Method for producing functional proximal tubule epithelial cells

Abstract

The present invention relates to a method for producing functional proximal tubule epithelial cells, wherein the method of the present invention produces functional proximal tubule epithelial cells by preparing kidney organoids from human pluripotent stem cells, isolating proximal tubule epithelial cells therefrom, and culturing same under HSPG treatment conditions, and the proximal tubule epithelial cells produced by the method exhibit enhanced functionality such as polarization, selective excretion, and maturation of drug transporters, compared to conventional human proximal tubule epithelial cell lines or proximal tubule epithelial cells cultured by treatment with laminin alone.

Description

Method for producing functional proximal tubular epithelial cells

[0001] The present invention relates to a method for producing functional proximal tubular epithelial cells.

[0002] The kidney is a vital organ that maintains homeostasis in the body. It regulates body fluid volume, blood ion concentration, and pH, excretes metabolic waste products, toxins, and drugs, and performs blood pressure control and other metabolic and endocrine functions. It also activates vitamin D, which aids calcium absorption in the small intestine, and is involved in the synthesis of various hormones. The proximal tubules of the kidney are the main component of the nephron, which performs most of the renal reabsorption and secretion of sodium, water, amino acids, and other essential nutrients such as glucose and albumin. Because of the high complexity of the nephron, in vitro models are necessary to study its development and function.

[0003] Despite the elucidation of the influence of endothelial vasculature on the proximal tubule epithelium, conventional models utilize primary cells or immortalized cell lines, resulting in a lack of research optimizing the epithelial tissue itself. Several human in vitro model systems have proven useful for applying integrated systems biology methods to mechanism-driven toxicity assessments. For example, immortalized cell lines such as RPTEC or HK-2 cells have been used to elucidate mechanistic and functional aspects of drug exposure. However, because cell lines are derived from a single donor, they are limited in representing only a single genetic background. Recently, the idea of ​​generating renal organoids by differentiating human pluripotent cells has been proposed, and methods for directly differentiating human iPSCs into proximal tubule-like cells have been reported and applied to some extent. However, the lack of in vivo conditions has limited optimal performance.

[0004] The purpose of the present invention is to provide a composition for enhancing the function of proximal tubule epithelial cells.

[0005] In addition, it is an object of the present invention to provide a culture vessel for enhancing the function of proximal tubule epithelial cells.

[0006] In addition, an object of the present invention is to provide a composition for culturing proximal tubule epithelial cells.

[0007] In addition, it is an object of the present invention to provide a method for producing functional proximal tubule epithelial cells.

[0008] In addition, the purpose of the present invention is to provide functional proximal tubule epithelial cells.

[0009] To achieve the above purpose, the present invention provides a composition for enhancing the function of proximal tubule epithelial cells containing HSPG (heparan sulfate proteoglycans).

[0010] In addition, the present invention provides a culture vessel for enhancing the function of proximal tubule epithelial cells having a surface coated with the composition.

[0011] In addition, the present invention provides a composition for culturing proximal tubule epithelial cells containing HSPG.

[0012] Additionally, the present invention provides a method for producing functional proximal tubular epithelial cells.

[0013] In addition, the present invention provides functional proximal tubular epithelial cells manufactured by the above method.

[0014] In the present invention, renal organoids are prepared from human pluripotent stem cells, proximal tubular epithelial cells are isolated therefrom, and the organoids are cultured under HSPG treatment conditions, thereby producing functional proximal tubular epithelial cells having improved functionality, including polarization, selective excretion, and drug transporter maturation, compared to proximal tubular epithelial cells cultured only with laminin treatment and conventional human proximal tubular epithelial cell lines.

[0015] Figure 1 is a diagram showing the process of producing renal organoids from hiPSCs, isolating LTL-positive cells therefrom, and producing functional proximal tubular epithelial cells:

[0016] A: Schematic diagram of the manufacturing process of functional proximal tubular epithelial cells; and

[0017] B: Expression of proximal tubule cell-specific markers in LTL-positive cells.

[0018] Figure 2 is a diagram analyzing the enhancement of functionality of proximal tubule cells by HSPG for optimizing conditions for producing functional proximal tubule epithelial cells.

[0019] Figure 3 is a diagram analyzing the optimized passaging and freeze-thawing conditions for maintaining the differentiated and polarized phenotype of functional proximal tubule epithelial cells:

[0020] P1: Generation 1;

[0021] P2: Generation 2;

[0022] P3: Generation 3;

[0023] P9: Generation 9;

[0024] SB431542: TGF-β inhibitor;

[0025] A: Schematic diagram of 2D culture of LTL-positive proximal tubular epithelial cells and their morphology;

[0026] B: Cell images from passages 1 to 9; and

[0027] C: Multicellular domes formed by functional proximal tubule epithelial cells.

[0028] Figure 4 compares the functionality of functional proximal tubular epithelial cells with that of an immortalized human proximal tubular cell line:

[0029] LTL positive cells: functional proximal tubular epithelial cells;

[0030] A: Images of the tight junction protein ZO-1, the proximal tubule marker LTL, and the ECM glycoprotein laminin; and

[0031] B: mRNA levels of the solute transporter genes ATP1A1, SLC34A1, and SGLT2, the drug transporter genes ABCB1 and LRP2, and the ciliary genes PKD1, PKD2, and PKDH1.

[0032] Figure 5 is a diagram evaluating the leakage of functional proximal tubule epithelial cells:

[0033] A: TER of a cell monolayer of functional proximal tubular epithelial cells or RPTECs; and

[0034] B: Intercellular permeability measured using FITC-labeled inulin.

[0035] Figure 6 is a diagram analyzing the selective excretion of functional proximal tubule epithelial cells.

[0036] Figure 7 is a diagram confirming the 3D tubular structure of functional proximal tubular epithelial cells cultured in 3D using HSPG of LTL-positive proximal tubular cells.

[0037] Figure 8 is a diagram showing the effect of improving the expression and polarization of drug transporters in functional proximal tubular epithelial cells cultured using HSPG and SB431542 coated on LTL-positive proximal tubular cells and in 3D-cultured functional proximal tubular epithelial cells:

[0038] A: Schematic diagram of the manufacturing process of functionally enhanced kidney organoid-derived proximal tubule cells (FEKOPTCs) in an in vitro model.

[0039] Brightfield image: FEKOPTCs of the second passage;

[0040] B: Expression heatmap of genes associated with transporters, epithelial markers, maturation, and differentiation in early passage (passage 1 and 2) and late passage (passage 5) FEKOPTCs;

[0041] C: Morphology of 3D cultured functional proximal tubule epithelial cells;

[0042] D: 3D tubular structure of 3D cultured functional proximal tubule epithelial cells.

