Method for constructing pluripotent stem cell differentiated kidney proximal tube organ and application of pluripotent stem cell differentiated kidney proximal tube organ

Abstract

The invention provides a method for constructing a pluripotent stem cell differentiated kidney proximal canal organ and application thereof, a three-stage induction method is adopted, the three-stage induction method sequentially comprises mesoderm induction, renal progenitor cell induction and proximal canal differentiation and maturation stage induction, and a kidney proximal canal organ model with real metabolic characteristics is provided. Wherein in the renal progenitor cell stage, the flow cytometry is used for sorting a glomerular proximal tube progenitor cell population with proximal tube potential for the first time, so that the purity and differentiation consistency of target cells are remarkably improved; in a renal progenitor cell induction stage, low-dose window use of TGF-beta1 in promotion of differentiation rather than fibrosis is realized; in the proximal tube differentiation and maturation stage, mitochondrial metabolism function and transport capacity are promoted, brush marginal protein expression is induced through short-time stimulation, and epithelium polarity is improved; and the high-quality kidney proximal tube organ-like sphere is constructed.

Description

Technical Field

[0001] This application relates to the field of cell culture and differentiation technology, and in particular to a method for constructing pluripotent stem cells to differentiate into proximal renal tubule organoids and its application. Background Technology

[0002] Existing methods for constructing kidney organoids mostly focus on simulating the early embryonic development stage of whole nephrons, making it difficult to achieve specific enrichment of renal tubular subtype cells, especially lacking functionally mature proximal tubular structures and metabolic phenotypes. Since the proximal tubules are important targets for research on drug toxicity, diabetic nephropathy, and other diseases, there is an urgent need for an innovative method to construct highly functional renal proximal tubular organoids (RPTOs).

[0003] In recent years, significant progress has been made in the study of renal proximal tubule organoids, and related models have been widely used in drug toxicity screening, disease modeling, and developmental research. However, current construction strategies still face many challenges. First, existing methods have limited differentiation efficiency, and the proportion of proximal tubule cells in organoids is usually low, accompanied by a large number of distal convoluted tubules, collecting ducts, or mesenchymal-like cells, resulting in insufficient cell purity and limitations in studying proximal tubule pathogenesis mechanisms and drug screening. Second, the differentiation cycle is generally long, usually requiring two to three weeks or more, and the induction conditions are complex and the degree of standardization is low, making it difficult to achieve high-throughput applications. In addition, many studies only verify cell identity through immune markers, lacking systematic functional verification of transport function, structural morphology, etc. Given the core role of the renal proximal tubule in key physiological processes such as substance reabsorption and metabolic detoxification, establishing an efficient, stable, highly pure, and functionally characteristic renal proximal tubule organoid model is of great significance for advancing drug screening, toxicity evaluation, and research on the mechanisms of kidney disease. Summary of the Invention

[0004] This application provides a method for constructing pluripotent stem cells to differentiate into proximal renal tubule organoids and its application, in order to solve the problems existing in related technologies. The technical solution is as follows:

[0005] In a first aspect, embodiments of this application provide a method for constructing pluripotent stem cell-differentiated proximal renal tubule organoids, comprising the following steps:

[0006] S1. From day 0 to day 3, human pluripotent stem cells are cultured and induced to differentiate into early mesoderm;

[0007] S2. From day 3 to 8, early mesodermal cells are induced to differentiate into kidney progenitor cells. The culture medium is gradually replaced with kidney progenitor cell induction medium and cultured for 3 to 8 days. The kidney progenitor cell induction medium is Advanced RPMI1640 medium containing B27minus insulin (Gibco, #A1895601, 1×), GlutaMAX (Gibco, #35050061, 1×), and inducing factors. The inducing factors include FGF9, retinoic acid, and BMP7.

[0008] The inducing factor TGF-β1 was further added on days 5 to 8.

[0009] S3. On day 8, CD24+ / CD10+ double-positive cell populations were sorted to obtain sorted and purified renal proximal tubule progenitor cells (RPTPCs).

[0010] S4. The sorted and purified proximal tubular renal progenitor cells were treated with a Collagen IV / Laminin-521 mixture.

[0011] S5. From day 8 to 14, renal proximal duct progenitor cells were induced to differentiate into renal proximal duct organoids (RPTOs) using DMEM / F12 medium containing ITS (Gibco #51500056, 1×), GlutaMAX (Gibco #35050061, 1×), ascorbic acid (StemCell, #72132, 50 μg / mL) and added factors. The added factors included dexamethasone, HGF, EGF, FGF9 and retinoic acid.

[0012] In one embodiment, human pluripotent stem cells include human induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

[0013] In one embodiment, the process of culturing and inducing differentiation of human pluripotent stem cells into early mesoderm includes the following steps:

[0014] S01. Human pluripotent stem cells were cultured using mTeSR on a Matrigel-coated culture device.

[0015] S02. On days 0 to 3, human pluripotent stem cells were inoculated into RPMI 1640 medium supplemented with 1×B27 (Minus insulin); mesodermal induction was performed by adding inducing factors CHIR99021 and Activin A to obtain mesodermal precursor cells.

[0016] In one embodiment, when inducing the differentiation of mesodermal precursor cells into renal progenitor cells, CD24+ / CD10+ double-positive cell populations are sorted using BD AriaIII to purify RPTPCs. The flow cytometry sorting process includes:

[0017] Kidney progenitor cells were digested with Accutase and filtered through a 40 μm filter to obtain a single-cell suspension; the concentration was adjusted to approximately 1 × 10⁻⁶ cells / cells. 7 Cells / mL ~ 2×10⁻⁶ 7 cells / mL;

[0018] Take an appropriate amount of cells and incubate them at 37°C in the dark for 30 min using CD24-APC and CD10-FITC antibodies. During incubation, the cells are placed in a cell culture incubator for further culture. After incubation, wash the cells twice with PBS and resuspend them in PBS containing 1% FBS.

