Induced differentiation method of pluripotent stem cell derived kidney organoid

Abstract

The invention provides an induced differentiation method of a pluripotent stem cell derived kidney organoid, which is realized through five key stages: firstly, inoculating pluripotent stem cells into a 24-pore plate coated with matrigel, so that the cells form a single cell adherent state with proper density; a WNT agonist and a Noggin factor are added in the Day 0-4 stage to induce original stripe differentiation; the cells are inoculated into the low-adhesiveness U-shaped bottom plate in the Day4 stage to form spheres; in the Day 4-7 stage, FGF9 and Activin A factors are added to induce an intermediary mesoderm; at the Day 7-11 stage, adding FGF9 to induce kidney mesenchymal; and factors do not need to be added to promote the formation of renal units in the Day 11-18 stages. According to the method, the whole differentiation period does not exceed 20 days, matrigel embedding is not needed in the whole process, the organoid capable of simultaneously expressing LRP2, PODXL and E-cadherin can be obtained, and the organoid contains glomerular-like clusters and renal tubule-like structures. The method has the characteristics of simplicity and convenience in operation, short period and low cost, and can be used for kidney development research, drug screening and disease model construction.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell engineering, in particular to a method for inducing differentiation of pluripotent stem cell-derived kidney organoids. BACKGROUND

[0002] Kidney organoids, as a three-dimensional in vitro model that mimics human nephron development, disease and drug response, have attracted increasing attention. Pluripotent stem cells (PSCs) have been widely used in the construction of kidney organoids. By simulating the activation and inhibition of signaling pathways during embryonic development, PSCs can be directed to differentiate into kidney organoids.

[0003] In recent years, researchers have developed various methods for inducing differentiation of kidney organoids from human pluripotent stem cells (hPSCs). These methods usually regulate Wnt, TGF-β and FGF signaling pathways to direct the differentiation of human pluripotent stem cells into kidney progenitor cells, and then form kidney organoids through self-assembly. For example, CN113943695A discloses a method for inducing differentiation of human pluripotent stem cells into kidney progenitor cells by regulating Wnt, TGF-β and FGF signaling pathways, and then preparing the progenitor cells into a single cell suspension to form kidney organoids through self-assembly.

[0004] However, the existing published PSCs-derived kidney organoid differentiation protocols usually have the following shortcomings:

[0005] 1. High culture cost: Most methods rely on a large amount of expensive growth factors and Matrigel® embedding, resulting in high culture cost;

[0006] 2. Complex process: Existing methods usually require multiple adhesion / resuspension or embedding operations, which are complex, frequent manual intervention, increasing the difficulty of operation and the risk of contamination;

[0007] 3. Long cycle: It usually takes 23-30 days to form mature nephron structure, which is not conducive to rapid access to experimental materials and clinical application;

[0008] 4. Limited scale: Matrigel embedding makes it difficult to achieve high-throughput parallel production in 96-well plates or bioreactors.

[0009] Therefore, it is of great significance to develop a kidney organoid induction method with lower cost, simpler steps, shorter cycle and suitable for automation and large-scale production. SUMMARY

[0010] In view of the above-mentioned disadvantages of the prior art, the purpose of the present application is to provide an induced differentiation method of pluripotent stem cell-derived kidney organoids, for solving the problems of high culture cost, complex process, long cycle and scale limitation of PSCs-derived kidney organoid differentiation scheme in the prior art.

[0011] To achieve the above-mentioned purpose and other related purposes, the present application provides an induced differentiation method of pluripotent stem cell-derived kidney organoids, the overall process is as shown in Figure 1 , comprising the following steps:

[0012] (1) Day-1 stage: pluripotent stem cells are inoculated in a 24-well plate coated with Matrigel, and cultured using a culture medium A containing a ROCK inhibitor, so that the cells form a single cell adherent state with a suitable cell density;

[0013] (2) Day0-4 stage: culture medium B containing WNT agonist and Noggin factor is used for culture to induce primitive streak differentiation;

[0014] (3) Day4 stage: the cells are inoculated in a low-adhesion U-bottom plate to form a sphere, and the culture medium of Day4-7 stage is used for culture;

[0015] (4) Day4-7 stage: culture medium B containing FGF-9 and Activin A factors is used for culture to induce mesoderm;

[0016] (5) Day7-11 stage: culture medium B containing FGF-9 factor is used for culture to induce metanephric mesenchyme;

[0017] (6) Day11-18 stage: culture medium B without additional factors is used for culture to promote the formation of nephron;

[0018] Wherein the entire differentiation cycle is not more than 20 days, and the whole process of the organoid does not need to be embedded in Matrigel.

