CN113943695A - Method for establishing kidney organoid by stem cell induced differentiation - Google Patents
Method for establishing kidney organoid by stem cell induced differentiation Download PDFInfo
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Abstract
The invention belongs to the field of cell engineering, and particularly relates to a method for establishing kidney organoids by stem cell induced differentiation. The invention directionally induces and differentiates the human pluripotent stem cells into the kidney progenitor cells by regulating Wnt, TGF-beta and FGF signal channels, then prepares the progenitor cells into single cell suspension, and forms the kidney organoid through self-assembly. The method for inducing differentiation of the stem cells can efficiently and stably establish kidney organoids, and can provide a strong platform for researching kidney diseases and evaluating the kidney toxicity.
Description
Technical Field
The invention belongs to the field of cell engineering, and particularly relates to a method for establishing kidney organoids by stem cell induced differentiation.
Background
The kidney is an important human organ and has the physiological functions of eliminating in-vivo metabolic waste, regulating electrolyte balance, secreting renin, erythropoietin and the like. Studies have shown that adult kidneys filter approximately 200L of fluid a day. However, after birth, kidney tissue gradually loses its regenerative function. The kidney is irreversibly damaged by aging, diseases, environment and other factors, and the function of the kidney is gradually impaired, thereby causing kidney diseases. As the disease progresses, eventually leading to end-stage renal disease, dialysis or kidney transplantation is required. Studies have shown that about 10% of the world's population suffers from kidney disease. Kidney diseases seriously harm public health and cause huge loss on economic and social development.
The stem cell is a cell with the capability of self-replication and renewal, and can directionally induce and differentiate in vitro to form various body cells so as to establish corresponding organoids. In recent years, organoids established by stem cell induced differentiation provide new techniques and methods for studying pathogenic mechanisms of organ diseases and exploring sources of organ transplantation. The team of the medical Harvard USA establishes a method for inducing stem cells to differentiate into kidney organoids, and related patents are applied. However, this method has problems of unstable differentiation and low differentiation efficiency.
Disclosure of Invention
The invention aims to provide a method for establishing kidney organoids by inducing stem cells to differentiate.
A method for establishing a kidney organoid by inducing differentiation of stem cells according to an embodiment of the present invention comprises the steps of:
(1) preparing human induced pluripotent stem cells into a single cell suspension;
(2) adding 10 μ M Rock inhibitor to the single cell suspension obtained in step (1) at 1.5X 104/cm2Inoculating cells at the cell density of (1) and culturing;
(3) when the cells cultured in the step (2) grow to 50% of the density, adding a differentiation culture solution, a wnt signal pathway activator and a TGF-beta signal pathway inhibitor, counting as 0 day of differentiation, and changing the solution every day;
(4) on the 4 th day of differentiation, the culture was continued for 72 hours using the differentiation medium, and the medium was changed every day;
(5) on the 7 th day of differentiation, FGF9 was added to the differentiation medium and cultured for 48 hours, with the medium changed every day;
(6) on the 9 th day of differentiation, FGF9 and wnt signaling pathway activators were added to the differentiation medium;
(7) when the cells are differentiated to the 11 th day, the cells are changed into a differentiation culture solution containing FGF9, and the cells are continuously cultured, wherein the solution is changed every day; if 3D kidney organoid is not established, maintaining the differentiated cells in a differentiation culture solution containing FGF9 for culture until the differentiation is finished;
if 3D kidney organoids are to be established, the cultured cells are digested from day11 to day 14 of differentiation to prepare a single cell suspension, and then cultured in a differentiation medium containing Rock inhibitor and FGF9, and then cultured in a differentiation medium containing FGF9 the next day until differentiation is completed.
According to the method for establishing the kidney organoid by inducing stem cells to differentiate, 1-20 mu M wnt signal pathway activator and 0.1-2 mu M TGF-beta signal pathway inhibitor are added into a differentiation culture solution.
