Method for regulating human induced pluripotent stem cells to be differentiated into kidney organoid

Abstract

The invention relates to the technical field of preparation of organoids, and discloses a method for regulating and controlling human induced pluripotent stem cells to differentiate into kidney organoids, which comprises the following steps: resuspending a cell cluster formed by human induced pluripotent stem cells by using a basic differentiation culture solution containing a GSK-3beta inhibitor, inoculating the cell cluster into an ultralow adsorption cell culture plate, and starting to culture, marking the time as the 0th hour of differentiation; and after culturing for 70-76 hours, replacing the basic differentiation culture solution containing the GSK-3beta inhibitor with a basic differentiation culture solution containing a Knockout TM serum substitute (KSR), and continuing to carry out differentiation culture. By adopting the method disclosed by the invention, the human induced pluripotent stem cells can be efficiently, quickly, simply, conveniently and stably subjected to directional induced differentiation into kidney organs containing podocytes, renal tubules and other structures, and the time for constructing a kidney in-vitro model can be shortened, so that the research on kidney diseases, kidney development and the like can be greatly assisted; and a powerful platform is provided for kidney toxicity assessment.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation of organoids, and in particular to a method for regulating the differentiation of human induced pluripotent stem cells into kidney organoids. Background Art

[0002] The kidney is the most important organ in the human urinary system. In the blood circulation system, it undertakes the important mission of filtering metabolic wastes and excreting them out of the body, as well as reabsorbing various nutrients. The nephron is the basic unit of the structure and function of the kidney and an important system for material exchange and energy transmission in the kidney, including the process of glomerular filtration of blood to form primary urine and the reabsorption of nutrients by the renal tubules and collecting ducts and their capillary material exchange. Therefore, the nephron is the basic unit for the kidney microcirculation system to function and plays a decisive role in the normal operation of various kidney functions.

[0003] To study the pathogenesis or regulatory mechanism of kidney development, a powerful in vitro model is needed, and kidney organoids have emerged. Kidney organoids have a three-dimensional structure, contain various cells, results and spatial arrangements of the kidney, and can highly simulate the functions and development of the kidney in vivo. Currently, there are mainly two sources of kidney organoids. One is kidney organoids derived from adult stem cells, which are obtained by shearing, digesting and culturing human tissues. However, the structure of this kind of kidney organoids is mainly composed of renal tubules and hardly contains glomerular structures, so it cannot highly simulate the kidney organs in vivo. The other is kidney organoids derived from human induced pluripotent stem cells. By simulating the changes in the in vivo environment during embryonic development and changing the culture system of human induced pluripotent stem cells, they are induced into kidney organoids. This kind of kidney organoids contains renal tubules and glomerular structures and can simulate kidney organs to a greater extent.

[0004] There have been some reports on the method for regulating the differentiation of human induced pluripotent stem cells into kidney organoids: adding fibroblast growth factor 9 (FGF9) to the culture medium of human induced pluripotent stem cells can regulate the differentiation of human induced pluripotent stem cells in the direction of kidney organoids. However, this method requires the preparation of multiple culture media and daily medium replacement, which is relatively cumbersome; adding spinal cord explants to the culture system of human induced pluripotent stem cells can also regulate the differentiation of human induced pluripotent stem cells in the direction of kidney organoids. However, this method has high requirements for the preparation of experimental materials and is not suitable for large-scale promotion; there are also researchers who perform gene editing on human induced pluripotent stem cells to overexpress genes that develop towards the kidney and induce their differentiation in the direction of kidney organoids. However, this method has high technical requirements for researchers. It is necessary to first have the technical means of gene editing before it is possible to differentiate human induced pluripotent stem cells into kidney organoids. None of the above methods can differentiate kidney organoids quickly, efficiently and simply. Therefore, there is still an urgent need in the current biomedical technology field for a method that can stably, efficiently and conveniently regulate the differentiation of human induced pluripotent stem cells into kidney organoids. Summary of the Invention

[0005] In order to solve the above technical problems, that is, the prior art cannot quickly, efficiently, and simply obtain kidney organoids from human induced pluripotent stem cells, the present invention provides a method for regulating the differentiation of human induced pluripotent stem cells into kidney organoids. Using this method, human induced pluripotent stem cells can be efficiently, quickly, simply, and stably directed to differentiate into kidney organoids.