[0043] Figure 9 shows the gene expression of early passage (1st and 2nd passage) and late passage (5th passage) FEKOPTCs:

[0044] A: Expression of ABCB1 mRNA, a drug transporter gene;

[0045] B: Expression of ATP1A1 mRNA, a tubular epithelial solute transporter;

[0046] C: Expression of SLC5A2 mRNA, a glucose transporter;

[0047] D: Expression of SLC2A1 mRNA, a glucose transporter;

[0048] E: Expression of SLC22A3 mRNA, an organic cation transporter 3.

[0049] Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are provided as examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0050] Additionally, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the customs of the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout this specification, when a part is said to "include" a certain component, unless specifically stated otherwise, this does not mean that other components are excluded, but rather that other components may be included.

[0051] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited herein as references are incorporated herein by reference.

[0052] Throughout this specification, '%' used to indicate the concentration of a particular substance is (w / w) % for solid / solid, (w / v) % for solid / liquid, and (v / v) % for liquid / liquid, unless otherwise stated.

[0053]

[0054] In one aspect, the present invention relates to a composition for enhancing the function of proximal tubule epithelial cells, comprising HSPG (heparan sulfate proteoglycans).

[0055] In one embodiment, the composition may further comprise laminin.

[0056] In one embodiment, the proximal tubule epithelial cells may be LTL (Lotus tetragonolobus lectin) positive cells and may be cells expressing GGT1, ABCB1 or SGLT2.

[0057] In one embodiment, the enhancement of proximal tubule epithelial cell function may be through enhanced polarization or drug transporter maturation.

[0058] In one embodiment, the composition can increase the expression of LTL, SLC34A1, SLC5A2, SLC2A1, SLC22A3, ABCB1, ATP1A1, SGLT2, LRP2, PKD1 or PKD2.

[0059] In one embodiment, the composition may be a coating composition.

[0060] In one embodiment, HSPG may be included at a concentration of 0.01 to 10 μg / mL.

[0061] In one embodiment of the present invention, when HSPG and laminin are mixed and used, the HSPG and laminin may be mixed and used in a weight ratio of 1:1 to 5, but is not limited thereto. Preferably, the HSPG and laminin may be mixed and used in a weight ratio of 1:1 to 4, 1:1 to 3, 1:1 to 2, 1:2, 1:2 to 5, 1:2 to 4, 1:2 to 3, 1:3 to 5, 1:3 to 4, or 1:4 to 5, and more preferably, the HSPG and laminin may be mixed and used in a weight ratio of 1:3.8, but is not limited thereto.

[0062] In one embodiment of the present invention, it was confirmed that proximal tubular epithelial cells grew best when cultured by mixing 0.66 μg / mL of HSPG and 2.5 μg / mL of laminin.

[0063] In one embodiment, the composition may further comprise a TGF-β inhibitor.

[0064] In one embodiment, the TGF-β inhibitor may be, but is not limited to, SB431542.

[0065] The above TGF-β inhibitor may be added to HSPG at a weight ratio of 1:1 to 20, but is not limited thereto. Preferably, the HSPG and the TGF-β inhibitor are mixed in a ratio of 1:1 to 19, 1:1 to 18, 1:1 to 17, 1:1 to 16, 1:1 to 15, 1:1 to 14, 1:1 to 13, 1:1 to 12, 1:1 to 11, 1:1 to 10, 1:1 to 9, 1:1 to 8, 1:1 to 7, 1:1 to 6, 1:1 to 5, 1:1 to 4, 1:1 to 3, 1:1 to 2, 1:1, 1:2 to 20, 1:2 to 19, 1:2 to 18, 1:2 to 17, 1:2 to 16, 1: 2 to 15, 1:2 to 14, 1:2 to 13, 1:2 to 12, 1:2 to 11, 1:2 to 10, 1:2 to 9, 1:2 to 8, 1:2 to 7, 1:2 to 6, 1:2 to 5, 1:2 to 4, 1:2 to 3, 1:3 to 20, 1:3 to 19, 1:3 to 18, 1:3 to 17, 1:3 to 16, 1:3 to 15, 1:3 to 14, 1:3 to 13, 1:3 to 12, 1:3 to 11, 1:3 to 10, 1:3 to 9, 1:3 to 8, 1:3 to 7, 1:3 to 6, 1:3 to 5, 1:3 to 4, 1:4 to 20, 1:4 to 19, 1:4 to 18, 1:4 to 17, 1:4 to 16, 1:4 to 15, 1:4 to 14, 1:4 to 13, 1:4 to 12, 1:4 to 11, 1:4 to 10, 1:4 to 9, 1:4 to 8, 1:4 to 7, 1:4 to 6, 1:4 to 5, 1:5 to 20, 1:5 to 19,1:5 to 18, 1:5 to 17, 1:5 to 16, 1:5 to 15, 1:5 to 14, 1:5 to 13, 1:5 to 12, 1:5 to 11, 1:5 to 10, 1:5 to 9, 1:5 to 8, 1:5 to 7, 1:5 to 6, 1:6 to 20, 1:6 to 19, 1:6 to 18, 1:6 to 17, 1:6 to 16, 1:6 to 15, 1:6 to 14, 1:6 to 13, 1:6 to 12, 1:6 to 11, 1:6 to 10, 1:6 to 9, 1:6 to 8, 1:6 to 7, 1:7 to 20, 1:7 to 19, 1:7 to 18, 1:7 to 17, 1:7 to 16, 1:7 to 15, 1:7 to 14, 1:7 to 13, 1:7 to 12, 1:7 to 11, 1:7 to 10, 1:7 to 9, 1:7 to 8, 1:8 to 20, 1:8 to 19, 1:8 to 18, 1:8 to 17, 1:8 to 16, 1:8 to 15, 1:8 to 14, 1:8 to 13, 1:8 to 12, 1:8 to 11, 1:8 to 10, 1:8 to 9, 1:9 to 20, 1:9 to 19, 1:9 to 18, 1:9 to 17, 1:9 to 16, 1:9 to 15, 1:9 to 14, 1:9 to 13, 1:9 to 12, 1:9 to 11, 1:9 to 10, 1:10 to 20, 1:10 to 19, 1:10 to 18, 1:10 to 17, 1:10 to 16, 1:10 to 15, 1:10 to 14, 1:10 to 13, 1:10 to 12, 1:10 to 11, 1: 11 to 20, 1:11 to 19, 1:11 to 18,1:11 to 17, 1:11 to 16, 1:11 to 15, 1:11 to 14, 1:11 to 13, 1:11 to 12, 1:12 to 20, 1:12 to 19, 1:12 to 18, 1:12 to 17, 1:12 to 16, 1:12 to 15, 1:12 to 14, 1:12 to 13, 1:13 to 20, 1:13 to 19, 1:13 to 18, 1:13 to 17, 1:13 to 16, 1:13 to 15, 1:13 to 14, 1:14 to 20, It can be mixed and used in a weight ratio of 1:14 to 19, 1:14 to 18, 1:14 to 17, 1:14 to 16, 1:14 to 15, 1:15 to 20, 1:15 to 19, 1:15 to 18, 1:15 to 17 or 1:15 to 16, and more preferably, it can be mixed and used in a weight ratio of 1:15.16, but is not limited thereto.