Currently commercially available activators of the Wnt signaling pathway are: CHIR-99021, KY19382, CP21R7(CP21), CGP 57380 and the like, and the preferred CHIR-99021 is used as an activator of the Wnt signal pathway.
Currently commercially available TGF- β signalling pathway inhibitors: a-83-01, LDN-193189, SD-208, GW788388, SRI-011381, LY2109761, SB431542 and the like, with SB431542 being preferred as a TGF-beta signaling pathway inhibitor in the present invention.
According to the method for establishing the kidney organoid by inducing the differentiation of the stem cells, 40-80 ng/mL FGF9 is added into a differentiation culture solution on the 7 th day of differentiation.
According to the method for establishing the kidney organoid by inducing the differentiation of the stem cells, 40-80 ng/mL FGF9 and 1-5 mu M wnt signaling pathway activator are added into a differentiation culture solution on the 9 th day of differentiation.
Wherein, FGF9 can be selected from 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, or FGF9 can be selected from any non-integer between 40-80 ng/mL, such as 40.1ng/mL, 40.2ng/mL, 40.3ng/mL, 40.4ng/mL, 40.5ng/mL, 40.6ng/mL, 40.7ng/mL, 40.8ng/mL, 40.9ng/mLv, 50.1ng/mL, 50.2ng/mL, 50.3ng/mL, 50.4ng/mL, 50.5ng/mL, 50.6ng/mL, 50.7ng/mL, 50.8ng/mL, 50.9ng/mL, etc.
Alternative concentrations of wnt signaling pathway activators include 1 μ M, 2 μ M, 3 μ M, 4 μ M, or 5 μ M.
According to the method for establishing the kidney organoid by inducing the differentiation of the stem cells, in the step (7), the cells are changed to the differentiation culture solution containing 40-80 ng/mL FGF9 for continuous culture until the 11 th day of differentiation.
The method for establishing kidney organoids by inducing stem cell differentiation according to an embodiment of the present invention comprises the step (1) of culturing human induced pluripotent stem cells in mTesR medium at 37 ℃ with 5% CO2The culture box is changed every day; when the cell density reaches 70-80%, digesting to form single cell suspension.
The method for establishing kidney organoids by inducing stem cell differentiation according to an embodiment of the present invention comprises the step (2) of inoculating the cells in 5% CO2After culturing at 37 ℃ for 24 hours, the culture medium was changed and the culture was continued for 48 hours.
According to the method for establishing kidney organoids by inducing stem cell differentiation of the present invention, in step (7), 10 μ M Rock inhibitor and 50ng/mL FGF9 are added to the obtained single cell suspension in a differentiation medium according to a ratio of 3.5X 104The number of cells of (2) the single cell suspension was transferred to a 96-well low adsorption plate, centrifuged, and transferred to 5% CO at 37 ℃2In an incubator.
The invention has the beneficial effects that:
the invention establishes a brand-new stem cell differentiation mode, directionally induces and differentiates human pluripotent stem cells into kidney progenitor cells by regulating Wnt, TGF-beta and FGF signal channels, then prepares the progenitor cells into single cell suspension, and forms kidney organoids through self-assembly. The established kidney organoids are shown to contain structures such as glomeruli, proximal tubules, distal tubules, interstitial cells and the like through immunofluorescence staining. The content of various kidney cell components was identified using flow cytometry.
The method for inducing differentiation of the stem cells can efficiently and stably establish kidney organoids, and can provide a strong platform for researching kidney diseases and evaluating the kidney toxicity. The method has stable differentiation and high differentiation efficiency.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a morphogram and immunofluorescence map of human induced pluripotent stem cells;
FIG. 2 is a photo-scoping of kidney organoids established by differentiation of human induced pluripotent stem cells under 2D culture conditions;
FIG. 3 is a 3D kidney organoid light map and immunofluorescence staining showing organoid cell composition;
FIG. 4 is a graph showing the results of screening for differentiation conditions;
FIG. 5 is a comparison of light before and after optimization of the differentiation process;
FIG. 6 shows flow cytometry results before and after condition optimization;
FIG. 7 shows the results of comparing the stability of the method of the present invention with that of the prior art differentiation method.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
Accutase: a cell digest; accutase is a cell dissociation reagent, a collagenase and proteolytic enzyme based cell digest.