[0019] Further, in step (1), the pluripotent stem cells are selected from human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSC).

[0020] Further, in step (1), the inoculation density is 2.0×10 4 cells / cm 2 ~3.0×10 4 cells / cm 2 ; preferably 2.5×10 4 cells / cm 2 , 500 μL / well.

[0021] Further, in step (1), the pluripotent stem cells are seeded in a 24-well plate coated with Matrigel, and the pluripotent stem cells form a single cell adherent state with appropriate density, rather than the sheet colony growth in the conventional maintenance culture, laying a foundation for subsequent differentiation.

[0022] Further, the Matrigel is selected from Matrigel®, and is a Matrigel product that is quality verified and specifically suitable for pluripotent stem cell culture.

[0023] Further, the culture medium A in step (1) is a pluripotent stem cell culture medium; the pluripotent stem cell culture medium includes but is not limited to mTeSR1 culture medium.

[0024] Further, in step (1), the culture time is 24 h.

[0025] Further, in step (1), the ROCK pathway inhibitor includes but is not limited to Y-27632; the concentration of the ROCK pathway inhibitor in the mTeSR1 culture medium is 10 μM.

[0026] Further, the pluripotent stem cells are pre-cultured before seeding.

[0027] The pre-culture process is specifically as follows: the pluripotent stem cells are cultured in a 6-well plate coated with Matrigel, and 2 mL / well of mTeSR1 culture medium is used for daily medium replacement; when the cell confluence is about 60%-70%, 1 ml / well of Gentle Cell Dissociation Reagent is used for 5-minute digestion, and the adherent cells are separated using a cell scraper in the mTeSR1 culture medium, and the small sheet-shaped cells are subcultured at a ratio of 1:6-8; when the cell confluence is about 80%, the Day-1 operation is performed.

[0028] Further, the culture medium B in steps (2)-(6) is a cell differentiation culture medium; the cell differentiation culture medium includes but is not limited to Advanced RPMI 1640 + 1X GlutaMAX culture medium.

[0029] Further, in step (2), the WNT agonist is selected from CHIR99021, and the concentration thereof is 8 μM; the concentration of the Noggin factor is 5 ng / mL.

[0030] Further, in the Day 0 to Day 4 stage, 500 μL / well of the culture medium for the Day 0-4 stage is used for daily medium replacement operation.

[0031] Further, in step (2), the high expression of the primitive streak marker TBXT and MIXL1 is detected on the 4th day to confirm the phased differentiation.

[0032] In the Day 0-4 stage, the pluripotent stem cells are induced to differentiate into primitive streak under the action of WNT agonist and Noggin factor. Fresh culture medium and factors are replaced every day to maintain a stable induction environment. Among them, the Day-1~Day4 stage is 2D adherent culture, and after the primitive streak is detected and confirmed on the 4th day, the cells are inoculated into a low-adhesion U-shaped bottom plate to form 3D spheres, and the subsequent whole-suspension process.

[0033] Further, in the Day 4-7 stage culture medium, the concentration of FGF-9 is 20 ng / mL; and the concentration of Activin A factor is 10 ng / mL.

[0034] Further, in step (3), the cells induced in step (2) are gently digested, collected and centrifuged; and then resuspended in a non-adhesion 96-well low-adhesion U-shaped bottom plate using the Day 4-7 culture medium. The cells aggregate to form spheres in the U-shaped bottom and spontaneously form organoid precursor structures; no Matrigel embedding or microencapsulation step is used.

[0035] Further, 1×10 5 cells, 200 μL / well.

[0036] Further, in step (4), the expression of PAX2, LHX1 and other IM markers is detected on the 7th day.

