2 mL of STEMdiff APEL2 medium containing FGF-9 (final concentration of FGF-9 was 200 ng / mL) and Heparin (final concentration of Heparin was 1 μg / mL) was directly added to continue differentiation culture.

[0102] During this process, the organoid spheres further differentiate. Microscopically, cells at the edge of the spheres can be observed to gradually differentiate, forming radiating antennae, and then gradually developing tubular structures.

[0103] On day 15 of the experiment, when numerous tubule-like structures were observed under the microscope, extending radially from the center to the edge of the sphere, it was determined that the kidney organoids had initially formed, kidney-specific genes began to be expressed, and tubules were formed. At this time, fresh STEMdiff APEL2 medium was replaced and cultured for another 9 days to allow the kidney organoids to further mature.

[0104] On the 24th day of the experiment, differentiation was completed and mature kidney organoids were obtained.

[0105] Effects of different cells on the differentiation of human kidney organoids

[0106] The induced differentiation culture method is the same as that in Example 1, except that: in the example, the stem cells used for induced differentiation are human urine-derived induced pluripotent stem cells, human skin fibroblast pluripotent stem cells, and human umbilical cord blood mesenchymal pluripotent stem cells.

[0107] Human urine-derived induced pluripotent stem cells, human skin fibroblast-derived pluripotent stem cells, and human umbilical cord blood mesenchymal stem cells all have the potential to be used to differentiate into human kidney organoids. The inventors selected stem cells from these three sources for induced differentiation based on the methods in the above examples, and compared the differences in indicators such as the formation of organoid structures and the expression levels of human kidney marker proteins.

[0108] The results are as follows Figures 4-5 shown.

[0109] It can be found that under the same differentiation conditions as in Example 1, human urine-derived induced pluripotent stem cells differentiated into kidney organoids with uniform structure, compact and regular tubule aggregation, and abundant tubules. Mesenchymal pluripotent stem cells can also differentiate into kidney organoids with some characteristics. Although they have tubule-like structures, the number of tubules is significantly less than that of urine-derived stem cells, and their structure is also looser than the former. However, the edge cells of the kidney organoids differentiated from skin fibroblast stem cells did not show obvious tubular structures, forming compact clumps, and no other obvious differentiated cells were seen. Further immunofluorescence identification results showed that the human kidney organoids differentiated from human urine-derived stem cells specifically and highly expressed the renal tubular cell marker protein LTL, the collecting duct cell marker protein GATA3, and the podocyte marker protein synaptopodin. The stem cells from the other two sources can only express the tubular cell marker protein LTL, and the expression levels of GATA3 and synaptopodin are very low, indicating that their differentiation is incomplete, indicating that the above embodiments are mainly aimed at human induced pluripotent stem cells, especially human urine-derived induced pluripotent stem cells differentiated into human kidney organoids.

[0110] Effect of stem cell plating density on the differentiation of human kidney organoids

[0111] The induction differentiation culture method is the same as that in Example 1, except that: in this example, on day 0, the stem cell plating density is 0.6×10 5 cells / well and 1.0×10 5 cells / well.

[0112] During the differentiation process of kidney organoids, since digestion and passage are not performed after the first stage of differentiation induction (i.e., days 0 to 7), the cells are in a continuous proliferation process. This can easily lead to excessive cell proliferation and accumulation in the later stages of this stage, resulting in malnutrition, poor cell health, and even extrusion and death. Therefore, the initial stem cell seeding density is particularly important for the effectiveness of differentiation induction.

[0113] In this embodiment, the inventors based on 0.6×10 5 cells / well and 1.0×10 5 The cell / well seeding density was adjusted to observe the cell density on the 7th day and the stem cell differentiation structure morphology on the 17th day under a microscope to evaluate the effect of stem cell plating density on the induced differentiation effect of human kidney organoids.

[0114] The results are as follows Figure 6 shown.

[0115] It can be found that when the seeding density is 0.6×10 5 cells / well, the cell density on the 7th day was just close to filling the culture plate, about 95%, with clear cell outlines and cell tentacles. Organoids were formed on the 17th day of differentiation, with obvious tubule structures, abundant numbers and compact structures. However, when the seeding density was 1.0×10 5 cells / well, the cell density on day 7 was relatively crowded, and some cells were squeezed out and died, the cell outlines were unclear, and cell tentacles could not be observed. On day 17 of differentiation, the spheroids did not show obvious differentiated structures, and no tubule-like structures were observed. Therefore, it can be shown that at the beginning of stem cell differentiation of human kidney organoids, the seeding density was 0.6×10 5 cells / well (6-well plate) is conducive to the induction of differentiation of human kidney organoids.

[0116] Effect of culture medium on the differentiation of human kidney organoids

[0117] The induction differentiation culture method was the same as that in Example 1, except that: in this example, the cells were treated differently on day 8 and then plated separately, that is, after collecting the cell suspension on day 7, group A was counted and centrifuged to obtain cell clusters that were directly inoculated on transwell culture plates; after group B was counted, the cells were first inoculated on a V-bottom 96-well culture plate for 3D suspension culture for 24 h before the spheres were inoculated on a transwell culture plate.

[0118] The results are as follows Figure 7 and as shown in Table 1.

