Composition for preventing or treating kidney diseases

Abstract

The kidney tissue-derived stem cells or organoids according to the present invention are easy to treat for treatment, can be supplied in large quantities with high self-proliferation ability, have excellent differentiation ability into kidney cells, and have low possibility of tumor formation, and when directly injected into a lesion, have a very excellent ability to regenerate damaged tissue. Therefore, among such stem cells, they can be very suitably used for adult stem cell-based regenerative therapy. In addition, the composition according to the present invention can specifically select only kidney stem cells, particularly kidney stem cells, among kidney cells. Furthermore, kidney stem cells expressing the Lrig1 protein or a gene encoding it have excellent regenerative ability and pluripotency, and have the ability to divide into nephrons, so they can be very effectively used in the prevention or treatment of kidney diseases.

Description

Technical Field

[0001] The present invention relates to a composition for preventing or treating kidney diseases.

Background Art

[0002] The kidney is an important organ for maintaining the homeostasis of the living body. It regulates the body fluid volume, ion concentration, and pH in the blood, excretes waste products such as metabolic waste products, toxins, and drugs, regulates blood pressure, and performs other metabolic and endocrine functions. It also assists in activating vitamin D so that calcium is absorbed in the small intestine and is involved in the synthesis of various hormones. A state in which the kidney cannot perform its excretion, regulation, metabolism, and endocrine functions normally and the overall function deteriorates or abnormalities occur is called a kidney disease. A decrease in function due to kidney damage leads to an increase in the kidney and related structures, atrophy of the kidney, changes in body fluid volume, electrolyte imbalance, metabolic acidosis, gas exchange disorder, anti-infection function damage, accumulation of uremic toxins, etc. In particular, acute kidney injury is a refractory disease with a high fatality rate due to a decrease in kidney function caused by various reasons. Even if the kidney function recovers, it may progress to chronic and end-stage renal failure if it recurs or is not treated depending on the cause and degree of the damage.

[0003] Despite the development of modern medicine, many patients admitted to hospitals suffer a lot due to a decrease in kidney function. In particular, patients with a high severity of illness often require renal replacement therapy due to a decrease in kidney function. The morbidity rate of acute kidney injury is about 5% of inpatients and is reported to be about 30 - 50% of patients admitted to the intensive care unit. Such a morbidity rate has a tendency to increase steadily despite the development of new treatment methods.

[0004] Currently, preclinical reports and clinical trials on stem cells are being actively conducted to treat such kidney diseases. Specifically, various types of stem cells, such as mesenchymal stem cells, adipose-derived stem cells, amniotic fluid stem cells, and kidney progenitor cells, have been extensively studied for kidney recovery. Among these stem cells, cell therapy agents using adult stem cells derived from adult kidneys in particular have potential advantages such as improved kidney engraftment and differentiation, and can be very usefully applied to autologous therapy.

[0005] In this context, in the case of the kidney, it has been reported that adult stem cells exist in the distal renal tubule ends, glomeruli, and renal papilla, and various studies on cell therapy agents that can induce the regeneration of damaged kidney cells by such stem cells are continuing.

[0006] However, currently, research on identifying such kidney stem cells, identifying the stem cells for application as therapeutic agents, and their applicability for clinical use is still insufficient.

Summary of the Invention

Problems to be Solved by the Invention

[0007] One object of the present invention is to provide a pharmaceutical composition for preventing or treating kidney diseases containing kidney tissue-derived stem cells as an active ingredient.

[0008] Another object of the present invention is to provide a method for producing a kidney organoid; a kidney organoid produced thereby; and a pharmaceutical composition for preventing or treating kidney diseases containing the kidney organoid as an active ingredient.

[0009] Still another object of the present invention is to provide a composition for detecting kidney tissue-derived stem cells; and a kit for detecting kidney stem cells containing the same.

[0010] Still another object of the present invention is to provide a method for detecting kidney tissue-derived stem cells; and a method for separating kidney stem cells.

[0011] Still another object of the present invention is to provide a method for culturing kidney tissue-derived stem cells.

[0012] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and still other problems not mentioned will be clearly understood by those having ordinary knowledge in the art from the following description.

Means for Solving the Problems

[0013] 1. A pharmaceutical composition containing kidney tissue-derived stem cells as an active ingredient In one embodiment of the present invention, a pharmaceutical composition for preventing or treating kidney diseases containing kidney tissue-derived stem cells as an active ingredient is provided.

[0014] The kidney stem cells of the present invention express the Lrig1 (Leucine Rich Repeats And Immunoglobulin Like Domains 1) protein or the gene encoding the same.

[0015] The kidney epithelial cells expressing the Lrig1 or the gene encoding the same of the present invention are kidney tissue-derived stem cells having stem cell ability, different from other cells, and in particular, can have the ability to divide into nephrons by participating in tubulogenesis, which is a late developmental stage of the kidney. Therefore, for the purpose of the present invention, when the kidney epithelial cells expressing the Lrig1 having the stem cell ability or the gene encoding the same are used, kidney diseases can be very effectively prevented or treated.

[0016] The "Lrig1" of the present invention is a transmembrane protein that interacts with receptor tyrosine kinases such as EGFR-family, MET, and RET proteins. The Lrig1 may be derived from mammals including primates such as humans and monkeys, and rodents such as mice and rats. For example, it may be human Lrig1 (a polypeptide encoded by accession number: NM_015541 or NP_056356, a polypeptide represented by SEQ ID NO: 1), but is not limited thereto.

[0017] In addition, in the present invention, the kidney tissue-derived stem cells can additionally express Klf6 (Krueppel-like factor 6) protein or a gene encoding the same.

[0018] The "Klf6" of the present invention is a protein encoded by the KLF6 gene corresponding to a tumor suppressor gene. The Klf6 may be derived from mammals including primates such as humans and monkeys, and rodents such as mice and rats. For example, it may be human Lrig1 (a polypeptide encoded by accession number: NP_001153596.1 or NM_001160124.1; a polypeptide encoded by NP_001153597.1 or NM_001160125.1 [Q99612-3]; a polypeptide encoded by NP_001291.3 or NM_001300.5 [Q99612-1], a polypeptide represented by SEQ ID NO: 2), but is not limited thereto.