[0037] In the Day 4-7 stage, the primitive streak cells are induced to differentiate into intermediate mesoderm under the joint action of FGF-9 and Activin A. The morphological changes of the cell spheres are observed every day without medium replacement.

[0038] Further, in step (5), the concentration of FGF-9 is 20 ng / mL.

[0039] From the 7th day to the 11th day, the culture medium is replaced with Advanced RPMI 1640 + 1X GlutaMAX medium, and only FGF9 is added. In this stage, Activin A is removed, and only FGF9 is used to induce the intermediate mesoderm to further differentiate into the nephridium lineage. Among them, the medium is replaced once on the 9th day.

[0040] Further, the expression of SIX2, WT1 and other MM markers is detected on the 11th day.

[0041] Further, in step (6), in the Day 11-18 stage, the induction factors are removed, and the culture medium used is Advanced RPMI 1640 + 1X GlutaMAX basic medium.

[0042] Further, in step (6), the culture medium is used in an amount of 200 μL / well, and the medium is replaced once every 2-3 days, and the culture is continued.

[0043] Further, in step (6), the total positive rate of PODXL, E-cadherin, and LRP2 is greater than 50% on Day 18; and a clear nephron-like structure is formed.

[0044] From the 11th day to the 18th day, the culture medium is replaced with an Advanced RPMI1640 + 1X GlutaMAX basal medium without any growth factors. In this stage, the cells spontaneously differentiate to form a nephron structure. The basal medium is replaced every two to three days to provide the necessary nutritional support for the cells while avoiding the interference of exogenous factors in the spontaneous differentiation process. On the 18th day, the organoids exhibit glomerulus-like clusters and tubular lumens, and express mature markers such as PODXL, E-cadherin, and LRP2.

[0045] As described above, the pluripotent stem cell-derived kidney organoid induction and differentiation method of the present application has the following beneficial effects:

[0046] 1. Simple operation: The present application adopts a full-suspension process after the original stripe stage, avoiding the frequent manual intervention caused by the multiple adhesion / resuspension or embedding operations in the traditional method, greatly simplifying the operation process; at the same time, it avoids the problem of high cell density and poor cell state caused by long-time, such as 7-9 days of 2D culture, improving the stability and success rate of the scheme.

[0047] 2. Significant cost reduction: Compared with the classic Takasato scheme, the types and doses of growth factors required by the present application are reduced by more than 50%, and Matrigel is not required for organoid embedding, significantly reducing the culture cost.

[0048] 3. Cycle compression: The present application only needs 18 days to obtain mature nephron structures, which is better than the 23-30 day culture cycle required by the traditional method, improving the research and application efficiency.

[0049] 4. Scale-friendly: The method of the present application can be realized in a 96-well plate, which is convenient for parallel preparation of dozens or even hundreds of organoids, suitable for automated high-throughput culture, and provides the possibility for large-scale production.

[0050] 5. Wide application range: The present application has been proven successful on human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC), and in theory is also applicable to other pluripotent stem cells.

[0051] 6. The generated organoids have complete morphology and accurate expression of molecular markers, and can be widely used in disease modeling and drug screening application fields. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 Overall flow chart for the induced differentiation method of pluripotent stem cell derived kidney organoids disclosed in the present invention.

[0053] Figure 2 Schematic diagram of organoid morphology at each stage in Example 1 of the present invention.

[0054] Figure 3 Immunofluorescence image of organoids at Day 18 stage in Example 1 of the present invention. DETAILED DESCRIPTION

[0055] The present invention will be described by specific working examples, and other advantages and effects of the present invention can be easily understood by those skilled in the art from the contents disclosed in the specification.

[0056] Example 1

[0057] Step 1: Cell preparation and adherent culture (~Day-1)

[0058] Pre-culture of starting cells:

[0059] First, human embryonic stem cells (hESC) were cultured in a 6-well plate coated with hESC-qualified-Matrigel, using mTeSR1 medium 2mL / well for daily replacement; when the cell confluence was about 60%-70%, use Gentle Cell Dissociation Reagent 1ml / well to digest for 5 minutes, use a cell scraper to separate adherent cells in mTeSR1 medium, 1:6-8 to subculture small cell pieces; the cell morphology is good, and when the cell confluence is about 80%, perform Day-1 operation.