[0019] Start the BD Aria III flow cytometer and select Purity mode to sort cells.

[0020] RPTPCs are early renal epithelial progenitor cells with differentiation potential induced from the mesoderm, capable of differentiating into functional proximal renal tubule cells. They are characterized by the expression of certain levels of stem / progenitor cell markers (such as CD24) and proximal lineage bias (such as CD10 positivity). Pluripotent stem cell differentiation systems often suffer from mixed cell lineages, low purity of proximal renal cells, and poor differentiation efficiency. The lack of specific cell surface marker combinations for targeted sorting and purification of RPTPC populations limits organoid purity and functional modeling capabilities. The CD24 / CD10 double-positive combination can be used to identify and purify proximal tubule-differentiating progenitor cell populations in human pluripotent stem cell differentiation systems. This combination exhibits high lineage specificity in renal developmental biology, superior to single-marker or traditional sorting methods. This application uses CD24 / CD10 dual markers to sort RPTPCs, improving differentiation purity and efficiency, and obtaining RPTPCs cell lines with high purity. This provides ideal RPTPCs cell lines for subsequent directed differentiation of RPTOs organoids, thereby improving the differentiation efficiency and experimental reproducibility of RPTOs organoids.

[0021] In one implementation, the BD Aria III flow cytometer uses an 85μm nozzle and sets the sheath fluid pressure to 45psi; system quality control is performed after checking that the fluid flow is stable.

[0022] The sorting rate is recommended to be controlled at 2000–3000 events / sec; the sorted cells should be placed immediately in pre-cooled culture medium containing FBS.

[0023] In one embodiment, step S4, the process of treating the sorted and purified proximal renal tubule progenitor cells with a Collagen IV / Laminin-521 mixture, includes the following steps:

[0024] The proximal renal tubule progenitor cells obtained in step S3 were placed in a culture medium containing FBS and centrifuged to obtain cell spheres. Then, a mixture of Collagen IV / Laminin-521 was added to coat the cell spheres with Collagen IV / Laminin-521.

[0025] In one embodiment, the FBS content is 5-10% by mass; preferably, the FBS content is 10% by mass.

[0026] In one embodiment, the mass concentrations of Collagen IV and Laminin-521 in the Collagen IV / Laminin-521 mixture are 20–50 μg / mL, respectively. Further, the Collagen IV / Laminin-521 mixture is prepared by mixing 50 μg / mL Collagen IV and 50 μg / mL Laminin-521 at a volume ratio of 1:1.

[0027] In one embodiment, the number-to-volume ratio of proximal renal tubular progenitor cells to the Collagen IV / Laminin-521 mixture is 2000 cells: (5-15) μL.

[0028] In one embodiment, proximal renal tubule progenitor cells are placed in a culture medium containing FBS, and then 5–10 μM of Y27632 is added.

[0029] In one implementation, the process of treating the RPTPCs sorted and purified in step S4 with Collagen IV / Laminin-521 is as follows:

[0030] RPTPCs in FBS-containing medium were transferred to 96-well plates with ultra-low adsorption within 1 hour, at a density of 2000 cells / well. 5-10 μM Y27632 was added, and the 96-well plates were centrifuged at 200g for 2-3 minutes. The cells aggregated at the bottom of the wells to form cell spheres.

[0031] Prepare a Collagen IV / Laminin-521 mixture in advance; add 10 μL / well to a 96-well plate to deposit Collagen IV / Laminin-521 at the bottom of the 96-well plate and coat the RPTPC cell spheres.

[0032] The extracellular matrix (ECM) not only provides structural support but also regulates stem cell fate and organoid structure self-organization through the integrin-FAK signaling pathway and polar protein networks. Collagen IV, derived from basement membrane skeletal proteins, promotes renal tubular basement membrane formation; Laminin-521, a heterotrimer composed of LAMA5 / LAMB2 / LAMC1, is expressed in embryonic kidney tissue and proximal tubule basement membrane, regulating stem cell attachment, self-renewal, and polarity formation. This application systematically introduces a Collagen IV and Laminin-521 composite matrix into the pluripotent stem cell-induced renal proximal tubule organoid construction, replacing traditional Matrigel or single ECM, thereby improving structural specificity and functional maturity. Collagen IV provides a stable scaffold, while Laminin-521 promotes PSC attachment and expansion by binding to integrin α6β1; Laminin-521 specifically mimics the renal developmental microenvironment, activating PI3K / Akt and β-catenin signaling to promote proximal tubule progenitor cell differentiation. This combination induces higher expression of functional genes such as AQP1, LRP2, CUBN, and SLC22A6, improving proximal transport capacity. Compared to Matrigel mixed with ECM, the purified Collagen IV + Laminin-521 combination is more "renal selective," inhibiting differentiation tendencies in other epidermal or neural lineages.

[0033] In one embodiment, in step S2, the concentration of FGF9 is 100-200 ng / mL, the concentration of retinoic acid is 0.1-0.2 μM, the concentration of BMP7 is 50-100 ng / mL, and the concentration of TGF-β1 is 0.5-1 ng / mL.