[0019] The "kidney tissue-derived stem cells" of the present invention refer to stem cells existing in the kidney tissue, which are self-renewing and pluripotent stem cells that can differentiate into all cell types of the kidney. Such stem cells can be involved in the regeneration and homeostasis of kidney injury.

[0020] The kidney tissue-derived stem cells of the present invention may be used as a cell therapy agent.

[0021] The "cell therapy agent" of the present invention is a living cell used in a treatment method of directly injecting into a patient, and means a pharmaceutical product manufactured by operating with physical, chemical or biological methods such as culturing, proliferating or selecting living autologous cells, allogeneic cells or xenogeneic cells in vitro. For the purpose of the present invention, since the kidney tissue-derived stem cells of the present invention are very excellent in stem cell ability in which the Lrig1 protein present in the patient's kidney or the gene encoding the same is expressed, there is an advantage that side effects such as transplant rejection reaction are very few.

[0022] The "kidney disease" of the present invention is a disease that causes weakening of kidney function, and includes acute kidney injury (AKI) and chronic kidney disease (CKD) depending on the rate at which the deterioration of kidney function progresses. For example, it may be acute kidney injury, but is not limited thereto.

[0023] The kidney disease of the present invention may be at least one selected from the group consisting of, for example, glomerulonephritis, chronic renal failure, acute renal failure, nephrotic syndrome, pyelonephritis, kidney stones and kidney cancer, but is not limited thereto.

[0024] The "prevention" of the present invention means a decrease in the occurrence of pathological cells or the degree of cell damage and disappearance in animals. The prevention may be complete or partial. In this case, it can mean a phenomenon in which the occurrence of pathological cells or abnormal immune action in the individual decreases as compared with the case where the composition for preventing and treating the kidney disease is not used.

[0025] The "treatment" of the present invention means all acts of clinically intervening to alter the natural processes of the subject or cells to be treated, and can be carried out during the progression of a clinical pathological condition or to prevent it. The intended therapeutic effect can include preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing all direct or indirect pathological consequences of the disease, preventing metastasis, reducing the progression rate of the disease, alleviating or temporarily relieving the disease state, or improving the prognosis. That is, the treatment is construed to include all acts by which the symptoms of the kidney disease are improved or cured by the composition.

[0026] The kidney tissue-derived stem cells of the present invention are 1×10 7 ~1×10 8 、1×10 8 ~2×10 8 、2×10 8 ~4×10 8 、4×10 8 ~6×10 8 、6×10 8 ~8×10 8 、8×10 8 ~1×10 9 、1×10 9 ~2×10 9 、2×10 9 ~4×10 9 、4×10 9 ~1×10 10 、2×10 8 ~6×10 8 、6×10 8 ~1×10 9 、1×10 8 ~2×10 8 、2×10 8 ~2×10 9 、1×10 7 ~1×10 8 、1×10 8 ~1×10 9 、1×10 9 ~1×10 10 Or can be administered at a dose of any one of 1×10 7 ~1×10 9 cells / kg, but is not limited thereto.

[0027] The pharmaceutical composition of the present invention is characterized by being in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition is characterized by targeting humans.

[0028] The pharmaceutical composition of the present invention is not limited to these, but can be formulated into oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories and sterile injection solutions by ordinary methods respectively. The pharmaceutical composition of the present invention can contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. for oral administration, and buffers, preservatives, soothing agents, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used for injections, and bases, excipients, lubricants, preservatives, etc. can be used for topical administration. The dosage forms of the pharmaceutical composition of the present invention can be manufactured in various ways by mixing with pharmaceutically acceptable carriers as described above. For example, for oral administration, it can be manufactured in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit-dose ampoules or multiple-dose forms. In addition, it can be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0029] On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, etc. In addition, fillers, anticoagulants, lubricants, wetting agents, flavors, emulsifiers, preservatives, etc. can be additionally included.

[0030] The administration routes of the pharmaceutical composition of the present invention are not limited thereto, and include oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal. For example, it may be oral or parenteral administration.

[0031] The "parenteral" of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. For the purpose of the present invention, the pharmaceutical composition can be administered by a method of directly injecting into the kidney, but is not limited thereto.

[0032] The pharmaceutical composition of the present invention can vary diversely depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug formulation, and severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but can be appropriately selected by those skilled in the art, and can be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The administration may be once a day or divided into several times. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention is formulated into tablets, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

[0033] 2. Organoid In the present invention, the kidney organoid described below is easy to operate for treatment, has a high self-renewal ability and can be supplied in large quantities, is excellent in the ability to differentiate into kidney cells, has a low possibility of tumor formation, and when directly injected into a lesion, the ability to regenerate damaged tissue is extremely excellent. Therefore, it can be very suitably used for adult stem cell-based regenerative therapy. In particular, such a kidney organoid has the merit that it is extremely excellent in the direct regeneration effect compared with cell therapy agents using existing mesenchymal stem cells, embryonic stem cells or induced pluripotent stem cells.

[0034] Hereinafter, the kidney organoid, a method for producing the kidney organoid, and its uses will be described in detail.

[0035] (1) Kidney organoid In another embodiment of the present invention, a kidney organoid is provided.

[0036] The kidney organoid of the present invention contains kidney tissue-derived stem cells that express the Lrig1 protein or the gene encoding the same.

[0037] The kidney epithelial cells expressing the Lrig1 or the gene encoding the same of the present invention are different from other cells and are kidney tissue-derived stem cells having stem cell ability. In particular, they can be involved in tubulogenesis, which is a late developmental stage of the kidney, and have the ability to divide into nephrons. Therefore, for the purpose of the present invention, the kidney epithelial cells expressing the Lrig1 having stem cell ability or the gene encoding the same can form organoids very effectively.

[0038] Also, in the present invention, the kidney tissue-derived stem cells can additionally express the Klf6 protein or the gene encoding the same.

[0039] In the kidney organoid of the present invention, the content regarding the Lrig1 or Klf6 protein or the gene encoded thereby, kidney tissue-derived stem cells, etc. is the same as that described in the above "1. Pharmaceutical composition containing kidney tissue-derived stem cells as an active ingredient", so the description is omitted.

