A method of induced differentiation of pluripotent stem cell-derived kidney organoids

Abstract

The application provides a method for inducing differentiation of pluripotent stem cell-derived kidney organoids, which is realized through five key stages: firstly, pluripotent stem cells are inoculated 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 agonists and Noggin factors are added in the Day 0-4 stage to induce primitive streak differentiation; in the Day 4 stage, the cells are inoculated in a low-adhesion U-shaped bottom plate to form spheroids; FGF9 and Activin A factors are added in the Day 4-7 stage to induce mesoderm; FGF9 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 the formation of kidney units. The whole differentiation cycle of the method is not more than 20 days, the whole process does not need to be embedded with Matrigel, the organoids can be obtained which express LRP2, PODXL and E-cadherin at the same time, and contain 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.

Description

Technical Field

[0001] This invention relates to the field of cell engineering technology, and in particular to a method for inducing differentiation of pluripotent stem cell-derived kidney organoids. Background Technology

[0002] Kidney organoids, as three-dimensional in vitro models that mimic the development, disease, and drug response of human nephrons, are attracting 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 directionally induced to differentiate into kidney organoids.

[0003] In recent years, researchers have developed various methods for inducing the differentiation of kidney organoids from human pluripotent stem cells (hPSCs). These methods typically involve regulating signaling pathways such as Wnt, TGF-β, and FGF to directionally induce the differentiation of hPSCs into kidney progenitor cells, which then self-assemble to form kidney organoids. For example, CN113943695A discloses a method for directionally inducing the differentiation of hPSCs into kidney progenitor cells by regulating the Wnt, TGF-β, and FGF signaling pathways, followed by preparing the progenitor cells into a single-cell suspension, which then self-assembles to form kidney organoids.

[0004] However, existing publicly available PSC-derived kidney organoid differentiation schemes generally have the following shortcomings:

[0005] 1. High cultivation cost: Most methods rely on the encapsulation of large amounts of expensive growth factors with substrate gels (such as Matrigel®), resulting in high cultivation costs;

[0006] 2. Complex process: Existing methods usually require multiple wall-mounting / re-suspending or embedding operations, which are complex, require frequent manual intervention, and increase the difficulty of operation and the risk of contamination.

[0007] 3. Long cycle: It usually takes 23 to 30 days to form a mature nephron structure, which is not conducive to the rapid acquisition of experimental materials and clinical applications;

[0008] 4. Limited scalability: Matrix gel 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 that is lower in cost, simpler in procedure, shorter in cycle, and suitable for automated and large-scale production. Summary of the Invention

[0010] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for inducing differentiation of pluripotent stem cell-derived kidney organoids, which solves the problems of high culture cost, complex process, long cycle and limited scalability of the existing PSC-derived kidney organoid differentiation scheme.

[0011] To achieve the above and other related objectives, this invention provides a method for inducing differentiation of pluripotent stem cell-derived kidney organoids, the overall process of which is as follows: Figure 1 As shown, it includes the following steps:

[0012] (1) Day-1 phase: Pluripotent stem cells were seeded into 24-well plates coated with matrix gel and cultured in medium A containing ROCK inhibitors to allow the cells to form a single cell adherent state with appropriate density.

[0013] (2) Day 0-4 stage: cultured in medium B containing WNT agonist and Noggin factor to induce differentiation of primitive stripes;

[0014] (3) Day 4 stage: The cells were seeded in a low-adhesion U-shaped base plate to form spheres and cultured using the culture medium of Day 4-7.

[0015] (4) Day 4-7 stage: Cultured in medium B containing FGF-9 and Activin A to induce interstitial mesoderm;

[0016] (5) Day 7-11 stage: cultured in medium B containing FGF-9 factor to induce renal mesenchymal tissue;

[0017] (6) Day 11-18 stage: Change to culture medium B without added factors for culture to promote nephron formation;

[0018] The entire differentiation cycle takes no more than 20 days, and the organoids do not require matrix gel embedding throughout the process.

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

[0020] Furthermore, in step (1), the inoculation density is 2.0 × 10⁻⁶. 4 cells / cm 2 ~3.0×10 4 cells / cm 2 Preferably 2.5×10 4 cells / cm 2 500 μL / well.

[0021] Furthermore, in step (1), pluripotent stem cells are seeded in a 24-well plate coated with matrix gel. The pluripotent stem cells form a single-cell adherent state with appropriate density, rather than the sheet-like community growth during conventional maintenance culture, thus laying the foundation for subsequent differentiation.

[0022] Furthermore, the matrix gel is selected from Matrigel® and is a quality-verified matrix gel product specifically designed for pluripotent stem cell culture.