[0006] The specific technical solution of the present invention is as follows: A method for regulating the differentiation of human induced pluripotent stem cells into kidney organoids, comprising the following steps: (1) Resuspend the cell mass composed of human induced pluripotent stem cells with a basic differentiation culture medium containing a GSK-3β inhibitor, and then inoculate it into an ultra-low attachment cell culture plate to start culturing, recorded as the 0th hour of differentiation; (2) After culturing for 70-76 hours, replace the basic differentiation culture medium containing a GSK-3β inhibitor with a basic differentiation culture medium containing Knockout TM serum replacement (KSR), and continue the differentiation culture.

[0007] The research team of the present invention found that by using a GSK-3β inhibitor in combination with a specific Knockout TM serum replacement (Knockout TM SR, KSR), and adopting a specific usage sequence and timing (that is, first using a GSK-3β inhibitor, and then removing the GSK-3β inhibitor after culturing for a specific time and replacing it with Knockout TM serum replacement (KSR)), human induced pluripotent stem cells can be directed to differentiate into kidney organoids containing podocytes, renal tubules and other structures. Moreover, only two culture media are required throughout the process, with simple and efficient operation, and the time for constructing an in vitro kidney model can be shortened.

[0008] Preferably, in step (1), the GSK-3β inhibitor is CHIR99021.

[0009] Preferably, in step (1), the addition amount of the GSK-3β inhibitor in the basic differentiation culture medium is 1-20 μM.

[0010] Preferably, in step (2), the addition amount of the Knockout TM serum replacement (KSR) in the basic differentiation culture medium is 1-10 wt%.

[0011] Preferably, in steps (1) and (2), the basic differentiation culture medium is composed of bovine serum albumin, L-ascorbic acid-2-phosphate, oleic acid and DMEM / F12 medium.

[0012] In combination with bovine serum albumin, L-ascorbic acid-2-phosphate, and oleic acid, it can promote the differentiation of human induced pluripotent stem cells into kidney organoids.

[0013] Furthermore, in the basic differentiation culture medium, the concentration of bovine serum albumin is 0.25 - 5 wt%.

[0014] Furthermore, in the basic differentiation culture medium, the concentration of L-ascorbic acid-2-phosphate is 5 - 500 μg / mL.

[0015] Furthermore, in the basic differentiation culture medium, the concentration of oleic acid is 0.2 - 20 μM.

[0016] Preferably, in step (2), the time of differentiation culture is 230 - 250 h, and the culture medium is changed every 45 - 50 h during this period.

[0017] Preferably, in step (1), the cell density inoculated into the ultra-low attachment cell culture plate is 1.0×10 4 ~2.0×10 4 cells / cm 2 .

[0018] Preferably, in step (1), the method for preparing the cell mass composed of human induced pluripotent stem cells includes the following steps: culturing human induced pluripotent stem cells in mTesR Plus medium, and when the cell density reaches 80% - 90%, scraping and cutting to obtain the cell mass composed of human induced pluripotent stem cells.

[0019] Compared with the prior art, the present invention has the following advantages: Using the method of the present invention, human induced pluripotent stem cells can be efficiently, rapidly, simply, and stably directed to differentiate into kidney organoids containing podocytes, renal tubules, etc., which can shorten the time for constructing an in vitro kidney model. Therefore, it has good application prospects and can greatly assist in the research of kidney diseases, kidney development, etc., providing a powerful platform for kidney toxicity assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a bright field image of the process of regulating the differentiation of human induced pluripotent stem cells into kidney organoids in Example 1. Among them, Figure 1 A is the bright field image at day 0 of differentiation; Figure 1 B is the bright field image at day 3 of differentiation; Figure 1 C is the bright field image at day 13 of differentiation.