[0066] In one embodiment of the present invention, when LTL-positive proximal tubular cells were cultured on a dish coated with a mixture of 0.66 μg / mL of HSPG and 2.5 μg / mL of laminin and treated with 10 μg / mL of SB431542, a selective TGF-β inhibitor, it was confirmed that the cells exhibited a cuboid phenotype that closely mimicked the cobblestone phenotype of an immortalized proximal tubular epithelial cell line and grew at a rapid rate.

[0067] The above SB431542 can be used to culture LTL-positive proximal tubule cells on a dish coated with a mixture of HSPG and laminin, and processed after 1 day, but is not limited thereto.

[0068] The term "expression" as used herein generally refers to the cellular process by which a biologically active polypeptide is generated from a DNA sequence and exhibits biological activity in a cell. In this sense, gene expression encompasses not only transcription and translation processes, but also post-transcriptional and post-translational processes that can affect the biological activity of a gene or gene product. These processes include, but are not limited to, RNA synthesis, processing, and transport, as well as polypeptide synthesis, transport, and post-translational modification of the polypeptide.

[0069] The term "increased expression" or "up-regulation" used in the present invention means that the amount of expression of a specific gene into mRNA or protein is significantly increased through intracellular transcription or translation.

[0070] In the present invention, the expression can be confirmed by measuring the expression level of a gene or mRNA using a nucleic acid sequence, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair, a probe, or a primer pair and a probe that specifically recognize a fragment of the nucleic acid sequence and the complementary sequence, a polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive RT-PCR, nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, Northern blot, or DNA chip method, and using an antibody, antibody fragment, aptamer, avidity multimer, or peptidomimetics that specifically recognizes the full-length protein or a fragment thereof, a Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), The expression level of the protein can be measured and confirmed using radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or protein microarray methods.

[0071] In one embodiment, the composition may be provided in the form of a kit. That is, the present invention may be provided as a kit for enhancing the function of proximal tubular epithelial cells including HSPG, and the kit may additionally include laminin. The kit of the present invention may further include instructions for use. The instructions for use may further include, but are not limited to, a description of a method for enhancing the function of proximal tubular epithelial cells using the composition of the present invention and a description of proximal tubular epithelial cells with enhanced function. The kit of the present invention may further include proximal tubular epithelial cells as a culture target, and may further include additional components for culturing the same.

[0072] In one aspect, the present invention relates to a culture vessel for enhancing the function of proximal tubule epithelial cells having a surface coated with the composition of the present invention.

[0073] In one aspect, the present invention relates to a composition for culturing proximal tubule epithelial cells comprising HSPG.

[0074] In one embodiment, the composition may further comprise L-Glutamine, insulin, Transferrin, Selenium, Laminin, TGF-β inhibitor, Epidermal Growth Factor (EGF) or steroid, and in one embodiment of the present invention, proximal tubular epithelial cells were cultured in a culture vessel coated with HSPG and laminin in DMEM / F12 Basal medium supplemented with Glutamax (2 mM, Gibco) 1% ITS (insulin-Transferrin-Selenium, Gibco), TGF-β / activin / NODAL pathway inhibitor (10 μM, SB431542), human EGF (10 ng / mL, Sigma-Aldrich) and Hydrocortisone (50 nM, Sigma-Aldrich).

[0075] In one embodiment, the composition can enhance polarization or functionality of proximal tubule epithelial cells.

[0076] In one embodiment, the composition can increase the expression of LTL, SLC34A1, SLC5A2, SLC2A1, SLC22A3, ABCB1, ATP1A1, SGLT2, LRP2, PKD1 or PKD2.

[0077] In one embodiment, the composition can maintain a differentiated and polarized phenotype even during subculture.

[0078] In one embodiment, proximal tubule epithelial cells cultured with the composition maintained ABCB1, ATP1A1, SLC5A2, SLC2A1, or SLC22A3 gene expression even after the 5th passage, and were significantly upregulated compared to the 1st passage.

[0079] In one embodiment, the composition may be for subculture.

[0080] In one aspect, the present invention relates to a method for producing functional proximal tubular epithelial cells, comprising the steps of: a) producing a renal organoid; b) dissociating the renal organoid into single cells; c) isolating proximal tubular epithelial cells; and d) enhancing the functionality of the proximal tubular epithelial cells.

[0081] In one embodiment, the step of producing a kidney organoid of step a) may include: i) differentiating human pluripotent stem cells (hPSCs) into intermediate mesoderm of nephron progenitor cells; and ii) 3D culturing.

[0082] In one embodiment, the human pluripotent stem cell may be a human embryonic stem cell (hESC) or a human induced pluripotent stem cell (iPSC).

[0083] In one embodiment, human induced pluripotent stem cells (iPSCs) can be produced by inducing reprogramming from peripheral blood mononuclear cells (PBMCs).

[0084] In one embodiment, the proximal tubule epithelial cells may be LTL (Lotus tetragonolobus lectin) positive cells.

[0085] In one embodiment, in step c), the proximal tubular epithelial cells can be separated by FACs or MACS (immunomagnetic cell separation), and it is more preferred that they are separated by MACS.

[0086] In one embodiment, the step of enhancing the functionality of the proximal tubular epithelial cells of step d) may be culturing the proximal tubular epithelial cells on a solid phase coated with laminin and HSPG.

[0087] In one embodiment, the solid phase may be a cell culture substrate or a cell culture carrier, and may be a dish, a plate, a flask, a bag, a bead, a membrane, or a slide glass.

[0088] In one embodiment, the culture may be a monolayer culture or a 3D culture.

[0089] In one embodiment, the culture may be cultured in a medium containing L-glutamine, insulin, transferrin, selenium, laminin, TGF-β inhibitor, epidermal growth factor (EGF), or steroid.

[0090] In one aspect, the present invention relates to functional proximal tubular epithelial cells produced by the method of the present invention.