Rock inhibitor: is a high-efficiency inhibitor of human ROCK-1 and ROCK-2 isoenzymes, and can avoid the apoptosis related to dissociation of stem cells after single cell planting.
Advanced RPMI: kidney differentiation basal medium.
mTesR medium: stem cells maintain the medium.
FGF 9: fibroblast growth factor 9(FGF9), promotes the differentiation of renal progenitor cells to form mature renal cells.
Example 1 establishment of Kidney organoids by induced differentiation of Stem cells
1.12 establishment of Kidney organoids
1. Culturing human induced pluripotent stem cells in mTesR medium at 37 deg.C with 5% CO2The culture chamber (2) was changed every day to maintain the cells in good growth state (as shown in FIG. 1, light microscope), and the marker expression state (as shown in FIG. 1, fluorescence staining) showing its multipotential state was good in immunofluorescence staining.
2. When the cell density reaches 70-80%, using Accutase to make digestion to form single cell suspension, adding 10 μ M Rock inhibitor according to 1.5 × 104/cm2Cell density of (a) seeding the cells. At 5% CO2After culturing at 37 ℃ for 24 hours, the culture medium was changed and the culture was continued for 48 hours.
3. When the cells grew to 50% density (day 0 of differentiation), wnt signaling pathway activator CHIR-99021 (10. mu.M) and TGF-signaling pathway inhibitor SB431542 (1. mu.M) were added to the differentiation and differentiation medium Advanced RPMI, and the medium was changed every day.
After 4.96 hours (day 4 of differentiation), culture was continued for 72 hours using differentiation medium Advanced RPMI, with daily fluid changes.
5. On day7 of differentiation, 50ng/mL FGF9 was added to the differentiation medium Advanced RPMI and cultured for 48 hours, with the medium changed daily.
6. Differentiation day9 FGF9 at 50ng/mL and 3. mu.M CHIR-99021 were added to the differentiation medium Advanced RPMI.
7. On day11 of differentiation, cells were changed to Advanced RPMI and cultured by adding 50ng/mL FGF9, and the medium was changed daily until differentiation was complete.
1.23D establishment of Kidney organoids
Steps 1-6 are the same as example 1.1;
and 7: when a sprout-like structure was observed under a light microscope during the period from day11 to day 14, a single cell suspension was prepared by digesting with Accutase, and 10. mu.M Rock inhibitor and 50ng/mL FGF9 were added to the differentiation medium Advanced RPMI as 3.5X 104Cell number of (2) transferring the single cell suspension to a 96-well low adsorption plate, centrifuging at 200g for 5 minutes, and then transferring the cells to 5% CO at 37 ℃2In an incubator.
And 8: the next day of 3D kidney organoid establishment until day20 of differentiation, kidney organoids were cultured in differentiation medium Advanced RPMI containing 50ng/mL FGF 9.
The stem cell differentiation was photographed at a critical time point of differentiation, and as a result, as shown in fig. 2, the morphological structure of the cells was significantly changed by day7 (day 7) of differentiation, and a small spot-like cell cluster was clearly observed due to the refractive index of the differentiated cells and other cells at this stage. By day 12 (day 12) of differentiation, a convex cell mass appeared under the light mirror. By day 21 (day 21) of differentiation, a very large number of structures consisting of kidney cells have been observed.