[0034] Preferably, in step S2, the concentrations of FGF9 and retinoic acid are 200 ng / mL, retinoic acid is 0.1 μM, BMP7 is 50 ng / mL, and TGF-β1 is 0.5–1 ng / mL. TGF-β1 (transforming growth factor-β1) is a member of the TGF-β family and plays a crucial regulatory role in various cell differentiation processes, particularly in embryonic development, nervous system formation, neural stem cell fate determination, and neuronal maturation. However, due to its pro-inflammatory and pro-fibrotic effects, TGF-β1 plays a central regulatory role, especially in chronic inflammation, organ fibrosis, and the tumor microenvironment. TGF-β1 is one of the most critical fibrotic factors, playing a central role in renal fibrosis; therefore, there are currently no reports of its application in differentiating renal cells.

[0035] RPTPCs originate from the mesoderm of the metamesoderm in the kidney, and their formation depends on a complex signaling regulatory network, including Wnt, FGF, BMP, and Retinoic Acid. Currently, the differentiation of pluripotent stem cells into proximal renal tubule progenitor cells (RPPCs) is inefficient and time-consuming. TGF-β1 may activate and promote the differentiation of pluripotent stem cells into RPTPCs. TGF-β1 is expressed in the mesoderm during kidney development, regulating the formation of epithelial-like structures, promoting cell polarity and tight junctions, modulating basement membrane assembly, and participating in the deposition of collagen IV and laminin. Therefore, during the induction of pluripotent stem cells into RPTPCs, moderate activation of TGF-β1 signaling can accelerate the transition from mesoderm to renal progenitors and optimize the polarity and functional maturation of RPTPCs. However, strict control of the TGF-β1 dosage and the window of opportunity is necessary.

[0036] In one embodiment, in step S5, the concentration of dexamethasone is 5-15 nM, the concentration of HGF is 50-100 ng / mL, the concentration of EGF is 10-50 ng / mL, the concentration of FGF9 is 100-200 ng / mL, and the concentration of retinoic acid is 1-2 μM.

[0037] Preferably, in step S5, the concentration of dexamethasone is 10 nM, the concentration of HGF is 50 ng / mL, the concentration of EGF is 10 ng / mL, the concentration of FGF9 is 200 ng / mL, and the concentration of retinoic acid is 1 μM.

[0038] In one embodiment, the concentration of CHIR99021 is 5–10 μM; the concentration of Activin A is 100–200 ng / mL; and CHIR99021 is added on days 0–2, while Activin A is added on days 1–3.

[0039] Preferably, the concentration of CHIR99021 is 8 μM; the concentration of Activin A is 100 ng / mL; and CHIR99021 is added on days 0 to 2, and Activin A is added on days 1 to 3.

[0040] In one embodiment, the culture conditions in step S5 are as follows: the culture medium substrate is pre-coated with Collagen IV / Laminin-521. Preferably, the Collagen IV / Laminin-521 mixture is prepared by mixing 50 μg / mL Collagen IV and 50 μg / mL Laminin-521 at a volume ratio of 1:1. In one embodiment, in step S5, the added factors further include sodium fumarate and / or linoleic acid; the concentration of sodium fumarate is 10–20 μM, and the concentration of linoleic acid is 20–50 μM.

[0041] Sodium fumarate, the sodium salt of fumarate, an intermediate product in the tricarboxylic acid (TCA) cycle, possesses various metabolic regulatory and cellular homeostasis-regulating functions. Using sodium fumarate as a metabolic regulator, it acts on the differentiation stage of renal progenitor cells or the maturation stage of the proximal tubules, improving mitochondrial metabolism, polarity establishment, and brush border formation. Sodium fumarate enhances mitochondrial function; proximal tubule cells highly depend on mitochondrial function for active transport. Sodium fumarate upregulates Na+ / K+-ATPase, OAT1 (SLC22A6), promoting the establishment of proximal tubule function; and activates the Nrf2 signaling pathway, regulating redox homeostasis and promoting the expression of brush border-related factors such as F-actin and Villin. In this application, the addition of sodium fumarate to RPTPCs during the differentiation of RPTOs significantly improved the differentiation efficiency of RPTOs.

[0042] In one embodiment, linoleic acid is added only on days 11 to 13.

[0043] The brush border is a microvilli-like structure at the apex of proximal tubular cells, supported by a large amount of F-actin as its backbone. Marker proteins include Villin, Ezrin, Moesin, and CD10. In renal tubular organoids or differentiation systems, poor brush border formation is an important marker of functional immaturity, limiting the ability of organoids to model drug metabolism and toxicity. Linoleic acid (LAA) is an essential polyunsaturated fatty acid (PUFA) that plays an important role in phospholipid synthesis, signal transduction, and energy metabolism. In the cell membrane, it affects membrane fluidity, lipid raft structure, and regulates the activity of transmembrane receptors and downstream signaling. Low doses (10–50 μM) can regulate lipid metabolism and promote cell polarity and membrane structure maturation. LAA can promote F-actin polymerization and microvilli formation by regulating small GTPase signaling pathways such as RhoA / ROCK, Rac1, and Cdc42. It also enhances the phosphorylation of Ezrin and Moesin, connects F-actin to the plasma membrane, and promotes brush border protein aggregation. Linoleic acid can regulate the ratio of PE, PC, PI, etc. in the phospholipid bilayer of the cell membrane, which is beneficial to the establishment of apical-basal polarity. This application proposes its "tissue maturation stimulation" effect, applying linoleic acid to the differentiation and maturation of RPTOs. Using a concentration of 50 μM on Day 11-13 effectively promotes brush border maturation.

[0044] Secondly, this application provides a renal proximal tubule organoid, which is prepared by the above-described method for constructing pluripotent stem cells to differentiate into renal proximal tubule organoids.