[0091] In one embodiment, the cells may have increased / upregulated expression of LTL, SLC34A1, SLC5A2, SLC2A1, SLC22A3, ABCB1, ATP1A1, SGLT2, LRP2, PKD1 or PKD2 compared to proximal tubule epithelial cells that do not have enhanced functionality.

[0092] In one embodiment, the cells may exhibit enhanced polarization, selective excretion, or drug transporter maturation compared to non-enhanced proximal tubular epithelial cells.

[0093] In one embodiment, the selective efflux may be mediated by multidrug resistance protein 2 and 4 (MRP2 / 4) efflux pumps.

[0094] In one aspect, the present invention relates to a proximal tubule model comprising a cell sheet or 3D structure of functional proximal tubule epithelial cells of the present invention.

[0095] In one aspect, the present invention relates to a bioink composition comprising functional proximal tubular epithelial cells of the present invention.

[0096] In one embodiment, the bio-ink composition may additionally contain one or more selected from the group consisting of cells, cell culture medium, bioactive substances, and additives.

[0097] The term "bioink" used in the present invention refers to a material that includes living cells or biomolecules and can be applied to bioprinting technology to produce a required structure. Bioink is a material that provides physical properties for 3D processing and a biological environment for cells to perform a desired function, and has excellent cell affinity. The bioink composition may include a liquid, semi-solid, or solid composition containing a plurality of cells. The bioink may include a liquid or semi-solid cell solution, a cell suspension, or a cell concentrate. The bioink composition is produced by 1) mixing a plurality of cells or cell aggregates with a biocompatible liquid or gel at a predetermined ratio to produce bioink, and 2) densifying the bioink to produce bioink having a desired cell density and viscosity.

[0098] In the present invention, the bio-ink composition may further include a substance that promotes cell adhesion and / or an antioxidant. In addition, the bio-ink composition of the present invention may further include a substance that inhibits cell death (e.g., necrosis, apoptosis, or autophagy). Non-limiting examples of agents that inhibit cell death include small molecules, antibodies, peptides, peptibodies, anti-TNF agents, agents that inhibit the activity of interleukins, agents that inhibit the activity of interferons, agents that inhibit the activity of granulocyte colony-stimulating factor (GCSF), agents that inhibit the activity of macrophage inflammatory proteins, agents that inhibit the activity of transforming growth factor B (TGF-B), agents that inhibit the activity of matrix metalloproteinases (MMPs), agents that inhibit the activity of caspases, agents that inhibit the activity of MAPK / JNK signaling cascades, agents that inhibit the activity of Src kinases, agents that inhibit the activity of JAKs (Janus kinases), or combinations thereof.

[0099] As used herein, the term "bioprinting" refers to the use of automated, computer-aided, three-dimensional prototyping devices (e.g., bioprinters) and commercially available methodologies for precise three-dimensional cell deposition (e.g., cell solutions, cell-containing gels, cell suspensions, cell concentrates, multicellular aggregates, multicellular bodies, etc.). The bioprinting invention may be continuous or substantially continuous. A non-limiting example of a continuous bioprinting method is to dispense bioink from a bioprinter via a dispense tip (e.g., a syringe, capillary, etc.) that is connected to a reservoir of bioink. A continuous bioprinting method dispenses bioink in a repeating pattern of functional units. The repeating functional units have any suitable geometry, including, for example, circles, squares, rectangles, triangles, polygons, and irregular geometries. In one embodiment, the bioprinting may be 3D bioprinting.

[0100] The term "3D bioprinting" used in the present invention refers to the creation of artificial living tissues and organs using 3D printing, and may mean inserting bioink containing cells extracted from human tissue into a 3D printer and creating cell structures in a three-dimensional space.

[0101]

[0102] The present invention is described in more detail through the following examples. However, the following examples are intended only to concretize the content of the present invention and are not intended to limit the present invention.

[0103]

[0104] Example 1. Isolation and identification of proximal tubule epithelial cells from kidney organoids.

[0105] 1-1. Differentiation of human induced pluripotent stem cells (iPSCs) derived from peripheral blood mononuclear cells (PBMCs)

[0106] Human-induced pluripotent stem cells (PISCs) with pluripotency present in peripheral blood were isolated and cultured using peripheral blood from normal individuals. Specifically, peripheral blood from healthy individuals was collected in heparin-coated syringe tubes, diluted with phosphate-buffered saline (PBS), and centrifuged at 850 g for 30 minutes using a Ficoll gradient to obtain PBMCs. After transferring to new tubes, they were resuspended in StemSpan medium containing CC110 cytokine cocktail and cultured for 5 days in a 37°C, 5% CO2 incubator. The cultured PBMCs were seeded in cell culture dishes at a density of 3 × 10 5 After the initial culture, reprogramming was induced using CytoTuneTM-iPS Sendai Reprogram Kit to induce differentiation into human induced pluripotent stem cells (hiPSCs). The produced hiPSCs were cultured in 12-well plates coated with GelTrex using TeSR-E8 culture medium (Fig. 1A).

[0107]

[0108] 1-2. Manufacturing of kidney organoids

[0109] Human iPSCs differentiated from peripheral blood mononuclear cells in Example 1-1 were seeded at a density of 15,000 cells / well in 6-well plates supplemented with 1% GelTrex (Thermo Fisher Scientific) and mTeSR1 medium (Stem Cell Technologies) (day 0). The plates were cultured by replacing with mTeSR1 medium containing 1.5% GelTrex (day 1), and then replaced with RPMI medium (Thermo Fisher Scientific) containing 12 μM CHIR99021 (Tocris) to induce differentiation of nephron progenitor cells into the intermediate mesoderm (day 4.5). Then, the plates were replaced with RPMI medium containing B27 supplement (Thermo Fisher Scientific) and VEGF growth factor to perform 3D culture for further differentiation into kidney organoids (day 16).

[0110] Through this, nephron-rich kidney organoids expressing glomerular, proximal tubule, and distal tubule were formed at day 16 (Fig. 1A).