When stem cells differentiated to the progenitor stage, they were fabricated into 3D kidney organoids, and the results are shown in the light mirror image in fig. 3. According to the invention, immunofluorescence staining is carried out to identify the cell structure composition, and the 3D kidney organoid established by the invention is found to have a kidney proximal tubule (LTL positive structure), a kidney distal tubule (CDH1 positive structure) and a kidney glomerulus (PODXL positive structure), so that the 3D kidney organoid obtained by the invention is in accordance with the normal kidney organ physiological structure.
Example 2 comparison of the Effect of different agents on Stem cell induced differentiation
The invention compares the influence of different reagents on the differentiation of human induced pluripotent stem cells, and the specific experimental conditions are shown in the following table:
the differentiation results of the differentiation method 24 in the above table are shown in FIG. 4, and only by using the conditions 16, 17, and 18, the differentiation of the human-induced pluripotent stem cells was successful. In the differentiation conditions 16, the structures obtained by differentiation under the condition 16 are clearer and more mature than those obtained under the conditions 17 and 18.
Example 3 comparison of Stem cell induced differentiation results
Experimental groups: inducing the differentiated stem cells by adopting the method of the embodiment 1;
control group: differentiating day0-day4, adding CHIR-9902110. mu.M to the differentiation medium; day4-day7, adding ActivinA 10ng/ml in differentiation medium; day7-day9, FGF 910 ng/m added to the differentiation medium; day9-day11, FGF 910 ng/ml and CHIR-990213. mu.M were added to the differentiation medium; day9-day20, FGF 910 ng/ml was added to the differentiation medium.
When the differentiation method of the control group is used, the differentiation result is unstable, the differentiation failure (without any structure) often occurs at the late stage of differentiation or the partial incomplete development result occasionally occurs (as shown in the left graph of fig. 5), and the right graph of fig. 5 is the result obtained by the experimental formula method, the differentiation result is stable, and the number of the structures obtained by differentiation is large.
Renal progenitor cells are progenitor cells that differentiate into various renal structures, and are generally used as criteria for evaluating the success or failure of differentiation and the efficiency of differentiation. Therefore, the present invention performs flow cytometry on the kidney progenitor cells of the control group and the experimental group to determine the percentage of the kidney progenitor cells. SIX1 is a biomarker for kidney progenitor cells. As shown in fig. 6, the control group had a renal progenitor content of only 7.59%, whereas the experimental group had a renal progenitor content of 63.6%.
Five consecutive experiments were performed according to the methods of the experimental group and the control group to compare the stability of the method of the present invention with the existing differentiation method.
The results are shown in fig. 7, five successive differentiation experiments were carried out, the experimental group was successfully differentiated for 5 successive times, and a large number of kidney-like structures appeared, showing extremely strong stability and extremely high differentiation efficiency. In contrast, in the control group, 5 consecutive differentiation experiments showed few occasional differentiation results, indicating that the differentiation results were unstable.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (8)
1. A method for establishing a kidney organoid by inducing differentiation of stem cells, comprising the steps of:
(1) preparing human induced pluripotent stem cells into a single cell suspension;
(2) adding Rock inhibitor into the single-cell suspension obtained in the step (1) according to the proportion of 1.5 multiplied by 104/cm2Inoculating cells at the cell density of (1) and culturing;
(3) when the cells cultured in the step (2) grow to 50% of the density, adding a differentiation culture solution, a wnt signal pathway activator and a TGF-beta signal pathway inhibitor, counting as 0 day of differentiation, and changing the solution every day;
(4) on the 4 th day of differentiation, the culture was continued for 72 hours using the differentiation medium, and the medium was changed every day;
(5) on the 7 th day of differentiation, FGF9 was added to the differentiation medium and cultured for 48 hours, with the medium changed every day;
(6) on the 9 th day of differentiation, FGF9 and wnt signaling pathway activators were added to the differentiation medium;
(7) when the differentiation is completed to day11, the cells are changed into a differentiation culture solution containing FGF9, and the culture is continued, and the solution is changed every day until the differentiation is completed; or,
and (3) preparing the cultured cells into single cell suspension on the 11 th to 14 th days of differentiation, culturing the cells in a differentiation culture solution containing Rock inhibitor and FGF9, and changing the cells into the differentiation culture solution containing FGF9 for culturing the cells on the next day until the differentiation is finished.