[0045] Thirdly, the embodiments of this application provide the application of the aforementioned proximal renal tubule organoids in the construction of proximal renal tubule models, high-throughput toxicity testing, drug screening, and research on metabolic disorders.

[0046] The advantages or beneficial effects of the above technical solutions include at least the following:

[0047] The method for preparing renal proximal tubule organoids disclosed in this application employs a three-stage induction approach, sequentially including mesodermal induction, renal progenitor cell induction, and proximal tubule differentiation and maturation induction. This provides a renal proximal tubule organoid model with realistic metabolic characteristics, possessing the structural morphology and function of the proximal tubule, and exhibiting the pathological phenotype and functional deficiencies of proximal tubule lesions. Specifically, in the renal progenitor cell stage, flow cytometry was used for the first time to sort a population of glomerular proximal tubule progenitor cells with proximal tubule potential, significantly improving the purity and differentiation consistency of the target cells. In the renal progenitor cell induction stage, a low-dose window for TGF-β1 was achieved to promote differentiation rather than fibrosis. The proximal tubule differentiation and maturation stages promote mitochondrial metabolic function and transport capacity, and short-term stimulation induces brush border protein expression, improving epithelial polarity. This process constructs high-quality renal proximal tubule organoid spheroids.

[0048] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0049] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0050] Figure 1 ESC-H9 white light image (A) shows that the cells exhibit a typical clonal state; RPTPCs cell morphology image before sorting and purification (B) shows that the cells exhibit various morphologies and complex cell components; RPTPCs cell morphology image after sorting and purification (C) shows that the cells exhibit a single morphology, are polygonal, have large nuclei with a high nucleus-to-cytoplasm ratio, loose intercellular connections, and lack polarization features and brush border structures.

[0051] Figure 2 The image shows the results of flow cytometry-sorted purification of CD24 / CD10 double-positive cell populations, which account for approximately 18.8%.

[0052] Figure 3 The graph shows the expression levels of specific genes SIX2, OSR1, HOXD11 and pluripotency genes NANOG and OCT4 in Ctrl-H9 cells, cells before flow cytometry sorting and purification (RPTPCs Pre-Sorting), and cells after flow cytometry sorting and purification (RPTPCs Post-Sorting). It can be seen that before sorting, the expression of specific genes in RPTPCs was significantly higher than that in the H9 cell line (n=3, ***P<0.001), while the expression of pluripotency genes was significantly lower than that in H9 (n=3, **P<0.01), indicating that cells can be induced to differentiate into RPTPCs. After sorting and purification, compared with before purification, the expression of specific genes in RPTPCs was significantly increased (n=3, *P<0.05, **P<0.01), while the expression of pluripotency genes was almost non-existent (n=3, **P<0.01, ***P<0.001), indicating that flow cytometry sorting and purification can obtain RPTPCs with higher purity.

[0053] Figure 4Under 3D culture conditions, the white light image (A) of the directed differentiation of proximal tubular organoids (RPTOs) shows the presence of the proximal tubule structure; the H&E image (B) shows the prominent proximal tubule structure and the presence of the brush border; the TEM image (C) shows the morphology of the proximal tubule cells and the brush border (red arrow).

[0054] Figure 5 Immunofluorescence staining of directed-differentiated proximal tubular organoids (RPTOs) under 3D culture system conditions; LRP2 (green) and LTL (red) are proximal tubule-specific genes. Immunofluorescence data show that RPTOs co-express LRP2 and LTL and exhibit proximal tubule-like morphology.

[0055] Figure 6 The image shows the results of real-time quantitative qPCR of directed differentiation of proximal tubular organoids (RPTOs) under the conditions of Ctrl-H9 cells, flow cytometry-sorted purified cells (RPTPCs post-sorting), and a 3D culture system. The results showed that the expression of proximal tubule-specific genes (LRP2, CUBN, SLC22A6, AQP1, SLC34A1, ABCC2, SLC22A8) in RPTPCs was significantly higher than that in H9 (n=2, **P<0.01, ***P<0.001), while the expression of these genes in RPTOs was significantly higher than that in RPTPCs (n=3, ***P<0.001), indicating that after induction, RPTPCs can be directed to differentiate into proximal tubular organoids.

[0056] Figure 7 The image shows the monitoring results of dextran in RPTOs using fluorescently labeled dextran (dextran-Alexa488). Using LRP2 (red) and dextran-Alexa488 (green), LRP2 labeled renal proximal tubular cells. The results showed that all RPTOs cells could be labeled with LRP2 and exhibited a tubular morphology. Dextran showed typical green fluorescence in most proximal tubules, indicating that renal tubules can actively absorb dextran into the lumen.

[0057] Figure 8 The graph shows the uric acid (UA) concentration in RPTOs. Under medium-high uric acid (0.5 mM) and high uric acid (1.0 mM) culture conditions, the uric acid concentration in RPTOs was monitored over 300 min and a UA curve was plotted. It can be seen that the UA concentration in RPTOs exhibits a curve-like change. At the time points of 30–50 min and 200–230 min, the UA concentration in RPTOs decreased, indicating that RPTOs have the function of actively absorbing and excreting UA, and that the rate of UA absorption is higher under high uric acid conditions.