[0060] Day -1: Induction of starting and 24-well plate seeding

[0061] Use Accutase 1mL / well to recover and digest hESC from a 6-well plate for 7-10 minutes, centrifuge at 200g x 4 minutes, resuspend with mTeSR1 medium + 10μM Y27632; seed in a 24-well plate coated with hESC-qualified-Matrigel at a density of about 2.5x10 4 cells / cm², 500μL / well, and culture for 24h.

[0062] Step 2: Primary streak differentiation stage (Day 0-4)

[0063] From day 0 to day 4 after cell seeding into 24-well plates, Advanced RPMI 1640 + 1X GlutaMAX medium was used, with the addition of WNT agonist CHIR99021 (8 μM) and Noggin factor (5 ng / mL). The medium was changed daily at 500 μL / well for a total of 4 days.

[0064] On day 4, high expression of the original stripe markers TBXT and MIXL1 was detected to confirm the staged differentiation.

[0065] Step 3: Entering the 3D spheroidization stage (Day 4)

[0066] On day 4, cells in 24-well plates were digested with 300 μL Accutase per well for 7-10 minutes and centrifuged at 200g for 4 minutes. The culture medium was then replaced with Advanced RPMI 1640 + 1X GlutaMAX medium, with the addition of FGF-9 (20 ng / mL) and Activin A (10 ng / mL). Cells were then resuspended in low-adhesion 96-well U-shaped plates, with 1 × 10⁶ cells per well. 5 Cells, 200 μL / well; centrifuged at 200 g for 30 seconds in a 96-well plate to allow cells to aggregate into spheres in a U-shaped bottom, spontaneously forming organoid precursor structures. No Matrigel embedding or microencapsulation steps were used.

[0067] Step 4: Intermediate Mesodermal (IM) Induction Stage (Day 4-7)

[0068] No fluid change is required during Days 4-7.

[0069] On day 7, the expression of IM markers such as PAX2 and LHX1 was detected.

[0070] Step 5: Metarenal Mesenchymal (MM) Induction Phase (Days 7-11)

[0071] From day 7 to day 11, the culture medium was replaced with Advanced RPMI 1640 + 1X GlutaMAX medium, with only FGF9 (20 ng / mL) added at 200 μL / well, and the cells were continued in suspension culture. Activin A was removed during this stage, and only FGF-9 was used to induce further differentiation of the mesenchyme into metanephric mesenchyme. The medium was changed once on day 9 to induce cell differentiation into the metanephric lineage.

[0072] On day 11, the expression of MM markers such as SIX2 and WT1 was detected.

[0073] Step Six: Nephron Formation Stage (Days 11-18)

[0074] From day 11 to day 18, the medium was changed to Advanced RPMI1640 + IX GlutaMAX basal medium without any growth factors, 200 μL / well, 2-3 days of liquid exchange, and continued to culture. At this stage, the cells spontaneously differentiated to form kidney unit structure.

[0075] The whole differentiation process lasted for 18 days, not more than 20 days, and the organoids did not need to be embedded in Matrigel during the whole process.

[0076] Results: At day 18, the total positive rate of PODXL, E-cadherin, LRP2 was > 50%; as shown in the figure, the kidney unit-like structure was clear. The cell morphology of organoids at each stage was as shown in the figure. Figure 3 Figure 2

[0077] Conclusion: At day 18, the kidney organoids obtained expressed LRP2, PODXL, E-cadherin markers, and contained glomerulus-like clusters and tubular structures, which indicated that organoids with kidney characteristics were successfully obtained.

[0078] Example 2

[0079] Step one: cell preparation and spheroid formation (~Day-1)

[0080] Pre-culture of starting cells:

[0081] First, human induced pluripotent stem cells (hiPSC) were cultured in a 6-well plate coated with hiPSC-qualified-Matrigel, using mTeSR1 medium 2 mL / well, and the liquid was changed every day; when the cell confluence was about 60%-70%, use Gentle Cell Dissociation Reagent 1 ml / well to digest for 5 minutes, use cell scraper to separate adherent cells in mTeSR1 medium, 1:6-8 to subculture small piece of cells; the cell morphology is good, and when the cell confluence is about 80%, the Day-1 operation is performed.