[0111]

[0112] 1-3. Isolation of proximal tubule epithelial cells

[0113] Proximal tubule epithelial cells expressing Lotus tetragonolobus lectin (LTL), a proximal tubule marker, were isolated from the kidney organoids prepared in Example 1-2 using immunomagnetic cell separation (MACS) selection. Specifically, the kidney organoids prepared in Example 1-1 were washed twice with DPBS excluding calcium and magnesium, treated with Accutase solution, and incubated at 37°C for 10 minutes. The organoids were mechanically dissociated by vortexing in the solution every 5 minutes and repeating pipetting (20 times for 30 seconds), and the pipetting frequency was increased to perform an additional 20 times to completely dissociate the organoids into single cells. Thereafter, the cells were centrifuged at 220 g for 4 minutes, the supernatant was removed, and the cells were resuspended in 1 mL of MACS separation buffer (cold PBS containing 1% FBS). This was sequentially filtered through a 40 μm cell strainer to remove any remaining cell aggregates, and the single-cell suspension was centrifuged at 220 g for 4 minutes and resuspended in 100 μL of biotinylated-LTL antibody dilution (1:100 B-1325 biotinylated LTL). The solution was kept on ice for 30 minutes, 3 mL of MACS separation buffer was added, and centrifuged at 220 g for 4 minutes. The supernatant was removed, and the cells were washed with 3 mL of MACS separation buffer, centrifuged at 220 g for 4 minutes, the supernatant was removed, and the cells were resuspended in 90 μL of MACS separation buffer, followed by the addition of 10 μL of streptavidin magnetic beads and incubated on ice for 30 minutes. After 30 minutes, 1000 μL of MACS separation buffer was added, centrifuged at 220 g for 4 minutes, the supernatant was removed, and washed with 3 mL of MACS separation buffer. Centrifuged at 220 g for 4 minutes and resuspended in 500 μL of MACS separation buffer.MACS selection was performed by placing an MS column (Miltenyi Biotec 130-042-201) on the provided MACS magnet. 500 μL of MACS separation buffer was applied to the magnetic column and collected in a conical tube labeled “LTL-negative.” The organoid-derived cell solution was then passed through the magnetic column, and 500 μL of MACS separation buffer was added to the conical tube containing the cell solution to collect the remaining cells and then applied back to the magnetic column. 500 μL of MACS separation buffer was then applied to the column. The magnetic column was then placed in a new conical tube labeled “LTL-positive,” and 1 mL of MACS separation buffer was added to the column and manually applied. The LTL-positive cells thus obtained were diluted with 5 mL of pre-warmed renal epithelial cell-based medium, centrifuged at 220 g for 4 minutes, the supernatant was removed, and resuspended in 1 mL of culture medium. The cell numbers of each LTL-positive and LTL-negative fraction were counted.

[0114]

[0115] 1-4. Identification of proximal tubule epithelial cells

[0116] To identify proximal tubule cells isolated from renal organoids, the expression levels of GGT1, ABCB1, and SGLT2, which are specific markers of proximal tubule cells, were determined in LTL-positive and LTL-negative cells, respectively, by real-time quantitative PCR analysis. Specifically, cells were collected, and total RNA was isolated using TRIzol Reagent (Life Technologies, Carlsbad, CA). RNA was quantified spectrophotometrically, and cDNA was synthesized using 2 μg of RNA and SuperScript III Reverse Transcriptase (Life Technologies). For qPCR, 100 ng of cDNA was used as a template for PCR using Brilliant SYBR green QPCR master mix (FastStart DNA Master SYBR Green I; Roche Molecular Biochemicals, Mannheim, Germany) with a LightCycler Instrument (Roche Molecular Biochemicals). At this time, cDNA was serially diluted from 1 ng / μL to 1 fg / μL and used as a template to generate a standard curve. Overlapping primers were used to amplify the standard and extended cDNA samples. The relative expression level of the target mRNA for each sample, expressed as a percentage of the GAPDH mRNA level, was determined using the comparative Ct method. Ct ratios were analyzed using LightCycler software (Version 4.05).

[0117] As a result, the expression of GGT1, ABCB1, and SGLT2, which are markers specific for proximal tubule cells, was found to be significantly increased in LTL-positive cells (LTL+) compared to LTL-negative cells (LTL-) (Fig. 1B).

[0118]

[0119] Example 2. Preparation and optimization of functional proximal tubule epithelial cells.

[0120] 2-1. Manufacturing functional proximal tubular epithelial cells

[0121] To confirm the synergistic effect of the combination of laminin and HSPG (heparan sulfate proteoglycans) on LTL-positive cell differentiation, LTL-positive proximal tubular epithelial cells isolated by MACS selection in Example 1 were cultured in a 2D monolayer on a dish coated with laminin and HSPG to produce functional proximal tubular epithelial cells (Fig. 1A). Specifically, a 12-well plate was coated with a solution containing 2.5 μg / mL laminin-511 (Biolamina LN-511) and 0.66 μg / mL HSPG (heparan sulfate proteoglycans) (Sigma, H4777) dissolved in DPBS (1× Dulbelco's phosphate buffered saline with calcium and magnesium), and incubated at 37°C for 1 hour. Then, the LTL-positive cells isolated above were seeded in the wells pretreated with laminin + HSPG at a density of 50,000-100,000 cells / cm. 2 After seeding at a density of , the medium was replaced with new medium every 24 hours and cultured for 7 days.

[0122]

[0123] 2-2. Analysis of functional enhancement by HSPG

[0124] The effects of media supplementation conditions and laminin coating with or without HSPG on 2D monolayer cultures of LTL-positive proximal tubular cells were analyzed. As described above, LTL-positive proximal tubular cells were cultured in DPBS medium without calcium and magnesium (excluding) on ​​dishes coated with ECM consisting of 2.5 μg / mL laminin and 0.66 μg / mL HSPG. Therefore, LTL-positive proximal tubular cells were cultured on surfaces coated with various concentrations of HSPG at 0.12, 0.3, or 0.6 μg / mL. In addition, to determine whether the concentration of HSPG affects the expression and polarization of major drug transporters, the mRNA levels of solute transporters, drug transporters, and ciliary genes were analyzed by Real-Time Quantitative PCR.

[0125] As a result, the expression of the tubular epithelial solute transporter SLC34A1, and the glucose and albumin transporters SGLT2 and LRP2 was significantly upregulated in LTL-positive proximal tubular cells cultured on substrates treated with HSPG at a concentration of 0.6 μg / mL and laminin at a concentration of 2.5 μg / mL (Fig. 2), confirming that the functionality of proximal tubular cells was improved under these conditions.