[0058] Figure 9 Toxicity testing of RPTOs for the application of hyperuricemic kidney injury; Figure 9 H&E staining data of A showed that the hyperuricemic injury group (H-UA) had pathological manifestations of significant tubular dilation and brush border loss compared with the normal RPTOs (Ctrl) group. Figure 9 Quantitative statistical data from the Paller renal tubular injury scoring system showed that the H-UA group had significantly more renal tubular damage than the Ctrl group (n=5, ***P<0.001). Detailed Implementation

[0059] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0060] This application provides an innovative method for constructing renal proximal tubule organoids through pluripotent stem cell differentiation, which incorporates specific optimized techniques during the differentiation process; at the renal tubular progenitor cell stage, a population of CD24 renal proximal tubule progenitor cells with proximal tubule potential is sorted and purified by flow cytometry. + / CD10 + This method significantly improves the purity and differentiation consistency of target cells; it activates proximal tubule differentiation-related signaling pathways through a combination of novel cytokines and small molecule compounds, promoting cell morphological and functional maturation; and it simulates the organoid microenvironment through a three-dimensional suspension culture system. The method in this application can stably and efficiently generate proximal tubule organoids rich in brush border structures, expressing typical proximal tubule marker genes (such as AQP1, LRP2, and SLC22A6), and possessing transport functions, providing a new approach for establishing high-quality in vitro kidney models.

[0061] The manufacturers and models of the materials used in the embodiments of this application are shown in Table 1:

[0062] Table 1

[0063]

[0064]

[0065] The qPCR primers used in the embodiments of this application are shown in Table 2:

[0066] Table 2

[0067]

[0068]

[0069] Example:

[0070] 1. Pluripotent stem cell culture: following the mTeSR+Matrigel culture system;

[0071] Human embryonic stem cells (hESC(H9)) were resuspended in mTeSR (StemCell, #5850) medium, then seeded into Matrigel (Corning, #354277) culture dishes and cultured at 37°C and 5% CO2; pluripotent stem cells were cultured to quantify cell status; white light images of ESC-H9 cells are shown below. Figure 1 As shown in Figure A.

[0072] 2. Mesodermal induction (Day 0-3):

[0073] From day 0 to day 3, induced pluripotent stem cells (PSCs) were seeded in RPMI 1640 (Gibco, #11875093) medium supplemented with 1×B27 (Gibco, #A1895601). From day 0 to day 2, 8 μM CHIR99021 (Selleck, #S2924) was added to the medium; from day 1 to day 3, 100 ng / mL Activin A (Peprotech, #120-14E) was added to the medium. This induced PSCs to transdifferentiate into mesodermal cells. Mesodermal progenitor cells were then induced to differentiate into kidney progenitor cells from day 3 to day 8.

[0074] 3. Renal progenitor induction (Days 3-8):

[0075] (1) Culture medium: Advanced RPMI 1640 (Gibco, #12633012) + B27 (Gibco, #A1895601) + GlutaMAX (Gibco, #35050061);

[0076] (2) Inducing factors: FGF9 (200 ng / mL) (Peprotech, 100-23), Retinoic acid (0.1 μM) (Sigma-Aldrich, #R2625), BMP7 (50 ng / mL) (Peprotech, 120-07), TGF-β1 (0.5-1 ng / mL) (PeproTech, #100-21), Day 5-8;

[0077] (3) Objective: To promote the differentiation of mesoderm into kidney progenitor cells.

[0078] The detailed operating instructions are as follows:

[0079] On day 3 of inducing mesodermal progenitor cells, half of the culture medium was replaced with renal progenitor cell induction medium. Specifically, half of the mesodermal induction medium was aspirated, and half was added to the renal progenitor cell induction medium. For a 6-well plate, each well contained 3 mL of medium; 1.5 mL of mesodermal induction medium was aspirated, and 1.5 mL of renal progenitor cell induction medium was added. On day 4, the process of day 3 was repeated to allow the mesodermal progenitor cells to adapt to the renal progenitor cell induction medium. On day 5, all the culture medium was aspirated, and 3 mL of renal progenitor cell induction medium was added. On days 6 and 7, the medium was changed daily, with half of the old medium aspirated and half of the new medium added. The cells were cultured at 37°C and 5% CO2 until day 8. Cells exhibiting various morphologies, complex cell composition, and dense cell structure were observed. Some cells showed polygonal shapes, large nuclei with a high nucleus-to-cytoplasm ratio, and loose intercellular connections; these were renal progenitor cells (RPTPCs).

[0080] 4. Sorting and purification of proximal renal tubule progenitor cells (RPTPCs) (Day 8):

[0081] BD Aria III sorting of CD24+ / CD10+ double-positive cell populations: RPTPCs morphology before sorting and purification as follows Figure 1 As shown in Figure B;

[0082] Kidney progenitor cells to be sorted were digested with Accutase and filtered through a 40 μm filter to obtain a single-cell suspension. After cell counting, the concentration was adjusted to approximately 1 × 10⁻⁶ cells / cells. 7 cells / mL ~2×10 7 cells / mL.

[0083] Take an appropriate amount of cells and incubate them with CD24-APC and CD10-FITC antibodies for 30 minutes (37°C, protected from light). Wash the cells twice and resuspend them in PBS containing 1% FBS.

[0084] Start the BD Aria III flow cytometer using an 85μm nozzle and set the sheath fluid pressure to 45psi. After confirming stable flow, perform system quality control (CS&T beads). Select Purity mode. The sorting rate should be controlled within 2000–3000 events / sec to ensure purity and cell viability. Immediately place the sorted cells in pre-cooled medium containing 10% FBS. The morphology of the sorted and purified RPTPCs is as follows. Figure 1 As shown in Figure C; CD24 was purified by flow cytometry. + / CD10 + Double-positive cell results as follows Figure 2 As shown.