[0021] Figure 2 It is the morphological image and RT-qPCR identification image of human induced pluripotent stem cells. Among them,Figure 2 Bright-field image of human induced pluripotent stem cells; Figure 2 RT-qPCR images of human induced pluripotent stem cells and human non-small cell lung cancer cell line A549 Figure 3 Pathological staining and immunofluorescence staining images of the kidney organoids obtained in Example 1. Among them, Figure 3 A is the pathological staining image of the kidney organoids; Figure 3 B is the immunofluorescence staining image of the kidney organoids. Note: After converting to a colorless form, Figure 3 the different fluorescent colors in B cannot be distinguished, and the blue fluorescence is difficult to show, so Figure 3 a color picture is used for B.

[0022] Figure 4 Bright-field image at day 13 of differentiation in Example 2.

[0023] Figure 5 Bright-field image at day 13 of differentiation in Example 3.

[0024] Figure 6 Bright-field image at day 3 of differentiation in Comparative Example 1.

[0025] Figure 7 Bright-field image at day 5 of differentiation in Comparative Example 2.

[0026] Figure 8 Bright-field image at day 4 of differentiation in Comparative Example 3.

[0027] Figure 9 Bright-field image at day 4 of differentiation in Comparative Example 4.

[0028] Figure 10 Bright-field image at day 4 of differentiation in Comparative Example 5. Detailed implementation methods

[0029] The present invention will be further described below in conjunction with the embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0030] General embodiments A method for regulating the differentiation of human induced pluripotent stem cells into kidney organoids, comprising the following steps: (1) Resuspend the cell mass composed of human induced pluripotent stem cells with a basic differentiation culture medium containing a GSK-3β inhibitor, and then inoculate it into an ultra-low attachment cell culture plate to start culturing, recorded as hour 0 of differentiation; (2) After culturing for 70 - 76 hours, replace the basic differentiation culture medium containing the GSK-3β inhibitor with a basic differentiation culture medium containing Knockout TM serum replacement (KSR), and continue the differentiation culture.

[0031] As a specific implementation manner, in step (1), the GSK-3β inhibitor is CHIR99021.

[0032] As a specific implementation manner, in step (1), the addition amount of the GSK-3β inhibitor in the basic differentiation culture medium is 1 - 20 μM.

[0033] As a specific implementation manner, in step (2), the addition amount of the Knockout TM serum replacement (KSR) in the basic differentiation culture medium is 1 - 10 wt%.

[0034] As a specific implementation manner, in steps (1) and (2), the basic differentiation culture medium is composed of bovine serum albumin, L-ascorbic acid-2-phosphate, oleic acid, and DMEM / F12 medium, wherein the concentration of bovine serum albumin is 0.25 - 5 wt%, the concentration of L-ascorbic acid-2-phosphate is 5 - 500 μg / mL, and the concentration of oleic acid is 0.2 - 20 μM.

[0035] As a specific implementation manner, in step (2), the time of the differentiation culture is 230 - 250 h, and the culture medium is replaced every 45 - 50 h during this period.

[0036] As a specific implementation manner, in step (1), the cell density inoculated into the ultra-low attachment cell culture plate is 1.0×10 4 ~2.0×10 4 cells / cm 2 .

[0037] As a specific implementation manner, in step (1), the preparation method of the cell mass composed of human induced pluripotent stem cells includes the following steps: culturing human induced pluripotent stem cells with mTesR Plus medium, and when the cell density reaches 80% - 90%, scraping and cutting to obtain a cell mass composed of human induced pluripotent stem cells.