[0126]

[0127] 2-3. Optimizing cell maintenance conditions

[0128] An optimized passaging and freeze-thawing protocol for maintaining the differentiated and polarized phenotype of functional proximal tubular epithelial cells manufactured in Example 2-1 was established, in which LTL-positive proximal tubular cells were passaged on dishes coated with laminin and HSPG, and then treated with SB431542, a selective TGF-β inhibitor, after 1 day. Specifically, LTL-positive proximal tubule cells isolated from renal organoids were cultured in 6-well plates coated with DPBS containing dissolved laminin-511 and HSPG, and cultured in DMEM / F12 Basal medium supplemented with Glutamax (2 mM, Gibco), 1% ITS (insulin-Transferrin-Selenium, Gibco), TGF-β / activin / NODAL pathway inhibitor (10 μM, SB431542), human EGF (10 ng / mL, Sigma-Aldrich), and Hydrocortisone (50 nM, Sigma-Aldrich) at passages 0 and 1. For comparison, immortalized cell line Human Renal Proximal Tubule Epithelial Cells (Lonza, CC-2553) were also cultured in REGM™ Renal Epithelial Cell Growth Medium BulletKit™ (Lonza, CC-3190). Upon reaching passage 2, cells reaching ~90% confluency were washed with calcium- and magnesium-free DPBS, treated with 0.05% trypsin / EDTA for 5 minutes, treated with culture medium, centrifuged at 220 g for 4 minutes, resuspended in culture medium, and dispensed into each dish. Each cell was then observed under a microscope.

[0129] As a result, cells not treated with SB431542 rapidly transformed from a cobblestone-like cell morphology to a fibroblast-like morphology within 1 day of culture (Fig. 3A). In addition, LTL-positive proximal tubular cells showed the fastest growth rate on the Laminin+HSPG-coated substrate, and became proximal tubular cells that closely mimicked the cobblestone phenotype of cultured immortalized proximal tubular epithelial cell lines, and were cultured up to passage 9 on the Laminin+HSPG-coated substrate (Fig. 3B). In addition, functional proximal tubular epithelial cells were cultured up to 2 × 10 using STEMCELL BANKER™. 6 We confirmed that functional proximal tubular epithelial cells could be cryopreserved at a density of 10 cells / mL. In addition, functional proximal tubular epithelial cell sheets cultured under the above conditions and growth factors or in conditioned media (CM) exhibited a typical epithelial dome-like structure, and this single-layer dome formation indicates functional plasma membrane polarization, tight junctions, and transepithelial solute transport. This dome formation was more prominent in functional proximal tubular epithelial cells than in primary RPTECs as a control (Fig. 3C).

[0130]

[0131] Example 3. Evaluation of reproducibility and applicability of functional proximal tubule epithelial cells.

[0132] To evaluate the reproducibility and applicability of the functional proximal tubule epithelial cells manufactured in Example 2, the functional LTL-positive proximal tubule cells cultured in 2D monolayer were compared with immortalized renal proximal tubule epithelial cells (RPTEcs). Specifically, each cell was fixed with 4% paraformaldehyde (in PBS) at pH 7.4 for 20 minutes and permeabilized with 0.3% Triton X-100 dissolved in PBS for 30 minutes at room temperature. Afterwards, each cell was washed and incubated overnight at 4°C with anti-LTL antibody (Vector Labs, Catalog no. B-1325-2), anti-ZO-1 antibody (Thermo Fisher, Catalog no. 33-9100), and anti-Laminin antibody (Sigma Aldrich, Catalog no. L9393) dissolved in PBS, reacted with fluorescence-conjugated secondary antibodies (Invitrogen, Carlsbad, CA), washed, and stained with VECTASHIELD H-1000. Images of each cell were observed using a Zeiss LSM 700 confocal microscope (Carl Zeiss, Germany) and analyzed with ZEN 3.1 software. In addition, mRNA levels of solute transporter, drug transporter, and ciliary genes were analyzed by Real-Time Quantitative PCR.

[0133] Apical-to-basal confocal scans revealed that functional proximal tubular epithelial cells expressed apical markers (zonula occludens protein 1, ZO-1) and proximal tubular markers (LTL), and that LTL-positive proximal tubular cells, which secrete their own ECM laminin, were densely clustered around areas where ZO-1 antibody was strongly expressed (Fig. 4A). In addition, the brush border marker LTL was found to be significantly higher expressed in functional proximal tubular epithelial cells of the present invention than in RPTEC (Fig. 4A). Furthermore, analysis of mRNA levels revealed that tubular epithelial solute transporters SLC34A1, ABCB1, and ATP1A1, glucose and albumin transporters SGLT2 and LRP2, and ciliary genes PKD1 and PKD2 were upregulated in functional proximal tubular epithelial cells compared to RPTECs, consistent with polarization and progressive morphogenesis (Fig. 4B), suggesting that co-treatment with laminin+HSPG contributed to the enhancement of proximal tubular cell functionality.

[0134]

[0135] Example 4. Polarization and Leakage Analysis of Functional Proximal Tubule Epithelial Cells

[0136] The first segment of the nephron's tubular network is known as the complex proximal tubule (PT), and human renal tubular epithelial cells (RPTECs) enable the continuous proliferation of primary renal epithelium by exerting signals related to physiological transition and regeneration in the adult kidney. However, immortalized renal proximal tubular epithelial cells generally show limited transporter expression, polarization, and loss of function in the absence of physiologic cues induced by the extracellular matrix. Therefore, we analyzed whether the functional proximal tubular epithelial cells of the present invention can generate leak-tight 2D monolayer apical access required for functional assessment of luminal transport and related diseases. Specifically, the functional proximal tubular epithelial cells of the present invention and RPTECs were each cultured in a Transwell TM Monolayer cultures were performed on filters (diameter 24 mm, pore size 0.4 μM, Corning, NY), and the integrity of the paracellular pathways of the monolayer to small ions was assessed by transepithelial electrical resistance (TER) using an EVOM™ device (World Precision Instruments, Sarasota, FL). Paracellular permeability was assessed by leakage of 2–5 kDa inulin-fluorescein isothiocyanate (FITC). Specifically, TER was normalized to the filter area after subtracting the resistance of the membrane support alone and then removing the background resistance of a blank filter measured with only medium without cells. TER was expressed in Ω cm 2 was measured in ohms × cm, with 0 being the case when there were no cells. 2 (Ω·cm 2) was calculated. For inulin leakage measurement, Transwell™ filters were washed with Hanks' Balanced Salt solution (HBSS, Gibco) containing 1% HEPES (Thermo Fisher Scientific), and 0.2 mL of 0.05 mg / mL 2–5 kDa inulin-FITC (Sigma) (in HBSS) was added to the apical side and incubated at 37°C for 90 min. Then, samples sampled twice from the basolateral compartment were transferred to a 96-well plate, and fluorescence (λex = 485 nm, λem = 530 nm) was measured using a fluorescence microplate reader (Gemini EM). The reference fluorescence value measured in HBSS containing 1% HEPES was subtracted, and the leakage rate (leakage %) was calculated using the following mathematical formula 1.