[0085] 5. 3D culture of proximal renal tubule cells (Day 8):

[0086] The purified RPTPCs were placed in pre-cooled medium containing 10% FBS and transferred to 96-well ultra-low adsorption round-bottom plates (Corning, #7007) within 1 hour, at a density of 2000 cells / well. To prevent apoptosis, it is recommended to add 10 μM Y27632 (Tocris, #1254) for the first 24 hours. The plates were then centrifuged (200×g, 2-3 minutes) to encourage cell aggregation at the bottom of the wells, forming cell spheres.

[0087] Prepare a Collagen IV (50 μg / mL) (Corning, #354233) and Laminin-521 (50 μg / mL) (Gibco, #A29249) (1:1) solution in advance. Add 10 μL of the Collagen IV / Laminin-521 mixture to a 96-well plate to deposit Collagen IV / Laminin-521 at the bottom of the 96-well plate and coat the RPTPC cell spheres.

[0088] 6. Directed differentiation to construct proximal tubular organoids (Days 8-14):

[0089] (1) Culture medium: DMEM / F12 (Gibco, #11330) + ITS-X (Gibco, #51500056) + GlutaMAX (Gibco, #35050061) + Ascorbic acid (StemCell, #72132);

[0090] (2) Added factors: Dexamethasone (10 nM) (Millipore, #D4902), HGF (50 ng / mL) (Peprotech, #100-39), EGF (10 ng / mL) (Peprotech, AF-100-15), sodium fumarate (10-20 μM) (Sigma-Aldrich, #F1506), linoleic acid (50 μM, Day 11-13 only) (Sigma-Aldrich, #L1012), FGF9 (200 ng / mL) (Peprotech, 100-23), Retinoic acid (1 μM) (Sigma-Aldrich, #R2625).

[0091] (3) Objective: To induce and promote the differentiation of RPTOs and to directionally differentiate proximal tubular organoids of the kidney under the conditions of a 3D culture system.

[0092] Specific procedures: Use ultra-low adsorption round bottom 96-well plates for culture. The cell culture medium in each well is 200 μL. Change half the volume of culture medium every day, that is, remove 100 μL of old culture medium and add 100 μL of new culture medium. Culture in a 37℃, 5% CO2 cell culture incubator until day 14.

[0093] 1. Validation of proximal tube progenitor cell purification:

[0094] Electron microscopy (white light) was used to observe ESC-H9 cells, RPTPCs cells before sorting and purification, and RPTPCs cells after sorting and purification. The white light image of ESC-H9 cells is shown below. Figure 1 As shown in Figure A; the morphology of RPTPCs before sorting and purification is as follows. Figure 1 As shown in Figure B; the morphology of the sorted and purified RPTPCs is as follows. Figure 1 As shown in Figure C.

[0095] from Figure 1 As can be seen, during the differentiation of PSCs into renal progenitor cells, the cells contain various cell morphologies. Figure 1 B), which includes undifferentiated PSC cells; via CD24 + / CD10 + After sorting the double-positive cell population, the cells showed a uniform morphology. Figure 1 C) It is polygonal in shape, with a large nucleus and a high nucleocytoplasmic ratio. The intercellular connections are loose, and it lacks polarization features and brush border structure.

[0096] The mRNA expression levels of specific genes SIX2, OSR1, HOXD11, NANOG, and OCT4 in Ctrl-H9 cells, pre-sorted cells (PTPCs pre-sorted), and post-sorted cells (PTPCs post-sorted) were detected using qPCR (primer sequences are shown in Table 2). The results are as follows: Figure 3 As shown.

[0097] qPCR method: Total RNA was extracted from cells using an RNA extraction kit (Qiagen) following the kit's instructions. cDNA was generated using a cDNA reverse transcription kit (Thermo Fisher Scientific) following the instructions, and analyzed by quantitative real-time PCR on a CFX-96 real-time PCR detection system (Bio-Rad) using iTaq Universal SYBR Green (Bio-Rac). A two-step detection method was used: pre-denaturation at 95℃ for 2–3 minutes; cyclic reaction (38–40 cycles): denaturation at 95℃ for 15 seconds, annealing at 60℃ for 30 seconds, and extension at 72℃ for 30 seconds. Three independent replicates were performed for each sample. An internal reference gene (GAPDH) was used as a control, and the relative expression level was calculated using the 2^-ΔΔCt method. Statistical analysis was performed using t-tests or ANOVA, with p < 0.05 considered statistically significant.

[0098] Figure 3 Compared with flow cytometry-sorted and purified cells, sorted and purified PTPCs highly expressed the cell-specific genes SIX2, OSR1, and HOXD11, while the pluripotency genes NANOG and OCT4 were significantly decreased (n=3, *P<0.05, **P<0.01, ***P<0.001), indicating that PTPCs with higher purity were obtained after sorting and purification.

[0099] 2. Verification of organoid structure and specific genes in the proximal renal tubules:

[0100] Electron microscopy (white light) was used to observe the directed differentiation of proximal tubular organoids (PTOs) in a 3D culture system. The white light image of the directed differentiation of PTOs is shown below. Figure 4 As shown in Figure B; after hematoxylin-eosin staining, the H&E diagram is as follows. Figure 4 As shown in Figure C; observation was performed using a transmission electron microscope (TEM), and the TEM image is shown below. Figure 4 As shown in Figure D.

[0101] White light imaging data showed that PTOs had abundant tubular folds; H&E data showed that PTOs had typical proximal tubular lumen and brush border structures; transmission electron microscopy data showed that proximal tubular organoids had abundant mitochondria, basement membrane and brush border structures (red arrows).