[0040] The "organoid" of the present invention means cells having a 3D three-dimensional structure and means a model similar to a tissue produced by an artificial culture process not collected or obtained from an animal or the like. Different from 2D culture, in 3D cell culture, cells can grow in all directions outside the body.

[0041] (2) Method for producing a kidney organoid In still other embodiments of the present invention, a method for producing kidney organoids is provided.

[0042] The production method of the present invention includes: (a) separating cells in which Lrig1 (Leucine Rich Repeats And Immunoglobulin Like Domains 1) protein or a gene encoding the same is expressed from kidney epithelial cells isolated from a target individual; (b) culturing the cells in which the Lrig1 protein or a gene encoding the same is expressed; and (c) placing the cultured cells in Matrigel to form organoids.

[0043] The kidney epithelial cells expressing Lrig1 or a gene encoding the same of the present invention are different from other cells and are kidney tissue-derived stem cells having stem cell ability, and in particular, can have the ability to divide into nephrons in relation to tubulogenesis in the late development stage of the kidney. Therefore, for the purpose of the present invention, when using kidney tissue-derived stem cells expressing Lrig1 having the stem cell ability or a gene encoding the same, kidney organoids can be produced at a very high yield.

[0044] Also, the cells separated in step (a) of the present invention may be those in which Klf6 (Krueppel-like factor 6) protein or a gene encoding the same is expressed.

[0045] In the method for producing kidney organoids of the present invention, the content regarding Lrig1 or Klf6 protein or a gene encoded thereby, kidney tissue-derived stem cells, organoids, etc. is the same as that described in the above "1. Pharmaceutical composition containing kidney stem cells as an active ingredient" and "(1) Kidney organoids", and thus the description is omitted.

[0046] In the step (a) of the present invention, the step of separating cells is carried out by dissociating a sample separated from a target individual and then by magnetic activated cell sorting (MACS) or flow cytometry analysis by a conventional method, but is not limited thereto.

[0047] The step of dissociating the sample of the present invention may use collagenase, but is not limited thereto.

[0048] In the step (b) of the present invention, the step of culturing the cells in which the gene is expressed may use a cell culture medium containing fetal bovine serum, growth factors and antibiotics.

[0049] In the step (c) of the present invention, the step of forming the organoid may use a cell culture medium containing a B27 supplement, a conditioned medium, growth factors, N-acetylcysteine and an ALK5 (TGFβ kinase / activin receptor-like kinase) inhibitor.

[0050] The "growth factor" of the present invention is a substance necessary for cell growth, and may be, for example, VEGF (Vascular endothelial growth factor), HGF (Hepatocyte growth factor), IGF (Insulin-like growth factor), EGF (Epidermal growth factor), FGF (Fibroblast growth factor), etc., but is not limited thereto.

[0051] The "conditioned medium" of the present invention may be at least one selected from the group consisting of Wnt3a conditioned medium, noggin conditioned medium, and Rspol conditioned medium. For example, it may contain Wnt3a conditioned medium, noggin conditioned medium, and Rspo1 conditioned medium. For example, it may contain 40% Wnt3a conditioned medium, 10% noggin conditioned medium, and 10% Rspo1 conditioned medium, but is not limited thereto.

[0052] The "cell culture medium" of the present invention contains basic components for the growth and maintenance of cell lines in vitro. For example, it may be DMEM (Dulbeco’s Modified Eagle’s Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI-1640, DMEM / F10, DMEM / F12, ADMEM / F12, GMEM (Glasgow’s Minimal essential Medium), IMDM (Iscove’s Modified Dulbecco’s Medium), etc. The cell culture medium in the step (b) may be RPMI-1640, and the cell culture medium in the step (c) may be ADMEM / F12, but is not limited thereto.

[0053] In a specific example of the present invention, in the step (b), the cell culture medium may contain 10% fetal bovine serum, 20 ng / ml of EGF (Epidermal growth factor), and 1% penicillin-streptomycin, but is not limited thereto.

[0054] In one specific example of the present invention, in the step (c), the cell culture medium may contain, but is not limited to, 1.5% B27 supplement, 40% Wnt3a conditioned medium, 10% noggin conditioned medium, 10% Rspo1 conditioned medium, 50 ng / ml EGF, 100 ng / ml FGF-10, 1.25 mM N-acetylcysteine, and 5 μM A8301 (CAS no. CAS Number 909910-43-6).

[0055] The Matrigel of the present invention may be, but is not limited to, growth factor-reduced Matrigel.

[0056] (3) Kidney organoids In another embodiment of the present invention, a kidney organoid produced by the method for producing a kidney organoid according to the present invention is provided.

[0057] The kidney organoid of the present invention is produced using kidney tissue-derived stem cells that express Lrig1 having very excellent stem cell ability or a gene encoding the same, preferably, Lrig1 and Klf6 proteins or genes encoding the same. It can not only very effectively realize the kidney and effectively screen a therapeutic agent for kidney injury, but also be used as a cell therapeutic agent directly used for kidney injury.

[0058] Regarding the content such as Lrig1 or Klf6 protein or the gene encoded thereby, the kidney organoid, and the production method in the kidney organoid of the present invention, it is the same as that described in the above "1. Pharmaceutical composition containing kidney stem cells as an active ingredient" and "(1) Kidney organoid", so the description is omitted.

[0059] (4) Pharmaceutical composition for preventing or treating kidney diseases In still another embodiment of the present invention, a pharmaceutical composition for preventing or treating kidney diseases containing a kidney organoid as an active ingredient is provided.

[0060] The kidney organoid of the present invention may be used as a cell therapeutic agent.

[0061] Regarding the Lrig1 protein or the gene encoded thereby, the pharmaceutical composition, prevention, treatment, kidney organoid, and manufacturing method in the pharmaceutical composition for preventing or treating the kidney disease of the present invention are the same as those described in the above "1. Pharmaceutical composition containing kidney tissue-derived stem cells as an active ingredient" and "(1) Kidney organoid", so the description is omitted.