2. The method for establishing kidney organoids by stem cell-induced differentiation according to claim 1, wherein in step (3), 1 to 20 μ M wnt signaling pathway activator and 0.1 to 2 μ M TGF- β signaling pathway inhibitor are added to the differentiation culture solution.
3. The method for establishing a kidney organoid by inducing differentiation of stem cells according to claim 1, wherein in step (5), 40 to 80ng/mL of FGF9 is added to the differentiation medium at day7 of differentiation.
4. The method for establishing kidney organoids by stem cell-induced differentiation according to claim 1, wherein in step (6), 40 to 80ng/mL FGF9 and 1 to 5 μ M wnt signaling pathway activator are added to the differentiation culture solution at day9 of differentiation.
5. The method for establishing kidney organoids by inducing differentiation of stem cells according to claim 1, wherein in step (7), the cells are cultured by replacing the cells with a differentiation medium containing 40-80 ng/mL of FGF9 until day11 of differentiation.
6. The method for establishing kidney organoids by stem cell-induced differentiation according to claim 1, wherein in step (1), the human induced pluripotent stem cells are cultured in mTesR medium at 37 ℃ with 5% CO2The culture box is changed every day; when the cell density reaches 70-80%, digesting to form single cell suspension.
7. The method for establishing kidney organoids by stem cell-induced differentiation according to claim 6, wherein in step (2), the seeded cells are cultured in 5% CO2After culturing at 37 ℃ for 24 hours, the culture medium was changed and the culture was continued for 48 hours.
8. Stem cell induction according to claim 1The method for establishing the kidney organoid through differentiation is characterized in that in the step (7), 10 mu M Rock inhibitor and 40-80 ng/mL FGF9 are added into the obtained single cell suspension in a differentiation culture solution according to the proportion of 3.5 multiplied by 104The number of cells of (2) the single cell suspension was transferred to a 96-well low adsorption plate, centrifuged, and transferred to 5% CO at 37 ℃2In an incubator.
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| WO2023146292A1 (en) * | 2022-01-28 | 2023-08-03 | 가톨릭대학교 산학협력단 | Composition for improving maturity of renal organoid comprising twist1 inhibitor as active ingredient |
| WO2023155921A1 (en) * | 2022-02-21 | 2023-08-24 | 广州华越肾科再生医学科技有限公司 | Method for preparing renal podocytes by discontinuous differentiation |
| CN117305222A (en) * | 2023-11-28 | 2023-12-29 | 广州华越肾科再生医学科技有限公司 | Method for differentiating pluripotent stem cells into kidney progenitor cells |
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| CN110468095A (en) * | 2019-09-05 | 2019-11-19 | 安徽中盛溯源生物科技有限公司 | A kind of renal epithelial cell and its preparation method and application |
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| CN110468095A (en) * | 2019-09-05 | 2019-11-19 | 安徽中盛溯源生物科技有限公司 | A kind of renal epithelial cell and its preparation method and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023146292A1 (en) * | 2022-01-28 | 2023-08-03 | 가톨릭대학교 산학협력단 | Composition for improving maturity of renal organoid comprising twist1 inhibitor as active ingredient |
| WO2023155921A1 (en) * | 2022-02-21 | 2023-08-24 | 广州华越肾科再生医学科技有限公司 | Method for preparing renal podocytes by discontinuous differentiation |
| CN117305222A (en) * | 2023-11-28 | 2023-12-29 | 广州华越肾科再生医学科技有限公司 | Method for differentiating pluripotent stem cells into kidney progenitor cells |
| CN117305222B (en) * | 2023-11-28 | 2024-02-20 | 广州华越肾科再生医学科技有限公司 | Method for differentiating pluripotent stem cells into kidney progenitor cells |
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