[0102] Under Ctrl-H9 conditions, after flow cytometry-purified cells (RPTPCs post-sorting) and a 3D culture system, directed differentiation of proximal tubular organoids (RPTOs) was performed. Immunofluorescence data analysis was conducted, and the fluorescence staining images are shown below. Figure 5As shown; Real-time quantitative qPCR results are as follows. Figure 6 As shown.

[0103] from Figure 5 and Figure 6 As can be seen, the differentiated organoids possess tubular structures and co-express the renal proximal tubule markers LRP2 and LTL (…). Figure 5 qPCR results showed that the renal proximal tubule organoid highly expressed proximal tubule-specific genes LRP2 (encoding Megalin, specific to the proximal tubule epithelium, responsible for protein reabsorption), CUBN (encoding Cubilin, which works in conjunction with Megalin to reabsorb filtered proteins), SLC22A6 (OAT1, an organic anion transporter expressed on the basolateral membrane of the proximal tubule), AQP1 (aquaporin 1, expressed in the proximal tubule and ascending limb segments, reflecting water reabsorption function), SLA34A1 (sodium-phosphate cotransporter, specifically expressed in the proximal tubule), ABCC2 (multidrug resistance-associated transporter, located at the brush border of the proximal tubule), and SLC22A8 (OAT3, specifically expressed in the proximal tubule, providing a marker for drug efflux). These data demonstrate that the constructed organoid possesses the specific structure and cell line of the renal proximal tubule.

[0104] 3. Functional verification of proximal renal tubule organoids:

[0105] LRP2 was used to label proximal renal tubular cells by co-staining with LRP2 (red) and dextran-Alexa488 (green); the dextran in RPTOs was monitored by electron microscopy using fluorescently labeled dextran (dextran-Alexa488);

[0106] Experimental procedure for RPTOs to absorb dextran: RPTOs were transferred to a medium containing fluorescently labeled dextran (dextran-Alexa488) and incubated at 37°C for 2–3 hours (concentration 0.5–1 mg / mL). After incubation, the probes were gently washed several times with PBS to remove unabsorbed fluorescent probes. Subsequently, the RPTOs were fixed with 4% paraformaldehyde for 20–30 min, followed by incubation with 30% sucrose for 2 hours, OCT embedding, freezing at -20°C, and sectioning into 10–20 μm sections. Sections were permeabilized with Triton X-100 for 10–20 min, and nonspecific binding sites were blocked with blocking buffer (such as buffer containing BSA or normal serum) for 1 hour. LRP2 primary antibody was added and incubated at room temperature for 2–3 hours. The primary antibody was washed away, and secondary antibody with red fluorescence and dyes such as DAPI were added to label cell nuclei. The sections were incubated at room temperature in the dark for 1–2 hours. The secondary antibody and excess DAPI were washed away, and the sections were mounted. The sections were then observed and imaged under a fluorescence or confocal microscope to observe the distribution of fluorescence signals within the renal tubular-like structures, thereby assessing the reabsorption function of the renal organoids. Results are as follows: Figure 7 As shown.

[0107] The uric acid concentration in RPTOs was monitored using urate culture medium to verify the uric acid reabsorption / excretion function.

[0108] Preparation and detection methods of urate culture medium:

[0109] Using the culture medium from day 14 as the basal medium, 16.81 mg of urate was weighed and dissolved in 100 mL of basal medium. The solution was shaken at 37°C for approximately 30 minutes to ensure complete dissolution of the urate. The solution was then filtered through a 0.1 μm filter to obtain RPTOs medium containing 1000 μmol / L UA. Kidney organoids were cultured using this medium, and the concentration of UA in the kidney organoids was measured at 1, 2, 3, and 24 hours of culture.

[0110] Method for detecting UA concentration in RPTOs in vivo: RPTOs were removed from the UA-containing culture medium, washed once with PBS, and the PBS was removed. Equal volumes of organoids (5 organoids of the same size) were resuspended in 100 μL PBS, sonicated, and centrifuged at 12000 × g and 4 °C for 5 minutes. The precipitate at the bottom of the centrifuge tube was removed, and the supernatant was used for UA detection. 5 μL of the supernatant was placed in a 300 μL buffer containing 50 mM Tris (pH 7.5, Sigma Aldrich Chemical Co.) and 1 mM sodium phosphate (Wako Pure Chemical Industries, Ltd.), and uric acid levels were determined using the uricase method (Uric acid C-test Wako, Wako Pure Chemical Industries, Ltd.). Results are as follows: Figure 8 As shown.

[0111] Figure 7 This demonstrates that the proximal renal tubule has the function of actively absorbing glucan. Figure 8 This demonstrates that the proximal renal tubule has the function of actively absorbing / excreting uric acid; this indicates that it possesses some of the specific functions of the proximal renal tubule.

[0112] 4. Proximal renal tubule hyperuricemia toxicity test:

[0113] The proximal renal tubules are the primary site of early action and pathological damage caused by hyperuricemia, exhibiting high sensitivity to uric acid levels, manifested as renal tubular dilation and brush border detachment. This study utilized the effects of hyperuricemia on proximal renal tubules (RPTOs) to observe and analyze their pathological phenotypic changes; and also conducted a toxicity test on the proximal renal tubules caused by hyperuricemia.

[0114] A normal control group (Ctrl) and a hyperuricemic uric acid (H-UA) group were set up. In the H-UA group, RPTOs were cultured in 1000 μmol / L UA medium for 3 days. The normal control group did not contain uric acid in its medium. H&E staining was used to analyze proximal tubular damage, and the degree of tubular damage was scored using the Paller scale: five diseased tubules were randomly selected, with the following criteria: significant tubular dilation and cell flattening scored 1 point; brush border damage scored 1 point, and detachment scored 2 points; casts scored 2 points; and the presence of detached or necrotic cells (not forming casts or cell fragments) within the tubular lumen scored 1 point. The tubular damage was quantitatively analyzed. Results are as follows: Figure 9 As shown.