[0119] Table 1 Effects of different treatment methods on the induction and differentiation of human kidney organoids

[0120]

[0121] Note: The long and short diameter data are normalized for easy comparison. Figure 8 )

[0122] Among the methods for kidney organoid differentiation, there are 2D planar culture and 3D suspension culture, but there is no research on the effect of the two on the proliferation of kidney organoid differentiation, nor has there been a comparison between suspension culture in a V-bottom 96-well plate and direct inoculation of cell aggregates after centrifugation. Figure 7 As shown in the figure, it can be found that the cell spheroids obtained after 24 hours of 3D suspension culture on day 8 can significantly improve the proliferation effect of organoids in the subsequent differentiation stage. On the 16th day of differentiation, the long and short diameters of the kidney organoids grew to 3.56±0.69 and 2.57±0.76 times that of the day 9, respectively. However, when the cells were directly collected and centrifuged on the 8th day and inoculated into transwell culture plates in the form of cell clusters, no significant proliferation occurred. Therefore, it is extremely important to obtain cell spheroids after 24 hours of 3D suspension culture on day 8 and then inoculate them for the induction of differentiation of human kidney organoids.

[0123] Effects of mechanical stimulation on the differentiation of human kidney organoids

[0124] The induction differentiation culture method was the same as in Example 1, except that: in this example, different mechanical stimulations were performed when the cells were seeded into a V-bottom 96-well culture plate for 3D suspension culture on day 8. The control group was cultured statically, and the experimental group was cultured for 24 hours with mechanical stimulation at 65 rpm. Subsequently, the spheres were seeded onto a transwell culture plate for continued culture.

[0125] The results are as follows Figure 8 shown.

[0126] Since the cells gradually formed spheres during 3D suspension culture on the 8th day, in order to increase the nutrients obtained by the spheres from the culture medium, the method in the embodiment of the present invention placed the V-shaped 96-well culture plate used for 3D suspension culture on a horizontal shaker for a certain degree of mechanical stimulation, and the stimulation intensity was preferably 65 rpm for 24 hours. The results showed that under the same cell inoculation conditions, the volume of the spheres in the 65 rpm mechanical stimulation group was significantly larger than that of the static cultured spheres, and the major and minor diameters of the spheres in the mechanical stimulation group were nearly 2 times that of the static control group, respectively. Therefore, by introducing 65 rpm mechanical stimulation for 24 hours during 3D suspension culture, the proliferation of organoid spheres can be effectively promoted.

[0127] Effects of transwell coating on the differentiation of human kidney organoids

[0128] The induction and differentiation culture method was the same as in Example 1, except that in this example, spheroids were plated onto transwell plates on day 9 and divided into two groups: a control group with no transwell coating, and an experimental group with transwells pre-coated with Matrigel for 30 minutes at 37°C. The proliferation and differentiation of renal organoids in each group were evaluated at day 9 and day 15, respectively.

[0129] The results are as follows Figure 9 shown.

[0130] It can be found that under the experimental conditions of pre-coating with matrix gel, the kidney organoids were significantly larger than the uncoated organoids on the 15th day, and the morphology and richness of tubular differentiation under coated conditions were also significantly higher than those without coating. This may be because matrix gel coating improves the nutritional level of cells during the differentiation process, resulting in better cell growth and differentiation effects.

[0131] Effect of culture time on the differentiation of human kidney organoids

[0132] The induction differentiation culture method was the same as in Example 1, except that: in this example, after the culture medium was replaced on the 15th day, the culture was continued to the 18th day, the 24th day, the 27th day, and the 30th day respectively to observe the differences in the maturation of the kidney organoids at different culture days.

[0133] The results are as follows Figure 10 shown.

[0134] It can be found that the renal tubular cell marker LTL is expressed from the 18th to the 30th day of culture, but the LTL expression level is significantly higher from the 24th to the 27th day than at other times, and begins to decrease on the 30th day. From the 18th to the 24th day, the expression of the collecting duct cell marker protein GATA3 gradually increases, begins to decrease on the 27th day, and there is almost no GATA3 expression on the 30th day. The podocyte marker protein is expressed most highly on the 24th day, and gradually decreases on the 27th and 30th days. It can be seen that based on the method in this embodiment, the 24th day is the optimal maturation time for kidney organoids, which is also significantly shorter than the time in the conventional method.

[0135] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for inducing differentiation of human kidney organoids, characterized in that: The steps include: (1) adding culture medium A containing a ROCK pathway inhibitor to human induced pluripotent stem cells for 18 to 24 hours; the culture medium A is mTeSR1 culture medium; (2) Change to medium B containing GSK-3 inhibitor for 1 to 4 days; (3) replacing the culture medium with culture medium B containing GSK-3 inhibitor, FGF-9 and Heparin for 1 to 3 days; the culture medium B is STEMdiff APEL2 culture medium; (4) Digest the cells to obtain a cell suspension, perform 3D culture, and obtain organoid spheroids; (5) Treat with medium B containing GSK-3 inhibitor for 50-70 min, transfer to a culture vessel, and culture with medium B containing FGF-9 and Heparin for 1-6 days; (6) Change to medium B without additives and culture for 1 to 10 days to obtain mature kidney organoids; The ROCK pathway inhibitor is Y-27632; The GSK-3 inhibitor is CHIR99021; (1) The inoculation volume of cells was 0.6×10 5 Cells / 9-10cm 2 ; The human induced pluripotent stem cells described in (1) are human urine-derived induced pluripotent stem cells; The steps of 3D culture described in (4) are: culturing cells using a V-shaped 96-well culture plate and assisting with mechanical stimulation; The intensity of the mechanical stimulation is 50 to 150 rpm, and the duration of the mechanical stimulation is 20 to 50 hours; The culture vessel described in (5) is coated with Matrigel in advance; the coating time is 25 to 35 minutes, and the coating temperature is 35 to 37°C.

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