[0137]

[0138] As a result, immortalized RPTECs were grown on 12-well transwell filters compatible with leaky epithelia, with a pH of 10-20 Ω cm. 2 While functional proximal tubular epithelial cell monolayers exhibited a steady-state TER, functional proximal tubular epithelial cell monolayers exhibited a significantly increased TER compared to RPTECs (Fig. 5A). Furthermore, inulin permeability was significantly reduced in functional proximal tubular epithelial cells compared to RPTECs at 24 h (Fig. 5B).

[0139]

[0140] Example 5. Selective excretion analysis of functional proximal tubule epithelial cells

[0141] In order to evaluate whether the functional proximal tubular epithelial cells of the present invention are capable of selective excretion (excretion) that secretes and reabsorbs electrolytes, the compound CDFDA, which diffuses into the cell and is hydrolyzed into CDF, a substrate of the MRP2 / 4 (multidrug resistance protein 2 and 4) efflux pumps that remove xenobiotics in the kidney, was exposed to the basolateral side of the functional proximal tubular epithelial cell monolayer, and CDF was detected apically to analyze luminal efflux. Specifically, RPTEC and the functional proximal tubular epithelial cells of the present invention were seeded on a transwell filter and differentiated for 7 days to form a cell monolayer, and then the degree of leak-tightness was evaluated. Transwells were washed once with HBSS containing 1% HEPES, and 0.2 mL of 10 μM CDFDA (5(6)-carboxy-2',7'-dichlorofluorescein diacetate) (Sigma) in the presence or absence of 20 μM MK571 (Sigma) dissolved in HBSS containing 1% HEPES was added to the apical chamber, and HBSS containing 1% HEPES + / - 20 μM MK571 was added to the basolateral chamber. Cells were then incubated at 37°C for 45 min, and 100 μL of duplicate samples were collected from the basolateral compartment and transferred to black flat-bottom 96-well plates (Greiner). Fluorescence was measured (λex = 485 nm, λem = 530 nm) using a fluorescence microplate reader (Gemini EM), and the reference fluorescence value (HBSS containing 1% HEPES) was subtracted from the measured value, and the leakage % was calculated using the above mathematical equation 1.

[0142] As a result, unlike the immortalized cell line RPTEC, in which the luminal excretion of CDF was not altered by MK571, an MRP2 / 4 inhibitor, functional proximal tubular epithelial cells (LTL) showed a significant decrease in excretion by MK571 treatment compared to when MK571 was not present (Fig. 6), confirming that the functional proximal tubular epithelial cells of the present invention are capable of MRP2 / 4-mediated excretion.

[0143]

[0144] Example 6. 3D culture of functional proximal tubule epithelial cells

[0145] To determine whether culturing LTL-positive proximal tubular cells in a 3D extracellular matrix-like environment during the production of functional proximal tubular epithelial cells increases tubular morphology and the degree of cell differentiation compared to 2D monolayer culture, LTL-positive proximal tubular cells were seeded on HSPG-coated Transwell™ membranes and cultured to form branched, interconnected structures, and then stained with anti-LTL antibody (Vector Labs, Catalog no. B-1325-2), anti-ZO-1 antibody (Thermo Fisher, Catalog no. 33-9100), anti-ECAD antibody (abcam, Catalog no. ab11512), and anti-Na + / K + -Tubular structures were identified by immunofluorescence analysis using an ATPase antibody (Millipore, Catalog no. 05-369), and polarization of 3D cultured LTL-positive proximal tubule cells was confirmed by expression of typical proximal tubule epithelial membrane markers.

[0146] As a result, under the above 3D culture conditions, branched and branched structures were formed and maintained without morphological changes or overgrowth for 15 days of culture. In addition, immunofluorescence analysis showed that tubules were composed of a single layer of LTL-positive proximal tubule cells surrounding the lumen, and branched structures were observed (Fig. 7). yz-orthogonal projection analysis showed that LTL-positive proximal tubule cells included highly polarized cells surrounding the lumen. In addition, localization of ZO-1, a tight junction protein, to the apical membrane was confirmed, and Na + / K + Co-expression of ATPase (green) and LTL (white) revealed polarization of cells within the tubular structure (Fig. 7). This demonstrated that 3D culture of LTL-positive proximal tubule cells exhibited characteristics similar to those observed in vivo, confirming that the 3D approach further enhanced differentiation and functionality.

[0147]

[0148] Example 7. Improvement of expression and polarization of drug transporters in functional proximal tubule epithelial cells.

[0149] Based on the finding that HSPGs induce maturation of proximal tubular epithelial cells during renal organoid differentiation, we hypothesized that HSPGs could maintain the identity and enhance the function of proximal tubular epithelial cells isolated from renal organoids.

[0150] To confirm this, LTL-positive proximal tubule cells were sorted using the same method as in the previous example and cultured in wells coated with HSPG and laminin. Specifically, LTL-positive proximal tubule cells were isolated by MACS from renal organoids differentiated according to a protocol based on HSPG and VEGF treatment, and cultured in wells coated with HSPG and laminin. LTL-positive proximal tubule cells were found to grow best in an extracellular matrix consisting of calcium- and magnesium-free DPBS medium and dishes coated with 0.66 μg / mL HSPG and 2.5 μg / mL laminin. To maintain the differentiated and polarized phenotype of the cells after 1 day of culture, the addition of SB431542, a selective TGF-β receptor inhibitor, was also necessary. In the absence of SB431542, the cells rapidly transformed from cobblestone-like cells to fibroblast-like cells within 1 day of culture (Fig. 8A). As a result of optimizing the culture conditions as described above, LTL-positive proximal tubule cells exhibited a cuboid-like phenotype, closely mimicking the cobblestone phenotype of immortalized proximal tubule epithelial cell lines, and grew rapidly (Fig. 8A). Therefore, the cells cultured using the above protocol were named functionally enhanced kidney organoid-derived proximal tubule cells (FEKOPTCs).

[0151] To confirm the identity and gene expression patterns of the FEKOPTCs according to the number of passages, bulk RNA sequencing (RNA-seq) was performed on early passages (passages 1 and 2) and late passages (passages 5) (Fig. 8B). A heatmap of representative gene expression patterns showed that the expression of genes related to transporters, epithelial markers, maturation, and differentiation increased, whereas EMT and injury markers decreased in FEKOPTCs of passage 5 compared to FEKOPTCs of passages 1 and 2 (Fig. 8B).

[0152] Moreover, FEKOPTCs showed high expression of E-cadherin and N-cadherin without undergoing EMT. Consistent with the RNA-seq analysis, RT-PCR analysis showed that the gene expression of the drug transporter gene ABCB1 (Fig. 9A), the tubular epithelial solute transporter ATP1A1 (Fig. 9B), the glucose transporters SLC5A2 (Fig. 9C) and SLC2A1 (Fig. 9D), and the organic cation transporter 3 SLC22A3 (Fig. 9E) was significantly upregulated in the fifth-passage FEKOPTCs compared to the first- and second-passage FEKOPTCs.