[0115] like Figure 9 The H&E data shown in Figure A indicate that in the hyperuricemia-damaged group, RPTOs showed significant tubular dilation and brush border detachment. Figure 9 Paller score data from group B showed a highly significant difference in proximal tubular injury compared to the Ctrl group. This demonstrates that these RPTOs can be used to construct a hyperuricemic kidney injury model.

[0116] Therefore, proximal renal tubular organoids can not only simulate the early pathogenesis of hyperuricemia-related nephropathy, but also serve as an ideal toxicity testing platform and disease model construction tool, providing a reliable basis for drug screening and mechanism research.

[0117] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0118] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0119] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for constructing pluripotent stem cells to differentiate into proximal renal tubule organoids, characterized in that, Includes the following steps: S1. From day 0 to day 3, human pluripotent stem cells are cultured and induced to differentiate into early mesoderm; S2. From day 3 to 8, early mesodermal cells are induced to differentiate into kidney progenitor cells: the culture medium is gradually replaced with kidney progenitor cell induction medium and cultured for 3 to 8 days; the kidney progenitor cell induction medium is Advanced RPMI 1640 medium containing B27minus insulin, GlutaMAX and inducing factors; the inducing factors include FGF9, retinoic acid and BMP7; The inducing factor TGF-β1 was further added on days 5 to 8. S3. On day 8, CD24+ / CD10+ double-positive cell populations were sorted to obtain sorted and purified proximal renal tubule progenitor cells; S4. The sorted and purified proximal renal tubule progenitor cells were treated with a mixture of Collagen IV / Laminin-521. S5. From day 8 to 14, proximal renal tubule progenitor cells were induced to differentiate into proximal renal tubule organoids using DMEM / F12 medium containing ITS, GlutaMAX, ascorbic acid and added factors; the added factors included dexamethasone, HGF, EGF, FGF9 and retinoic acid.

2. The method for constructing pluripotent stem cells to differentiate into proximal renal tubule organoids according to claim 1, characterized in that: The process of culturing and inducing human pluripotent stem cells to differentiate into early mesoderm includes the following steps: S01. Human pluripotent stem cells were cultured using mTeSR on a Matrigel-coated culture device. S02. On days 0 to 3, human pluripotent stem cells were inoculated into RPMI 1640 medium supplemented with 1×B27Minus insulin; mesodermal induction was performed by adding inducing factors CHIR99021 and Activin A to obtain mesodermal precursor cells.

3. The method for constructing pluripotent stem cell-differentiated proximal renal tubule organoids according to claim 1 or 2, characterized in that: In step S4, the process of treating the sorted and purified proximal renal duct progenitor cells with a Collagen IV / Laminin-521 mixture includes the following steps: The proximal renal tubule progenitor cells obtained in step S3 were placed in a culture medium containing FBS and centrifuged to obtain cell spheres. Then, a Collagen IV / Laminin-521 mixture was added to coat the cell spheres with Collagen IV / Laminin-521.

4. The method for constructing pluripotent stem cell-differentiated proximal renal tubule organoids according to claim 3, characterized in that: The mass concentrations of Collagen IV and Laminin-521 in the Collagen IV / Laminin-521 mixture were 20–50 μg / mL, respectively. The ratio of proximal renal tubular progenitor cells to Collagen IV / Laminin-521 mixture was 2000 cells: (5–15) μL.

5. The method for constructing pluripotent stem cell-differentiated proximal renal tubule organoids according to claim 3, characterized in that: After placing proximal renal tubule progenitor cells in a culture medium containing FBS, 5–12 μM of Y27632 was added.

6. The method for constructing pluripotent stem cell-differentiated proximal renal tubule organoids according to claim 1 or 2, characterized in that: In step S2, the concentration of FGF9 is 100-200 ng / mL, the concentration of retinoic acid is 0.1-0.2 μM, the concentration of BMP7 is 50-100 ng / mL, and the concentration of TGF-β1 is 0.5-1 ng / mL. In step S5, the concentration of dexamethasone is 5–15 nM, the concentration of HGF is 50–100 ng / mL, the concentration of EGF is 10–50 ng / mL, the concentration of FGF9 is 100–200 ng / mL, and the concentration of retinoic acid is 1–2 μM.

7. The method for constructing pluripotent stem cell-differentiated proximal renal tubule organoids according to claim 2, characterized in that: The concentration of CHIR99021 was 5–10 μM; the concentration of Activin A was 100–200 ng / mL; and CHIR99021 was added on days 0–2, while Activin A was added on days 1–3.

8. The method for constructing pluripotent stem cell-differentiated proximal renal tubule organoids according to claim 1 or 2, characterized in that: In step S5, the added factors also include sodium fumarate and / or linoleic acid; the concentration of sodium fumarate is 10-20 μM, and the concentration of linoleic acid is 20-50 μM. Furthermore, linoleic acid was added only on days 11 to 13. The conditions for cultivation in step S5 are as follows: the culture medium substrate is pre-coated with Collagen IV / Laminin-521.

9. The renal proximal tubule organoid prepared by the method for constructing pluripotent stem cell differentiation into renal proximal tubule organoids according to any one of claims 1-8.

10. The application of the renal proximal tubule organoids as described in claim 9 in the construction of renal proximal tubule models, high-throughput toxicity testing, drug screening, and research on metabolic disorders.

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