[0062] The kidney organoid of the present invention can be administered at a dose of 1×10 7 ~1×10 8 、1×10 8 ~2×10 8 、2×10 8 ~4×10 8 、4×10 8 ~6×10 8 、6×10 8 ~8×10 8 、8×10 8 ~1×10 9 、1×10 9 ~2×10 9 、2×10 9 ~4×10 9 、4×10 9 ~1×10 10 、2×10 8 ~6×10 8 、6×10 8 ~1×10 9 、1×10 8 ~2×10 8 、2×10 8 ~2×10 9 、1×10 7 ~1×10 8 、1×10 8 ~1×10 9 、1×10 9 ~1×10 10 or 1×10 7 ~1×10 9 cells / kg of any one of the doses, but is not limited thereto.

[0063] 4. Detection Composition, Kit, and Detection Method (1) Composition for Detecting Kidney Tissue-Derived Stem Cells In another embodiment of the present invention, a composition for detecting kidney tissue-derived stem cells is provided.

[0064] The detection composition of the present invention includes a preparation for measuring the expression level of Lrig1 protein or a gene encoding the same.

[0065] Furthermore, the detection composition of the present invention may further include a preparation for measuring the expression level of Klf6 protein or a gene encoding the same.

[0066] Regarding the content related to Lrig1 or Klf6 protein and kidney tissue-derived stem cells in the detection composition of the present invention, it is the same as that described in the above "1. Pharmaceutical Composition Containing Kidney Tissue-Derived Stem Cells as an Active Ingredient", so the description is omitted.

[0067] The preparation for measuring the expression level of the gene of the present invention may include all those capable of measuring the expression level of DNA present in a biological sample or mRNA transcribed therefrom, for example, at least any one selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene, but is not limited thereto.

[0068] The "primer" of the present invention is a fragment that recognizes a target gene sequence and includes a primer pair in the forward and reverse directions. Preferably, it is a primer pair that provides analytical results with specificity and sensitivity. Since the nucleic acid sequence of the primer is a sequence that does not match the non-target sequence present in the sample, high specificity can be imparted when the primer amplifies only the target gene sequence containing complementary primer binding sites and does not induce non-specific amplification.

[0069] The "probe" of the present invention means a substance that can specifically bind to a target substance to be detected in a sample, and means a substance that can specifically confirm the presence of the target substance in the sample by the binding. The types of probes are not limited to substances commonly used in the art, but preferably may be PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, and most preferably PNA. More specifically, the probe includes substances derived from organisms as biological substances, those similar thereto, or those produced in vitro. For example, it may be an enzyme, protein, antibody, microorganism, animal and plant cells and organs, nerve cells, DNA, and RNA. DNA includes cDNA, genomic DNA, oligonucleotides, and RNA includes genomic RNA, mRNA, oligonucleotides. Examples of proteins can include antibodies, antigens, enzymes, peptides, etc.

[0070] The "LNA (Locked nucleic acids)" of the present invention means a nucleic acid analog containing a 2'-O, 4'-C methylene bridge. LNA nucleosides contain common nucleobases of DNA and RNA and can form base pairs according to the Watson-Crick base pair rule. However, due to the "locking" of the molecule by the methylene bridge, LNA cannot form an ideal shape with Watson-Crick binding. When LNA is included in a DNA or RNA oligonucleotide, LNA can pair with a complementary nucleotide strand faster and enhance the stability of the double helix.

[0071] The "antisense" of the present invention means an oligomer having a nucleotide base sequence and a backbone between subunits, in which the antisense oligomer hybridizes with a target sequence in RNA by forming Watson-Crick base pairs and allows the formation of an RNA:oligomer heterodimer, typically with mRNA, within the target sequence. The oligomer can have exact or approximate sequence complementarity to the target sequence.

[0072] Since the information of the gene of the present invention can be easily confirmed by an ordinary technician through a site that can be confirmed by the NCBI accession number, primers, probes or antisense nucleotides that specifically bind to the gene can be easily prepared based on the gene sequence derived by the ordinary technician.

[0073] The preparation for measuring the expression level of the protein of the present invention may include all those that can measure the amount of protein present in a biological sample. For example, it may be at least any one selected from the group consisting of an antibody, oligopeptide, ligand, PNA (peptide nucleic acid), and aptamer that specifically binds to the protein, but is not limited thereto.

[0074] The "protein" of the present invention includes not only the protein itself, but also protein isoforms or protein variants that can be generated by splicing and variable promoters, or by genetic changes such as mutations or polymorphisms.

[0075] The "antibody" of the present invention refers to a substance that specifically binds to an antigen to cause an antigen-antibody reaction. For the purpose of the present invention, the antibody means an antibody that specifically binds to the biomarker protein. The antibodies of the present invention include all polyclonal antibodies, monoclonal antibodies and recombinant antibodies. The antibodies can be easily produced using techniques widely known in the art. For example, polyclonal antibodies can be produced by methods widely known in the art, including the process of injecting the antigen of the protein into an animal, collecting blood from the animal, and obtaining serum containing the antibody. Such polyclonal antibodies can be produced from any animal such as goats, rabbits, sheep, monkeys, horses, pigs, cows, dogs, etc. Also, monoclonal antibodies can be produced using methods widely known in the art, such as the hybridoma method or phage antibody library technology. The antibodies produced by the above methods are separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, etc. In addition, the antibodies of the present invention include not only the complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of the antibody molecule. The functional fragment of the antibody molecule means a fragment that possesses at least the antigen-binding function, and includes all of Fab, F(ab’), F(ab’)2 and Fv, etc.

[0076] The "PNA" of the present invention refers to a polymer similar to artificially synthesized DNA or RNA, and was first introduced in 1991 by Professors Nielsen, Egholm, Berg and Buchardt of the University of Copenhagen in Denmark. While DNA has a phosphate-ribose sugar backbone, PNA has a repeating N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases the binding force and stability to DNA or RNA, and is used in molecular biology, diagnostic analysis and antisense therapy. The PNA can be further specified with reference to the background art widely known in the technical field of the present invention.

[0077] The "aptamer" of the present invention is an oligonucleic acid or peptide molecule, and the general content of the aptamer can be embodied with reference to the documents [Bock LC et al., Nature 355(6360):564 - 6(1992); Hoppe - Seyler F, Butz K “Peptide aptamers: powerful new tools for molecular medicine”. J Mol Med. 78(8):426 - 30(2000); Cohen BA, Colas P, Brent R. “An artificial cell - cycle inhibitor isolated from a combinatorial library”. Proc Natl Acad Sci USA. 95(24):14272 - 7(1998)].