[0082] Day -1: Induce starting and 24-well plate seeding

[0083] Use gentle enzyme Accutase 1 mL / well to recover and digest hiPSC from 6-well plate for 7-10 minutes, centrifuge at 200g x 4 minutes, resuspend with mTeSR1 medium + 10 μM Y27632; about 2.5 x 10 4 ​​cells / cm², 500 μL / well seeded in 24-well plates coated with hiPSC-qualified-Matrigel, cultured for 24 h.

[0084] Step Two: Primitive streak differentiation phase (Day 0-4)

[0085] From Day 0 to Day 4 of cell seeding in 24-well plates, Advanced RPMI 1640 + 1X GlutaMAX medium was used, with the addition of WNT agonist CHIR99021 (concentration of 8 μM) and Noggin factor (concentration of 5 ng / mL), 500 μL / well was used for medium exchange operation every day, for a total of 4 days.

[0086] On Day 4, primitive streak marker TBXT, MIXL1 high expression was detected to confirm the phase differentiation.

[0087] Step Three: Enter 3D spheroidization phase (Day 4)

[0088] On Day 4, the cells in the 24-well plate were digested with Accutase 300 μL / well for 7-10 minutes, centrifuged at 200g for 4 minutes; then the medium was changed to Advanced RPMI 1640 + 1X GlutaMAX medium, with the addition of FGF-9 (concentration of 20 ng / mL) and Activin A (concentration of 10 ng / mL); resuspended in low-attachment 96-well U-bottom plates, seeded 1x10 5 cells, 200 μL / well; 96-well plates were centrifuged at 200g for 30 seconds to allow cells to aggregate in the U-bottom to form spheroids, and spontaneously form organoid precursor structures. No Matrigel embedding or microencapsulation step was used.

[0089] Step Four: Intermediate mesoderm (IM) induction phase (Day 4-7)

[0090] Day 4-7 stage does not require medium exchange.

[0091] On Day 7, PAX2, LHX1, and other IM marker expression were detected.

[0092] Step Five: Metanephric mesenchyme (MM) induction phase (Day 7-11)

[0093] From Day 7 to Day 11, replace the medium with Advanced RPMI 1640 + 1X GlutaMAX medium, add only FGF9 (concentration of 20 ng / mL), 200 μL / well, continue to suspend culture. Remove Activin A in this stage, only use FGF-9 to induce mesoderm in the intermediate to further differentiate into the kidney mesenchyme. Change the medium once at Day 9 to induce the cells to differentiate into the kidney lineage.

[0094] At Day 11, detect the expression of MM markers such as SIX2, WT1, etc.

[0095] Step six: nephron formation stage (Day 11-18)

[0096] From Day 11 to Day 18, replace the medium with Advanced RPMI 1640 + 1X GlutaMAX basal medium without any growth factors, 200 μL / well, change the medium once every 2-3 days, continue to culture. In this stage, the cells spontaneously differentiate to form nephron structures.

[0097] The entire differentiation process lasts for 18 days, not more than 20 days, and the organoids do not need to be embedded in Matrigel during the whole process.

[0098] Results: At Day 18, the total positive rate of PODXL, E-cadherin, LRP2 is > 50 %; the nephron-like structure is clear.

[0099] Conclusion: At Day 18, the obtained kidney organoids express LRP2, PODXL, E-cadherin markers, and contain glomerulus-like clusters and tubular-like structures, which indicates that kidney organoids with kidney characteristics have been successfully obtained.

[0100] In summary, the induced differentiation method of the present application is achieved through five key stages: firstly, pluripotent stem cells are seeded in a 24-well plate coated with Matrigel, so that the cells form a single cell adherent state with a suitable cell density; then WNT agonist and Noggin factors are added in the Day 0-4 stage to induce primitive streak differentiation; in the Day 4 stage, the cells are seeded in a low-adhesion U-bottom plate to form spheroids; FGF-9 and Activin A factors are added in the Day 4-7 stage to induce mesendoderm; FGF-9 factor is added in the Day 7-11 stage to induce metanephric mesenchyme; and no factor is added in the Day 11-18 stage to promote nephron formation. The whole differentiation cycle of the method does not exceed 20 days, the whole process does not need to be embedded in Matrigel, and the organoid expressing LRP2, PODXL and E-cadherin at the same time can be obtained, and contains glomerulus-like clusters and tubular structures. The method has the characteristics of simple operation, short cycle and low cost, and can be used for kidney development research, drug screening and disease model construction. Therefore, the present application effectively overcomes the various shortcomings in the prior art and has a high industrial utilization value.