[0038] Example 1 By the following steps, regulate the differentiation of human induced pluripotent stem cells into kidney organoids: (1) Culturing of human induced pluripotent stem cells: Human induced pluripotent stem cells were cultured using mTeSR Plus medium purchased from Stemcell Technology, Canada, in an incubator at 37°C and 5% CO2, with the medium changed daily to maintain good cell growth status (as shown in the bright field image of Figure 2 A), and RT-qPCR showed good expression status of its stem cell markers (as shown in the RT-qPCR image of Figure 2 B). Compared with the human non-small cell lung cancer cell line A549, the gene expression levels of stem cell markers SOX2, NANOG, OCT4, and SSEA1 in human induced pluripotent stem cells were significantly increased by 62.70-fold, 6273.26-fold, 48.12-fold, and 4.84-fold, respectively.

[0039] (2) Preparation of basic differentiation culture medium: Bovine serum albumin, L-ascorbic acid-2-phosphate, and oleic acid were added to DMEM / F12 medium at addition amounts of 5 wt%, 500 μg / mL, and 20 μM, respectively, and mixed evenly to obtain the basic differentiation culture medium.

[0040] (3) Inductive differentiation: When the cell density of human induced pluripotent stem cells reached 83%, the human induced pluripotent stem cells were scraped off as a whole with a cell scraper and cut into small cell clusters, resuspended with the basic differentiation culture medium containing 20 μM GSK-3β inhibitor CHIR99021, and inoculated into an ultra-low attachment cell culture plate at a cell density of 1.5×10 4 cells / cm 2 to start culturing, recorded as day 0 (hour 0) of differentiation (the morphology of the small cell clusters is shown in Figure 1 A); embryoid bodies appeared on day 3 of differentiation, with a spherical shape (see Figure 1 B). At 72 hours of differentiation, the cells were completely replaced with the culture medium, and the culture medium was changed to the basic differentiation culture medium containing 10 wt% Knockout TM serum replacement (KSR), and continued to differentiate for 10 days, with the medium changed every 2 days until the differentiation ended. The structure of the embryoid bodies changed significantly, and tubular structures such as folds appeared inside the sphere (see Figure 1 C), and renal organoids were obtained.

[0041] The cell structure composition of the renal organoids obtained in this example was identified by pathological staining and immunofluorescence staining, and the results are shown in Figure 3 . It was found that it had renal tubules (structures positive for CK19) and glomeruli (structures positive for PODXL), indicating that the renal organoids obtained in this example conformed to the physiological structure of normal kidney organs.

[0042] Example 2 Regulate the differentiation of human induced pluripotent stem cells into kidney organoids through the following steps: (1) Culture of human induced pluripotent stem cells: Culture human induced pluripotent stem cells using mTeSR Plus medium purchased from Stemcell Technology, Canada. Culture them in an incubator at 37 °C and 5% CO2, and change the medium daily to maintain good cell growth status.

[0043] (2) Preparation of basic differentiation culture medium: Add bovine serum albumin, L-ascorbic acid-2-phosphate, and oleic acid to DMEM / F12 medium, with the addition amounts being 0.25 wt%, 250 μg / mL, and 0.2 μM respectively. Mix well to obtain the basic differentiation culture medium.

[0044] (3) Inductive differentiation: When the cell density of human induced pluripotent stem cells grows to 85%, use a cell scraper to scrape the human induced pluripotent stem cells as a whole and cut them into small cell clusters. Resuspend them with the basic differentiation culture medium containing 1 μM GSK-3β inhibitor CHIR99021, and inoculate them into an ultra-low attachment cell culture plate at a cell density of 1.0×10 4 cells / cm 2 to start the culture, which is recorded as day 0 (hour 0) of differentiation; embryoid bodies appear on day 3 of differentiation, with a spherical shape. At 76 hours of differentiation, perform a full medium change for the cells, and change the culture medium to the basic differentiation culture medium containing 5 wt% Knockout TM serum replacement (KSR), and continue the differentiation culture for 10 days. During this period, change the medium every 2 days until the differentiation ends. The structure of the embryoid bodies shows obvious changes, and tubular structures such as folds appear inside the sphere (see Figure 4 ), and kidney organoids are obtained.