[0023] Furthermore, 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] Furthermore, in step (1), the culture time is 24 hours.

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

[0026] Furthermore, pluripotent stem cells are pre-cultured before inoculation.

[0027] The pre-culture process is as follows: pluripotent stem cells are cultured in 6-well plates coated with matrix gel, and the medium is changed daily at 2 mL / well of mTeSR1 medium; when the cell confluence is about 60%-70%, the cells are digested with 1 mL / well of Gentle Cell Dissociation Reagent for 5 minutes, and adherent cells are separated in mTeSR1 medium using a cell scraper, and small cell sheets are passaged at a 1:6-8 ratio; when the cell confluence is about 80%, Day-1 operation is performed.

[0028] Furthermore, the culture medium B in steps (2) to (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] Furthermore, in step (2), the WNT agonist is selected from CHIR99021 and its concentration is 8 μM; the concentration of the Noggin factor is 5 ng / mL.

[0030] Furthermore, during the Day 0 to Day 4 phases, the culture medium used in Day 0-4 was changed at 500 μL / well per day.

[0031] Furthermore, in step (2), high expression of the original stripe markers TBXT and MIXL1 was detected on day 4 to confirm staged differentiation.

[0032] During Day 0-4, pluripotent stem cells were induced to differentiate into primitive stripes under the influence of WNT agonists and Noggin. Fresh culture medium and factors were introduced daily to maintain a stable induction environment. Days 1-4 involved 2D adherent culture. After confirming the primitive stripes on day 4, cells were seeded onto a low-adhesion U-shaped substrate to form 3D spheres, followed by a fully suspended culture process.

[0033] Furthermore, during the Day 4-7 culture period, the concentration of FGF-9 was 20 ng / mL, and the concentration of Activin A was 10 ng / mL.

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

[0035] Furthermore, inoculate 1×10 per well. 5 cells, 200 μL / well.

[0036] Furthermore, in step (4), the expression of IM markers such as PAX2 and LHX1 is detected on day 7.

[0037] During Days 4-7, primitive striped cells were induced to differentiate into mesenchymal cells under the combined action of FGF-9 and Activin A. The morphological changes of the cell spheroids were observed daily without changing the medium.

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

[0039] From day 7 to day 11, the culture medium was changed to Advanced RPMI 1640 + 1X GlutaMAX medium, with only FGF9 added. Activin A was removed during this stage, and only FGF9 was used to induce further differentiation of the mesenchyme into the metanephron lineage. The medium was changed once on day 9.

[0040] Furthermore, the expression of MM markers such as SIX2 and WT1 was detected on day 11.

[0041] Furthermore, in step (6), during the Day 11-18 phase, the inducing factor was removed, and the culture medium used was Advanced RPMI 1640 + 1X GlutaMAX basal medium.

[0042] Furthermore, in step (6), the culture medium volume is 200 μL / well, and the medium is changed every 2-3 days to continue culturing.

[0043] Furthermore, in step (6), the total positive rate of PODXL, E-cadherin, and LRP2 detected on Day 18 was >50%; the nephron-like structures were clear.

[0044] From day 11 to day 18, the culture medium was replaced with Advanced RPMI 1640 + 1X GlutaMAX basal medium, which contained no growth factors. During this stage, cells spontaneously differentiated to form nephron structures. The basal medium was changed every two to three days to provide necessary nutritional support for the cells while avoiding interference from exogenous factors in the spontaneous differentiation process. On day 18, the organoids showed glomerular clusters and tubular lumens, and expressed maturation markers such as PODXL, E-cadherin, and LRP2.

[0045] As described above, the method for inducing differentiation of pluripotent stem cell-derived kidney organoids of the present invention has the following beneficial effects:

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

[0047] 2. Significantly reduced costs: Compared to the classic Takasato method, the present invention requires more than 50% fewer types and doses of growth factors, and eliminates the need for Matrigel for organoid embedding, thus significantly reducing culture costs.

[0048] 3. Compressed cycle: This invention can obtain mature nephron structures in just 18 days, which is better than the traditional method that requires a culture cycle of 23 to 30 days, thus improving the efficiency of research and application.

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

[0050] 5. Wide range of applications: This invention has been successfully demonstrated in human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC), and theoretically it 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.

[0052] The pluripotent stem cells used in this application are selected from mature and commercially available human embryonic stem cell lines or human induced pluripotent stem cells. Attached Figure Description

[0053] Figure 1 This is an overall flowchart of the method for inducing differentiation of pluripotent stem cell-derived kidney organoids disclosed in this invention.