[0078] The antibody, oligopeptide, ligand, PNA, aptamer, etc. of the present invention can be easily prepared by an ordinary technician based on the amino acid sequence that can be confirmed by the site that can be confirmed by the NCBI accession number.

[0079] (2) Kit for detecting kidney tissue - derived stem cells In still another embodiment of the present invention, there is provided a kit for detecting kidney tissue - derived stem cells containing the detection composition according to the present invention.

[0080] In the kit of the present invention, the content regarding the Lrig1 protein or the gene encoding the same, kidney tissue - derived stem cells, the preparation for measuring the expression level of the protein, the preparation for measuring the expression level of the gene, etc. is the same as that described in the above “1. Pharmaceutical composition containing kidney tissue - derived stem cells as an active ingredient” and “(1) Detection composition for kidney stem cells”, so the description is omitted.

[0081] The kit of the present invention may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit, but is not limited thereto.

[0082] The kit of the present invention may further include one or more other component compositions, solutions, or devices suitable for the analysis method.

[0083] In one specific example of the present invention, the kit may further include essential elements necessary for performing reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes a primer pair specific to the gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of the gene, and can have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. In addition, it can include a primer specific to the nucleic acid sequence of a control group gene (for example, a housekeeping gene). Other reverse transcription polymerase reaction kits can include, but are not limited to, test tubes or other suitable containers, reaction buffers (with various pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor, DEPC-water, sterilized water, etc.

[0084] In another specific example of the present invention, the diagnostic kit of the present invention can include essential elements necessary for performing a DNA chip. The DNA chip kit can include a substrate to which cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, preparations, enzymes, etc. for preparing a fluorescently labeled probe. In addition, the substrate can include, but is not limited to, cDNA or oligonucleotide corresponding to a control group gene (for example, a housekeeping gene) or a fragment thereof.

[0085] In still other specific examples of the present invention, the diagnostic kit of the present invention can include essential elements necessary for performing ELISA. The ELISA kit includes an antibody specific for the protein. The antibody is an antibody with high specificity and affinity for the marker protein and almost no cross-reactivity with other proteins, and is a monoclonal antibody, polyclonal antibody or recombinant antibody. Further, the ELISA kit can include an antibody specific for a control group (e.g., housekeeping protein) protein. Other ELISA kits can include reagents capable of detecting the bound antibody, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to the antibody) and their substrates or other substances capable of binding to the antibody.

[0086] (3) Method for detecting kidney tissue-derived stem cells In still other embodiments of the present invention, a method for detecting kidney tissue-derived stem cells is provided.

[0087] The detection method of the present invention includes the step of measuring the expression level of Lrig1 protein or the gene encoding the same from a biological sample isolated from a subject individual.

[0088] Also, when measuring the expression level in the step of the present invention, the expression level of Klf6 protein or the gene encoding the same can be additionally measured from the biological sample.

[0089] In the detection method of the present invention, the content regarding kidney tissue-derived stem cells, Lrig1 or Klf6 protein or the gene encoding the same is the same as that described in the above “1. Pharmaceutical composition containing kidney tissue-derived stem cells as an active ingredient” and “(1) Composition for detecting kidney stem cells”, so the description is omitted.

[0090] The "biological sample" of the present invention means any substance, biological fluid, tissue or cell obtained from an individual or derived from an individual, and includes, for example, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, blood including plasma and serum, urine, semen, organ secretions, cells or cell extracts, and may be, for example, kidney tissue or kidney cells, but is not limited thereto.

[0091] The step of measuring the expression level of the gene of the present invention is carried out by reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting or DNA chip, etc., using a preparation for measuring the expression level of a gene which is at least any one selected from the group consisting of primers, probes and antisense nucleotides that specifically bind to the gene, but is not limited thereto.

[0092] The step of measuring the expression level of the protein of the present invention is performed by protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (liquid chromatography-Mass Spectrometry / Mass Spectrometry), Western blotting, ELISA (enzyme linked immunosorbent assay), etc., using a preparation for measuring the expression level of a protein which is at least any one selected from the group consisting of an antibody, oligopeptide, ligand, PNA (peptide nucleic acid), and aptamer that specifically binds to the protein, but is not limited thereto.

[0093] In the detection method of the present invention, when the expression level of the Lrig1 protein or the gene encoding the same is increased compared to the control group, the step of detecting as kidney tissue-derived stem cells can further be included.

[0094] 5. Method for isolating kidney tissue-derived stem cells In still another embodiment of the present invention, a method for isolating kidney tissue-derived stem cells is provided.

[0095] The separation method of the present invention includes the step of separating cells in which the Lrig1 protein or the gene encoding the same is expressed from a biological sample separated from a subject of interest.

[0096] In the step of separating the cells of the present invention, the cells may additionally express Klf6 protein or a gene encoding the same.

[0097] In the separation method of the present invention, the content related to Lrig1 or Klf6 protein or a gene encoding the same, kidney tissue-derived stem cells, biological samples, etc. is the same as that described in the above "1. Pharmaceutical composition containing kidney stem cells as an active ingredient", "(1) Composition for detecting kidney stem cells" and "(3) Method for detecting kidney tissue-derived stem cells", so the description is omitted.

[0098] The step of separating in the present invention is performed by Magnetic activated cell sorting (MACS) or Flow cytometry Analysis.

[0099] 6. Method for culturing kidney tissue-derived stem cells In still another embodiment of the present invention, a method for culturing kidney tissue-derived stem cells is provided.

[0100] The culturing method of the present invention includes a step of separating kidney tissue-derived stem cells that express Lrig1 protein or a gene encoding the same, and a step of culturing the separated kidney tissue-derived stem cells.

[0101] In the step of separating in the present invention, the kidney tissue-derived stem cells may additionally express Klf6 protein or a gene encoding the same.

[0102] In the separation method of the present invention, the content related to Lrig1 or Klf6 protein or a gene encoding the same, kidney tissue-derived stem cells, etc. is the same as that described in the above "1. Pharmaceutical composition containing kidney stem cells as an active ingredient", so the description is omitted.