[0045] Example 3 Regulate the differentiation of human induced pluripotent stem cells into kidney organoids through the following steps: (1) Culture of human induced pluripotent stem cells: Culture human induced pluripotent stem cells using mTeSR Plus medium purchased from Stemcell Technology, Canada. Culture them in an incubator at 37 °C and 5% CO2, and change the medium daily to maintain good cell growth status.

[0046] (2) Preparation of basic differentiation culture medium: Add bovine serum albumin, L-ascorbic acid-2-phosphate, and oleic acid to DMEM / F12 medium, with the addition amounts being 2.5 wt%, 5 μg / mL, and 10 μM respectively. Mix well to obtain the basic differentiation culture medium.

[0047] (3) Inductive differentiation: When the human induced pluripotent stem cells grow to a cell density of 88%, use a cell scraper to scrape the human induced pluripotent stem cells as a whole and cut them into small cell clusters, resuspend them with a basic differentiation culture medium containing 10 μM GSK-3β inhibitor CHIR99021, and inoculate them into an ultra-low attachment cell culture plate at a cell density of 2.0×10 4 cells / cm 2 to start the culture, which is recorded as day 0 (hour 0) of differentiation; embryoid bodies appear on day 3 of differentiation, with a spherical shape. At 70 hours of differentiation, perform a full medium change for the cells, and change the culture medium to a basic differentiation culture medium containing 1 wt% Knockout TM serum replacement (KSR), and continue the differentiation culture for 10 days. During this period, change the medium every 2 days until the differentiation ends. The structure of the embryoid bodies shows obvious changes, and tubular structures such as folds appear inside the spheres (see Figure 5 ), and renal organoids are obtained.

[0048] Comparative Example 1 In this comparative example, KSR and CHIR99021 were added together. The specific steps are as follows: (1) Culture of human induced pluripotent stem cells: Culture human induced pluripotent stem cells using mTeSR Plus medium purchased from Stemcell Technology, Canada, in an incubator at 37°C and 5% CO2, and change the medium every day to keep the cells in good growth condition.

[0049] (2) Preparation of basic differentiation culture medium: Add bovine serum albumin, L-ascorbic acid-2-phosphate, and oleic acid to DMEM / F12 medium, with the addition amounts being 5 wt%, 500 μg / mL, and 20 μM respectively, and mix well to obtain the basic differentiation culture medium.

[0050] (3) Inductive differentiation: When the human induced pluripotent stem cells grow to a cell density of 85%, use a cell scraper to scrape the human induced pluripotent stem cells as a whole and cut them into small cell clusters, resuspend them with a basic differentiation culture medium containing 20 μM GSK-3β inhibitor CHIR99021 and 10 wt% Knockout TM serum replacement (KSR), and inoculate them into an ultra-low attachment cell culture plate at a cell density of 1.5×10 4 cells / cm 2 to start the culture, which is recorded as hour 0 of differentiation; embryoid bodies appear on day 3 of differentiation, and their morphology is observed to be irregular and their sizes are uneven (see Figure 6 ), which will affect the efficiency of subsequent differentiation into renal organoids.

[0051] Comparative Example 2 In this comparative example, the addition time of KSR was delayed, and the specific steps are as follows: (1) Culture of human induced pluripotent stem cells: Human induced pluripotent stem cells were cultured using mTeSR Plus medium purchased from Stemcell Technology, Canada, in an incubator at 37°C and 5% CO2, with medium changed daily to maintain good cell growth status.

[0052] (2) Preparation of basic differentiation culture medium: Bovine serum albumin, L-ascorbic acid-2-phosphate, and oleic acid were added to DMEM / F12 medium, with the addition amounts being 5 wt%, 500 μg / mL, and 20 μM respectively. After mixing, the basic differentiation culture medium was obtained.