[0153] In summary, these transcription results show that FEKOPTCs can be continuously subcultured without losing their identity, and their maturity can be increased according to the culture conditions of the present invention, which confirms that they can be usefully utilized in polar drug absorption and toxicity studies.

[0154] Next, the morphology of FEKOPTCs was confirmed. Dome formation reflects functional plasma membrane polarization, formation of tight junctions, and transepithelial solute transport of proximal tubule cells. In this regard, the FEKOPTCs sheets of the present invention exhibited a structure similar to a typical epithelial dome. This confluent monolayer at the bottom was formed within 3 to 4 days. Furthermore, microscopic observation and immunofluorescence staining of N-cadherin, a marker of proximal tubules, confirmed that the multicellular dome at the top remained viable for up to 7 to 8 days (Fig. 8C).

[0155] A 3D environment is known to promote morphological changes and organoid-like functions. Therefore, FEKOPTCs were seeded onto HSPG-coated Transwell™ membranes, and cultured in a 3D extracellular matrix (ECM)-like environment, forming branched, interconnected structures. Under these 3D culture conditions, FEKOPTCs could be maintained for up to 15 days without significant morphological changes or excessive cell proliferation (Fig. 8D).

[0156] Confocal microscopy was used to further characterize the tubular structure. Analysis of various optical planes confirmed that the tubules consisted of a single layer of FEKOPTCs surrounding the lumen. Furthermore, orthogonal projection analysis revealed that FEKOPTCs were composed of highly polarized cells surrounding the lumen. Next, to assess the polarization of 3D-cultured LTL-positive proximal tubular epithelial cells, typical proximal tubular epithelial membrane markers were detected immunocytochemically. Apical membrane localization was examined by staining with an antibody against the cell junction protein zonula occludens protein 1 (ZO-1). As a result, the 3D model was confirmed to exhibit promising in vivo-like properties, which confirmed that the 3D approach could further enhance differentiation and enhance the functionality of LTL-positive proximal tubule epithelial cells (Fig. 8D).

Claims

1. A composition for enhancing the function of proximal tubule epithelial cells containing HSPG (heparan sulfate proteoglycans).

2. A composition for enhancing the function of proximal tubule epithelial cells, further comprising laminin in the first paragraph.

3. A composition for enhancing the function of proximal tubular epithelial cells, wherein the proximal tubular epithelial cells in paragraph 1 are LTL (Lotus tetragonolobus lectin) positive cells.

4. A composition for enhancing the function of proximal tubular epithelial cells, wherein the proximal tubular epithelial cells in paragraph 1 are cells expressing GGT1, ABCB1 or SGLT2.

5. A composition for enhancing the function of proximal tubular epithelial cells, wherein the enhancement of the function of proximal tubular epithelial cells in paragraph 1 is an enhancement of polarization or drug transporter maturation.

6. A composition for enhancing the function of proximal tubule epithelial cells, which increases the expression of LTL, SLC34A1, SLC5A2, SLC2A1, SLC22A3, ABCB1, ATP1A1, SGLT2, LRP2, PKD1 or PKD2 according to claim 1.

7. A culture vessel for enhancing the function of proximal tubule epithelial cells having a surface coated with the composition of paragraph 1.

8. Composition for culturing proximal tubule epithelial cells containing HSPG.

9. A composition for culturing proximal tubule epithelial cells, further comprising L-glutamine, insulin, transferrin, selenium, laminin, TGF-β inhibitor, epidermal growth factor (EGF), or steroid according to claim 8.

10. A composition for culturing proximal tubular epithelial cells, which improves polarization or functionality of proximal tubular epithelial cells in accordance with claim 8.

11. A composition for culturing proximal tubule epithelial cells, which increases the expression of LTL, SLC34A1, SLC5A2, SLC2A1, SLC22A3, ABCB1, ATP1A1, SGLT2, LRP2, PKD1 or PKD2 according to claim 8. 12.a) Step of preparing kidney organoids; b) a step of dissociating the kidney organoid into single cells; c) a step of isolating proximal tubule epithelial cells; and d) A method for producing functional proximal tubular epithelial cells, comprising a step of enhancing the functionality of proximal tubular epithelial cells.

13. In paragraph 12, the step of preparing a kidney organoid comprises: i) a step of differentiating human pluripotent stem cells (hPSCs) into the intermediate mesoderm of nephron progenitor cells; and ⅱ) A method for producing functional proximal tubular epithelial cells, comprising a 3D culturing step.

14. A method for producing functional proximal tubular epithelial cells, wherein the human pluripotent stem cell (hPSC) in claim 13 is a hiPSC (human-induced PSC) induced by reprogramming from peripheral blood mononuclear cells (PBMC).

15. A method for producing functional proximal tubular epithelial cells in claim 12, wherein the proximal tubular epithelial cells are LTL (Lotus tetragonolobus lectin) positive cells.

16. A method for producing functional proximal tubular epithelial cells, wherein the step of enhancing the functionality of proximal tubular epithelial cells in claim 12 is to culture proximal tubular epithelial cells on a solid phase coated with laminin and HSPG.

17. A method for producing functional proximal tubular epithelial cells in claim 16, wherein the solid phase is a cell culture substrate or a cell culture carrier.

18. A method for producing functional proximal tubular epithelial cells, wherein the culture is a monolayer culture or a 3D culture in accordance with claim 16.

19. A method for producing functional proximal tubular epithelial cells, wherein the culture is performed in a medium containing L-glutamine, insulin, transferrin, selenium, laminin, TGF-β inhibitor, epidermal growth factor (EGF), or steroid in accordance with claim 16.

20. Functional proximal tubular epithelial cells manufactured by the method of Article 12.

21. In claim 20, a functional proximal tubular epithelial cell having increased expression of LTL, SLC34A1, SLC5A2, SLC2A1, SLC22A3, ABCB1, ATP1A1, SGLT2, LRP2, PKD1 or PKD2 compared to a proximal tubular epithelial cell having no enhanced functionality.

22. In claim 20, a functional proximal tubular epithelial cell having improved polarization, selective excretion, or drug transporter maturation compared to a proximal tubular epithelial cell without enhanced functionality.

23. A functional proximal tubule epithelial cell in claim 22, wherein the selective excretion is efflux pump-mediated excretion by MRP2 / 4 (multidrug resistance protein 2 and 4) efflux pumps.

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