[0103] The method for culturing according to the present invention may be, but is not limited to, a method in which kidney tissue-derived stem cells are separated by the method described in the method for separating the kidney tissue-derived stem cells of the present invention, and then cultured, for example, in vitro (in a test tube).

[0104] In the step of culturing according to the present invention, "culturing" means culturing in any test tube of cells. For the culturing of the present invention, a cell culture medium can be used, and the cell culture medium means all types of media used in an environment for culturing cells, and can include, for example, amino acids, at least one carbohydrate as an energy source, trace elements, vitamins, salts, and possible additional components (e.g., to affect cell growth, productivity, or product quality), but is not limited thereto.

[0105] 7. Method for producing an animal model In still another embodiment of the present invention, a method for producing an animal model for screening a cell therapeutic agent for preventing or treating kidney diseases is provided.

[0106] The method for producing the animal model according to the present invention includes a step of inducing kidney injury in an animal in which a target gene is conditionally expressed by the CreERT2-LoxP system, and a step of treating the animal with an estrogen antagonist to induce the expression of the target gene.

[0107] In the method for producing the animal model according to the present invention, the content regarding kidney tissue-derived stem cells, kidney diseases, and cell therapeutic agents is the same as that described in the above "1. Pharmaceutical composition containing kidney tissue-derived stem cells as an active ingredient", and thus the description is omitted.

[0108] The "CreERT2-LoxP system" of the present invention refers to a system in which the Cre-ERT2 polypeptide expressed specifically by a promoter recognizes two LoxP nucleotide sequences and catalyzes site-specific recombination between the two LoxP nucleotide sequences to specifically express a gene downstream of the LoxP nucleotide. The construction method of such a system can be easily constructed by an ordinary technician according to the content described in Lab Anim Res. 2018 Dec; 34(4): 147-159, etc.

[0109] The "target gene" of the present invention is a gene that is specifically expressed or expected to be expressed in cells that may be used as a cell therapy agent, and for the purpose of the present invention, it may be a gene encoding Lrig1, preferably a gene encoding Lrig1 and a gene encoding Klf6, but is not limited thereto.

[0110] The "animal model" of the present invention means an animal that can show a form very similar to human diseases, etc. When using such an animal model, the causes and pathogenesis of various diseases can be studied, and basic data for judging the possibility of drug screening, toxicity testing, etc. can be obtained.

[0111] The animal of the present invention means any mammalian animal other than humans, including animals of all ages including embryos, fetuses, newborns, and adults. Such animals may be any one selected from the group consisting of rabbits, rodents (mice, rats, hamsters, gerbils or guinea pigs), cows, sheep, pigs, goats, horses, dogs, cats, birds (chickens, pigeons, ducks, geese) and primates (chimpanzees, monkeys, macaques), but is not limited thereto.

[0112] The step of inducing kidney injury of the present invention may be any one of the methods composed of intraperitoneal administration of folic acid; inducing ischemia / reperfusion injury; and inducing unilateral ureteral obstruction, but is not limited thereto.

[0113] The folic acid of the present invention may be intraperitoneally administered at 100 mg / kg to 300 mg / kg, for example, it may be administered at 150 mg / kg or 250 mg / kg, but is not limited thereto. When the administration dose of the folic acid is less than 100 mg / kg, acute kidney injury may not be induced in the kidney. When it exceeds 300 mg / kg, there is toxicity in the animal model, which may affect the analysis of the target experimental results, or the animal model may die.

[0114] The estrogen antagonist of the present invention may be tamoxifen, 4-hydroxytamoxifen, clomifene, raloxifene, or a combination thereof, but is not limited thereto.

[0115] 8. Animal model In still another embodiment of the present invention, there is provided an animal model for screening a cell therapy agent for preventing or treating kidney diseases produced by the production method of the present invention.

[0116] In the animal model of the present invention, the contents related to the production method, cell therapy agent, kidney disease, etc. are the same as those described in the above "1. Pharmaceutical composition containing kidney tissue-derived stem cells as an active ingredient" and "6. Production method of animal model", so the description is omitted.

Advantages of the invention

[0117] The kidney tissue-derived stem cells or organoids according to the present invention are easy to operate for treatment, have a high self-renewal ability and can be supplied in large quantities, are excellent in the ability to differentiate into kidney cells, have a low possibility of tumor formation, and when directly injected into a lesion, have an extremely excellent ability to regenerate damaged tissues. Therefore, among such stem cells, they are particularly suitable for use in adult stem cell-based regenerative medicine.

[0118] In addition, the composition according to the present invention can specifically select only kidney cells, particularly kidney stem cells. Furthermore, kidney stem cells that express the Lrig1 protein or the gene encoding the same are very excellent in regenerative ability and pluripotency and have the ability to divide into nephrons, so they can be very effectively used for the prevention or treatment of kidney diseases.

Brief Description of the Drawings

[0119]

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Mode for Carrying Out the Invention

[0120] The present invention is for overcoming the limitations in the treatment of kidney diseases such as conventional acute renal failure and developing more effective therapeutic agents. The present invention is for developing an effective therapeutic agent for kidney diseases using kidney tissue-derived stem cells or organoids. When only high-concentration folic acid (FA) was treated and when high-concentration folic acid was treated and PBS was injected (FA + PBS), the levels of blood BUN and creatinine were very high, whereas when a kidney organoid was injected, the levels of BUN and creatinine decreased to a level similar to that of normal. From such results, it can be directly understood that Lrig1-positive kidney organoids can be used very effectively for the treatment of damaged kidneys.

Examples

[0121] [Experimental Method] [Experimental Method 1] Experimental Animals All in vivo experiments conducted in this specification were carried out with the approval of the Institutional Animal Care and Use Committee (IACUC2017-0325) of Yonsei University in Korea.

[0122] Under a 12-hour light-dark cycle, the experimental animals were housed in a specific pathogen-free (SPF) barrier facility and fed with PicoLab Lab Rodent Diet 20 (LabDiet, St. Louis, MO, USA).