[0053] (3) Inductive differentiation: When the cell density of human induced pluripotent stem cells reached 83%, the human induced pluripotent stem cells were scraped off as a whole with a cell scraper and cut into small cell clusters, resuspended with the basic differentiation culture medium containing 20 μM GSK-3β inhibitor CHIR99021, and inoculated into an ultra-low attachment cell culture plate at a cell density of 1.5×10 4 cells / cm 2 to start the culture, which was recorded as day 0 (hour 0) of differentiation; embryoid bodies appeared on day 3 of differentiation, with a spherical shape; at 96 hours of differentiation, the cells were completely replaced with the culture medium, and the culture medium was changed to the basic differentiation culture medium containing 10 wt% Knockout TM serum replacement (KSR). After continuous differentiation culture for 1 day, it was observed that all embryoid bodies disintegrated and all the spheres died (see Figure 7 ), so renal organoids could no longer be obtained.

[0054] Comparative Example 3 In this comparative example, Knockout TM serum replacement (KSR) was replaced with serum replacement N2 supplement, and the specific steps are as follows: (1) Culture of human induced pluripotent stem cells: Human induced pluripotent stem cells were cultured using mTeSR Plus medium purchased from Stemcell Technology, Canada, in an incubator at 37°C and 5% CO2, with medium changed daily to maintain good cell growth status.

[0055] (2) Preparation of basic differentiation culture medium: Add bovine serum albumin, L-ascorbic acid-2-phosphate and oleic acid to DMEM / F12 medium, with the addition amounts being 5 wt%, 500 μg / mL and 20 μM respectively. Mix well to obtain the basic differentiation culture medium.

[0056] (3) Inductive differentiation: When the human induced pluripotent stem cells grow to a cell density of 87%, use a cell scraper to scrape off the human induced pluripotent stem cells as a whole and cut them into small cell clusters. Resuspend them with the basic differentiation culture medium containing 20 μM GSK-3β inhibitor CHIR99021, and inoculate them into an ultra-low attachment cell culture plate at a cell density of 1.5×10 4 cells / cm 2 to start the culture, which is recorded as day 0 (hour 0) of differentiation; embryoid bodies appear on day 3 of differentiation, with a spherical shape. At 72 hours of differentiation, perform a full medium change for the cells, and change the culture medium to the basic differentiation culture medium containing 10 wt% serum replacement N2 supplement. After continuing the differentiation culture for 1 day, it is observed that the embryoid bodies disintegrate, leaving only a few very small spheres (see Figure 8 ), resulting in the failure of differentiation.

[0057] Comparative Example 4 In this comparative example, the Knockout TM serum replacement (KSR) was replaced with fetal bovine serum (FBS), and the specific steps are as follows: (1) Culture of human induced pluripotent stem cells: Culture human induced pluripotent stem cells using mTeSR Plus medium purchased from Stemcell Technology, Canada. Culture them in an incubator at 37°C and 5% CO2, and change the medium every day to keep the cells in good growth condition.

[0058] (2) Preparation of the basic differentiation culture medium: Add bovine serum albumin, L-ascorbic acid-2-phosphate and oleic acid to DMEM / F12 medium, with the addition amounts being 5 wt%, 500 μg / mL and 20 μM respectively. Mix well to obtain the basic differentiation culture medium.

[0059] (3) Inductive differentiation: When the human induced pluripotent stem cells grow to a cell density of 84%, use a cell scraper to scrape off the human induced pluripotent stem cells as a whole and cut them into small cell clusters. Resuspend them with the basic differentiation culture medium containing 20 μM GSK-3β inhibitor CHIR99021, and inoculate them at a cell density of 1.5×10 4 cells / cm 2Cells were inoculated at a cell density into an ultra-low attachment cell culture plate to start culturing, denoted as day 0 (hour 0) of differentiation; embryoid bodies appeared on day 3 of differentiation, with a spherical shape. At 72 hours of differentiation, the cells were completely replaced with fresh medium, and the culture medium was changed to a basic differentiation medium containing 10 wt% fetal bovine serum (FBS). After continued differentiation culture for 1 day, it was observed that the edges of the embryoid bodies were blurred, the morphology collapsed, and only a few cell aggregates remained (see Figure 9 ), resulting in failed differentiation.