[0054] Figure 2 This is a schematic diagram of the organoid morphology at each stage in Embodiment 1 of the present invention.

[0055] Figure 3 This is an immunofluorescence image of organoids in the Day 18 stage of Example 1 of the present invention. Detailed Implementation

[0056] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0057] Example 1

[0058] Step 1: Cell preparation and adherent culture (Day 1)

[0059] Initiation of cell pre-culture:

[0060] First, human embryonic stem cells (hESCs) were cultured in 6-well plates coated with hESC-qualified-Matrigel, with 2 mL / well of mTeSR1 medium changed daily. When the cell confluence was about 60%-70%, the cells were digested with 1 mL / well of Gentle Cell Dissociation Reagent for 5 minutes. Adherent cells were then separated in mTeSR1 medium using a cell scraper, and small cell sheets were passaged at a 1:6-8 ratio. When the cells had good morphology and the cell confluence was about 80%, Day-1 was performed.

[0061] Day 1: Induction Initiation and 24-well Plate Inoculation

[0062] hESCs were recovered from 6-well plates using 1 mL / well of the mild digestive enzyme Accutase and digested for 7–10 minutes. The plates were then centrifuged at 200 g for 4 minutes and resuspended in mTeSR1 medium + 10 μM Y27632. The resuspending was repeated approximately 2.5 × 10⁻⁶ times. 4 The cells / cm² were seeded at a density of 500 μL / well in 24-well plates coated with hESC-qualified-Matrigel and incubated for 24 h.

[0063] Step Two: Primary Stripe Differentiation Stage (Day 0-4)

[0064] 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.

[0065] On day 4, high expression of the original stripe markers TBXT and MIXL1 was detected to confirm the staged differentiation.

[0066] Step 3: Entering the 3D spheroidization stage (Day 4)

[0067] 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.

[0068] Step 4: Intermediate Mesodermal (IM) Induction Stage (Day 4-7)

[0069] No fluid change is required during Days 4-7.

[0070] On day 7, the expression of IM markers such as PAX2 and LHX1 was detected.

[0071] Step 5: Metarenal Mesenchymal (MM) Induction Phase (Days 7-11)

[0072] 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.

[0073] On day 11, the expression of MM markers such as SIX2 and WT1 was detected.

[0074] Step Six: Nephron Formation Stage (Days 11-18)

[0075] From day 11 to day 18, the culture medium was replaced with Advanced RPMI 1640 + 1X GlutaMAX basal medium (200 μL / well) without any growth factors, and the medium was changed every 2-3 days. During this stage, cells spontaneously differentiated to form nephron structures.

[0076] The entire differentiation process lasts 18 days, not exceeding 20 days, and the organoids do not require matrix gel embedding throughout the process.

[0077] Results: At Day 18, the overall positive rate for PODXL, E-cadherin, and LRP2 was >50%; Figure 3 As shown, the nephron-like structures are clearly visible. The morphology of organoid cells at each stage is as follows: Figure 2 As shown.

[0078] Conclusion: At Day 18, the obtained kidney organoids simultaneously expressed LRP2, PODXL, and E-cadherin markers and contained glomerular clusters and tubular structures, indicating that kidney-characteristic organoids were successfully obtained.

[0079] Example 2

[0080] Step 1: Cell Preparation and Spheroid Formation (~Day-1)

[0081] Initiation of cell pre-culture:

[0082] First, human induced pluripotent stem cells (hiPSCs) were cultured in 6-well plates coated with hiPSC-qualified-Matrigel, with 2 mL / well of mTeSR1 medium changed daily. When the cell confluence was about 60%-70%, the cells were digested with 1 mL / well of Gentle Cell Dissociation Reagent for 5 minutes. Adherent cells were then separated in mTeSR1 medium using a cell scraper, and small cell sheets were passaged at a 1:6-8 ratio. When the cells had good morphology and the cell confluence was about 80%, Day-1 was performed.

[0083] Day 1: Induction Initiation and 24-well Plate Inoculation

[0084] HiPSCs were recovered from 6-well plates using 1 mL / well of the mild digestive enzyme Accutase and digested for 7–10 minutes. The plates were then centrifuged at 200 g for 4 minutes and resuspended in mTeSR1 medium + 10 μM Y27632. The culture was then repeated approximately 2.5 × 10⁻⁶ times. 4The cells / cm² were seeded at a density of 500 μL / well in 24-well plates coated with hiPSC-qualified-Matrigel and incubated for 24 h.

[0085] Step Two: Primary Stripe Differentiation Stage (Day 0-4)

[0086] 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.