[0123] For the experimental animals used in in vivo experiments, 1) Lrig1CreERT2 / + was provided by Robert J. Coffey of Vanderbilt University, 2) B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato) / Hze / J (R26R-LSL-tdTomato; The Jackson Laboratory, 007914) was provided by Professor Pok Jinwoon of Yonsei University, and 3) B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdToamto-EGFP)Luo / J (ACTB-mT / mG; The Jackson Laboratory, 007676) was provided by Professor Ki Hyongwoon of Yonsei University.

[0124] [Experimental Method 2] In Vivo Lineage Tracing Research Method As shown in Figure 1, Lrig1CreERT2 / + mice and R26R-LSL-tdTomato mice, which are reporter mice of the same type as in Experimental Method 1, were mated to produce heterozygous mice. After injecting 2 mg of tamoxifen (Sigma-Aldrich) contained in corn oil intraperitoneally for 3 consecutive days to induce Cre-loxp recombination in the mice, analysis was performed on the 1st, 3rd, 10th, 30th, 60th, 90th, 180th, or 365th day.

[0125] Also, as shown in Fig. 2, E9.5, E10.5, E13.5, and E18.5 embryos generated by mating Lrig1CreERT2 / + mice and R26R-LSL-tdTomato mice, which are isogenic reporter mice, were injected once with 2 mg of 4-hydroxytamoxifen (Sigma-Aldrich) contained in corn oil, and analyzed at 6 weeks postpartum.

[0126] [Experimental Method 3] In vitro 2D and Organoid Culture Methods For 2D and organoid culture, isogenic reporter mice produced by mating Lrig1CreERT2 / + mice and R26R-ACTB-mT / mG mice were bred for 6 to 10 weeks, and then primary kidney epithelial cells were collected. The collected primary kidney epithelial cells were placed with 2 mg / ml of type I collagenase and cultured with gentle stirring at 37 °C for 30 minutes. Then, the primary kidney epithelial cells were filtered using a filter, and the separated single cells were cultured. Thereafter, the cells expressing Lrig1 protein were placed in RPMI1640 medium (containing 10% fetal bovine serum (FBS), 20 ng / ml of EGF, and 1% penicillin-streptomycin) and cultured for 7 to 8 days under the conditions of 5% CO2 and 37 °C until the confluence reached about 80%. Thereafter, 1×10 3 number of the cultured cells were dispensed into wells containing growth factor-reduced Matrigel and culture medium and cultured. Here, the culture medium was based on ADMEM / F12 culture medium containing 1% penicillin-streptomycin, HEPES, and Glutamax, and contained 1.5% B27 supplement, 40% Wnt3a conditioned medium (produced using stably transfected L cells), 10% noggin conditioned medium, 10% Rspo1 conditioned medium, 50 ng / ml of EGF, 100 ng / mL of FGF-10, 1.25 mM of N-acetylcysteine, and 5 μM of A8301 (CAS no. CAS Number 909910-43-6).

[0127] After allowing the cells to polymerize sufficiently with Matrigel, an organoid culture medium was added and the organoid culture medium was changed every three days.

[0128] [Experimental method 4] Preparation of an animal model of acute kidney injury [4-1] Preparation of an animal model of high-dose folic acid-induced acute kidney injury As shown in Figure 3, in adult (8-10-week-old) Lrig1-CreERT2;LSL-tdTomato mice, during the process of injecting tamoxifen as described in Experimental method 2 above, Lrig1 protein expression was enabled. Thereafter, for induction of acute kidney injury, 250 mg / kg of folic acid (FA) was injected intraperitoneally.

[0129] Also, as shown in Figure 4, 150 mg / kg of folic acid was injected intraperitoneally into C57BL / 6 mice to induce acute kidney injury, and on the 3rd day after folic acid injection, the kidney organoids prepared by the above Experimental method 3 were transplanted in an orthotopic transplantation manner. Specifically, the C57BL / 6 mice (n = 3) in which the acute kidney injury was induced using isoflurane were anesthetized, the flank was incised to expose the kidney externally. Thereafter, about 40 PBS as a control group or kidney organoids prepared by the above Experimental method 3 were directly injected into the cortical region of the kidney at least 15 times. Thereafter, the mice were bred for 11 days for analysis. Thereafter, on the 14th day, blood samples were collected to measure the plasma creatinine and BUN values from the mice.

[0130] [4-2] Preparation of an animal model of acute kidney injury induced by ischemia / reperfusion injury As shown in Fig. 5, in adult (8 - 10 weeks old) Lrig1-CreERT2;LSL-tdTOmato mice, during the process of injecting tamoxifen as described in the above Experimental Method 2, Lrig1 protein was enabled to be expressed. Subsequently, acute kidney injury was induced by occluding the renal artery of the said mice with forceps for 20 minutes. Three days later, the kidney tissues of the said mice were collected and confirmed. As a result, it was confirmed that acute kidney injury had been induced.

[0131] [4 - 3] Preparation of an animal model of acute kidney injury induced by unilateral ureteral obstruction As shown in Fig. 6, in adult (8 - 10 weeks old) Lrig1-CreERT2;LSL-tdTomato mice, during the process of injecting tamoxifen as described in the above Experimental Method 2, Lrig1 protein was enabled to be expressed. Subsequently, acute kidney injury was induced by occluding the ureter with forceps for 7 days. Seven days later, the kidney tissues of the said mice were collected and confirmed. As a result, it was confirmed that acute kidney injury had been induced.

[0132] [Experimental Results] [Experimental Result 1] Lineage analysis of tamoxifen induction of Lrig1-tdTomato progeny in the mouse kidney To analyze the behavior patterns of cells expressing Lrig1 in the kidney and their progeny, lineage tracing analysis was performed using the R26R-LSL-tdTomato mouse model.

[0133] As shown in Fig. 7, on the first day, very low levels of tdTomato+ cells were observed in the Cortex, while on the 365th day, it was confirmed that the progeny cells induced by cells expressing Lrig1 generated 12 (±2.4)% of the tubular structures in the whole kidney.

[0134] As shown in Fig. 8, when quantifying the number of cells expressing Lrig1 (Lrig1tdT+) by date, such cell clones increased sequentially together as time went on, and such an increase continued until 365 days after Lrig1 expression.