[0060] Comparative Example 5 In this comparative example, the Knockout TM serum replacement (KSR) was replaced with serum replacement B27 additive. The specific steps are as follows: (1) Culture of human induced pluripotent stem cells: Human induced pluripotent stem cells were cultured using mTeSR Plus medium purchased from Stemcell Technology, Canada, in an incubator at 37 °C and 5% CO2, and the medium was changed daily to maintain good cell growth status.

[0061] (2) Preparation of basic differentiation medium: Bovine serum albumin, L-ascorbic acid-2-phosphate, and oleic acid were added to DMEM / F12 medium, with the addition amounts being 5 wt%, 500 μg / mL, and 20 μM, respectively. After mixing evenly, the basic differentiation medium was obtained.

[0062] (3) Inductive differentiation: When the cell density of human induced pluripotent stem cells reached 84%, the human induced pluripotent stem cells were scraped off as a whole with a cell scraper and cut into small cell clusters, resuspended with the basic differentiation medium containing 20 μM GSK-3β inhibitor CHIR99021, and inoculated into an ultra-low attachment cell culture plate at a cell density of 1.5×10 4 cells / cm 2 to start culturing, denoted as day 0 (hour 0) of differentiation; embryoid bodies appeared on day 3 of differentiation, with a spherical shape. At 72 hours of differentiation, the cells were completely replaced with fresh medium, and the culture medium was changed to a basic differentiation medium containing 10 wt% serum replacement B27 additive. After continued differentiation culture for 1 day, it was observed that the embryoid bodies aggregated and adhered, forming large cell clusters without rules, and the internal cells could not contact the culture medium, resulting in nutritional deficiency death (see Figure 10 ).

[0063] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the methods used herein are conventional methods unless otherwise specified; the reagents, biological materials, and equipment used are common reagents, biological materials, and equipment in the art and can be obtained commercially unless otherwise specified.

[0064] As described above, the above are only the preferred embodiments of the present invention and do not impose any limitation on the present invention. Any simple modifications, changes, and equivalent transformations made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for regulating the differentiation of human induced pluripotent stem cells into kidney organoids, characterized in that, It includes the following steps: (1) Resuspend the cell mass composed of human induced pluripotent stem cells with a basic differentiation culture medium containing a GSK-3β inhibitor, and then inoculate it into an ultra-low attachment cell culture plate to start culturing, which is recorded as the 0th hour of differentiation; (2) After culturing for 70 to 76 hours, replace the basic differentiation culture medium containing the GSK-3β inhibitor with the basic differentiation culture medium containing Knockout TM serum replacement, and continue the differentiation culture.

2. The method according to claim 1, wherein In step (1), the GSK-3β inhibitor is CHIR99021.

3. The method according to claim 1 or 2, characterized in that, In step (1), the addition amount of the GSK-3β inhibitor in the basic differentiation culture medium is 1-20 μM.

4. The method according to claim 1, characterized in that In step (2), the Knockout TM The addition amount of the serum substitute in the basic differentiation culture medium is 1-10 wt%.

5. The method according to claim 1, characterized in that, In steps (1) and (2), the basic differentiation culture medium is composed of bovine serum albumin, L-ascorbic acid-2-phosphate, oleic acid and DMEM / F12 medium.

6. The method according to claim 5, characterized in that, In the basic differentiation culture medium, the concentration of bovine serum albumin is 0.25-5 wt%.

7. The method according to claim 5, wherein In the basic differentiation culture medium, the concentration of L-ascorbic acid-2-phosphate is 5-500 μg / mL.

8. The method according to claim 5, characterized in that In the basic differentiation culture medium, the concentration of oleic acid is 0.2-20 μM.

9. The method according to claim 1, wherein In step (2), the differentiation culture time is 230-250 h, and the culture medium is changed every 45-50 h during this period.

10. The method according to claim 1, characterized in that In step (1), the cell density inoculated into the ultra-low attachment cell culture plate is 1.0×10 4 ~2.0×10 4 cells / cm 2 .

Images