[0087] On day 4, high expression of the original stripe markers TBXT and MIXL1 was detected to confirm the staged differentiation.

[0088] Step 3: Entering the 3D spheroidization stage (Day 4)

[0089] 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.

[0090] Step 4: Intermediate Mesodermal (IM) Induction Stage (Day 4-7)

[0091] No fluid change is required during Days 4-7.

[0092] On day 7, the expression of IM markers such as PAX2 and LHX1 was detected.

[0093] Step 5: Metarenal Mesenchymal (MM) Induction Phase (Days 7-11)

[0094] 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.

[0095] On day 11, the expression of MM markers such as SIX2 and WT1 was detected.

[0096] Step Six: Nephron Formation Stage (Days 11-18)

[0097] From day 11 to day 18, the culture medium was replaced with Advanced RPMI 1640 + 1X GlutaMAX basal medium (200 μL / well) without any growth factors, and the medium was changed every 2-3 days. During this stage, cells spontaneously differentiated to form nephron structures.

[0098] The entire differentiation process lasts 18 days, not exceeding 20 days, and the organoids do not require matrix gel embedding throughout the process.

[0099] Results: At Day 18, the total positive rate of PODXL, E-cadherin, and LRP2 was >50%; the nephron-like structures were clear.

[0100] Conclusion: At Day 18, the obtained kidney organoids simultaneously expressed LRP2, PODXL, and E-cadherin markers and contained glomerular clusters and tubular structures, indicating that kidney-characteristic organoids were successfully obtained.

[0101] In summary, the differentiation induction method of this invention is achieved through five key stages: First, pluripotent stem cells are seeded into 24-well plates coated with matrix gel, allowing the cells to form a single-cell adherent state at an appropriate density; then, during Day 0-4, WNT agonists and Noggin are added to induce primitive stripe differentiation; during Day 4, cells are seeded into a low-adhesion U-shaped substrate to form spheroids; during Day 4-7, FGF-9 and Activin A are added to induce mesenchymal differentiation; during Day 7-11, FGF-9 is added to induce renal mesenchymal differentiation; and during Day 11-18, no additional factors are needed to promote nephron formation. The entire differentiation cycle of this method does not exceed 20 days, requires no matrix gel embedding, and yields organoids simultaneously expressing LRP2, PODXL, and E-cadherin, containing glomerular clusters and tubular structures. This method is characterized by its simplicity, short cycle, and low cost, and can be used for kidney development research, drug screening, and disease model construction. Therefore, this invention effectively overcomes the various shortcomings of the prior art and has high industrial application value.

[0102] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for inducing differentiation of pluripotent stem cell-derived kidney organoids, characterized in that, Includes the following steps: (1) Day-1 phase: Pluripotent stem cells were seeded into 24-well plates coated with matrix gel and cultured in culture medium A containing ROCK inhibitors. The culture medium A is a pluripotent stem cell culture medium, and the pluripotent stem cell culture medium is mTeSR1 medium; The ROCK inhibitor is Y-27632, and the concentration of the ROCK inhibitor in mTeSR1 medium is 10 μM; (2) Day 0-4 stage: cultured in medium B containing WNT agonist and Noggin factor to induce differentiation of primitive stripes; The WNT agonist was selected from CHIR99021, with a concentration of 8 μM, and the Noggin factor concentration was 5 ng / mL. (3) Day 4 stage: The cells were seeded in a low-adhesion U-shaped base plate to form spheres and cultured using the culture medium from Day 4 to 7. (4) Day 4-7 stage: cultured in medium B containing FGF-9 and Activin A to induce interstitial mesoderm, wherein the concentration of FGF-9 is 20 ng / mL and the concentration of Activin A is 10 ng / mL; (5) Day 7-11 stage: cultured in medium B with only FGF-9 added to induce renal mesenchymal tissue. The concentration of FGF-9 was 20 ng / mL. (6) Day 11-18: The culture was changed to culture medium B without added factors to promote the formation of nephrons. Culture medium B is a cell differentiation medium. The cell differentiation medium includes Advanced RPMI 1640 + 1X GlutaMAX medium. The pluripotent stem cells are selected from mature and commercially available human embryonic stem cell lines or human induced pluripotent stem cells.

2. The method for inducing differentiation of pluripotent stem cell-derived kidney organoids according to claim 1, characterized in that, In step (1), the inoculation density is 2.5 × cells / .

3. The method for inducing differentiation of pluripotent stem cell-derived kidney organoids according to claim 1, characterized in that, In step (3), the 3D spheres are formed from the primitive striped cells of Day 4, with a seeding density of 1× cells / pores.

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