[0101] The above examples only illustrate the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical idea disclosed by the present application should be covered by the claims of the present application.

Claims

1. A method of induced differentiation of pluripotent stem cell-derived kidney organoids, characterized by, The method comprises the following steps: (1) Day-1 stage: pluripotent stem cells are inoculated in a 24-well plate coated with Matrigel, and adherent culture is carried out using culture medium A containing a ROCK inhibitor; (2) Day0-4 stage: culture is carried out using culture medium B containing a WNT agonist and a Noggin factor to induce primitive streak differentiation; (3) Day4 stage: the cells are inoculated in a low-adhesion U-bottom plate to form spheroids, and culture is carried out using culture medium of the Day4-7 stage; (4) Day4-7 stage: culture is carried out using culture medium B containing FGF-9 and Activin A factors to induce mesoderm intermediates; (5) Day7-11 stage: culture is carried out using culture medium B containing FGF-9 factors to induce metanephric mesenchyme; (6) Day11-18 stage: culture is carried out using culture medium B without added factors to promote the formation of nephron units.

2. The method of claim 1, wherein the pluripotent stem cell-derived kidney organoids are induced to differentiate by, The pluripotent stem cells are selected from human embryonic stem cells or human induced pluripotent stem cells.

3. The method of claim 1, wherein the pluripotent stem cell-derived kidney organoid is induced to differentiate by, In step (1), the inoculation density was 2.5 x 10 4 cells / cm 2 .

4. The method of claim 1, wherein the pluripotent stem cell-derived kidney organoids are induced to differentiate by, The culture medium A in step (1) is a pluripotent stem cell culture medium; the pluripotent stem cell culture medium includes but is not limited to mTeSR1 culture medium; the ROCK pathway inhibitor includes but is not limited to Y-27632; the concentration of the ROCK pathway inhibitor in the mTeSR1 culture medium is 10 μM.

5. The method of claim 1, wherein the pluripotent stem cell-derived kidney organoids are induced to differentiate by, The pluripotent stem cells are pre-cultured before inoculation; the pre-culture process is as follows: the pluripotent stem cells are cultured in a 6-well plate coated with Matrigel, and mTeSR1 culture medium 2 mL / well is used for daily medium replacement; when the cell confluence is about 60%-70%, Gentle Cell Dissociation Reagent 1 ml / well is used for digestion for 5 minutes, adherent cells are separated using a cell scraper in the mTeSR1 culture medium, and small piece-shaped cell passage is carried out at a ratio of 1:6-8; when the cell confluence is about 80%, the Day-1 operation is carried out.

6. The method of claim 1, wherein the pluripotent stem cell-derived kidney organoids are induced to differentiate by, The culture medium B is a cell differentiation culture medium; the cell differentiation culture medium includes but is not limited to Advanced RPMI 1640 + 1X GlutaMAX culture medium.

7. The method of claim 1, wherein the pluripotent stem cell-derived kidney organoids are induced to differentiate by, In step (3), the 3D spheroids were formed from Day 4 primary streak cells at a seeding density of 1 x 10 5 cells / well.

8. The method of claim 1, wherein the pluripotent stem cell-derived kidney organoids are induced to differentiate by, In step (2), the WNT agonist is selected from CHIR99021, and the concentration is 8 μM; the concentration of the Noggin factor is 5 ng / mL.

9. The method of claim 1, wherein the pluripotent stem cell-derived kidney organoids are induced to differentiate by, In step (4), the concentration of FGF-9 is 20 ng / mL; the concentration of the Activin A factor is 10 ng / mL.

10. The method of claim 1, wherein the pluripotent stem cell-derived kidney organoids are induced to differentiate by, In step (5), the concentration of FGF-9 is 20 ng / mL.

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