[0135] As shown in FIGS. 9 and 10, tdTomato+ cells (Lrig1tdT+) were not observed at E9.5 and E10.5, which are the ureteric bud branching stages. However, tdTomato+ cells were observed at E13.5, which is the nephrogenesis stage, and most of the cells were extended to form tube structures.

[0136] These results indicate that Lrig1 is a stem cell population that is involved in generating nephron divisions after developing in the initially mature kidney.

[0137] [Experimental Result 2] Confirmation of the therapeutic effect by orthotopic transplantation of kidney organoids in an animal model of acute kidney injury induced by high-dose folic acid As shown in FIG. 11, when the kidney organoids prepared by the method described in Experimental Method 3 were analyzed using a fluorescence microscope, a large number of green Lrig1 cells were observed on the 7th and 9th days of culturing the kidney organoids.

[0138] Also, as shown in FIG. 12, when the gene expression level was confirmed by performing real-time polymerase chain reaction (Real-time PCR) using the primers in Table 1 below by the usual method on the 17th day of culturing the kidney organoids, it was confirmed that not only the expression level of Lrig1 but also the expression levels of genes of kidney stem cells such as Sall1, Six2, Foxo1, Cited, Osr1, Hoxp7, Jagged1, and Gata3 increased together. Furthermore, as shown in FIG. 13, in the case of kidney organoids that are Lrig1 positive, the number of generated organoids increased to about 40 or more from the 9th day to the 19th day.

[0139] [Table 1]

[0140] As shown in Fig. 14, PBS (FA + PBS) or kidney organoids (FA + Organoid) were directly injected into the kidneys of an animal model of acute kidney injury induced by high - concentration folic acid described in [4 - 1] of the experimental method 4. After 14 days, H&E staining was performed by the usual method. As a result, when only PBS was injected (FA + PBS), the kidneys damaged by high - concentration folic acid did not recover, and the expression level of KIM1 was high. In contrast, when kidney organoids were injected, the kidneys damaged by high - concentration folic acid recovered, and the expression level of KIM1 decreased. From these results, it can be seen that the injection of kidney organoids can very effectively recover damaged kidneys.

[0141] Also, as shown in Fig. 15, when only high - concentration folic acid (FA) was treated, and when high - concentration folic acid was treated and PBS was injected (FA + PBS), the levels of blood BUN and creatinine were very high. In contrast, when kidney organoids were injected, the levels of BUN and creatinine decreased to a level similar to that of normal. From these results, it can be directly seen that Lrig1 - positive kidney organoids can be very effectively used for the treatment of damaged kidneys.

[0142] Also, as shown in Fig. 16, as a result of confirmation by a fluorescence microscope, it was found that the organoids injected into the kidneys were confirmed by immunofluorescence staining and that proliferation markers such as Ki - 67 were expressed. Therefore, it was confirmed that the organoids injected into the kidneys were proliferative.

[0143] [Experimental Result 3] Stem cell niche formation effect of Lrig1 and its progeny cells in adult kidneys Based on the above experimental results, it was found that Lrig1-positive cells survived for a long time in the proximal tubule (PT), corresponding to potential renal stem / progenitor cells, and the descendant cells derived from the Lrig1-positive cells substantially contributed to maintaining PT homeostasis. To confirm the cellular heterogeneity of Lrig1+ cells and their derived cells in the PT, as shown in Figure 17, the PT clusters were classified into four sub-clusters: PTS1, PTS2, PTS3, and PTQPs. At this time, the Slc5a12 and Slc5a2 markers were used for the classification of PTS1, the Slc13a3 and Ddah1 markers were used for the classification of PTS2, and the Slc16a9 and Slc7a13 markers were used for the classification of PTS3. Then, as shown in Figure 18, PTQPs, a sub-cluster rich in pyruvate dehydrogenase kidney 4 (Pdk4) and cystein-rich protein 61 (Cyr61), which are upregulated in the acute kidney injury state, was found. To define it more specifically, PTQPs was compared with other PT sub-clusters. As a result of confirming the expression levels of the top 50 genes expressed in PTQPs, as shown in Figure 19, it was confirmed that genes related to kidney injury and recovery were highly expressed in PTQPs, and it was also confirmed that 2 nephron progenitor genes were expressed. As a result of measuring the cell numbers in each PT sub-cluster, as shown in Figure 20, the cell numbers of PTS2 and PTQPs increased on the 365th day after Cre-loxp recombination. Also, as shown in Figure 21, it was confirmed that the cell number of the PTQPs cluster in the kidney on the 365th day after Cre-loxp recombination increased 5-fold compared to that of the kidney on the 1st day. From this, it was found that PT cells increased over time, and in particular, the cell numbers of the PTQPs and PTS2 clusters increased. Next, the PTQPs cluster of the kidney was defined using the adult stem cell gene module. As a result, a total of 650 genes were detected among the DEGs.As shown in Fig. 22, it was confirmed that stem - related genes were highly expressed in the PTQPs cluster, and self - renewal - related genes (except Klf5, Ptbp1, Ncl, Ctr9, and Cited2), quiescence - related genes (Hif1a, Myc, and Foxo3), and pluripotency or immature cell - related genes (Id2, Btg2, Tubb6, and Klf2) were up - regulated. Thus, it was found that the PTQPs cluster expressed stem - related genes including kidney injury - recovery - related genes and markers of mature PT cells, and based on such results, this cluster was named "PT quiescent progenitors (PTQPs)".

[0144] Next, as a result of identifying PTQPs markers distinct from PTS3, as shown in Fig. 23, genes known as stem cell niches that were highly expressed among the DEGs were identified. In the PTS3 cluster, PT segment 3 - specific genes were highly expressed, whereas they were not highly expressed in PTQPs. To verify the PTQPs markers, kidney sections were stained by IF using Jun, Klf6, and Cyr61. Jun and Cyr61 were highly expressed not only in PT but also in various nephron sections, and these were excluded. Next, to confirm whether they were expressed in PT, KLF6 and LTL were stained in kidney sections on day 1 and day 365. As shown in Fig. 24, Klf6 + LTL + PT tubules were weakly expressed in kidney sections on day 1, whereas Klf6 + LTL + PT tubules were strongly expressed in kidney sections on day 365. As a result of quantification, as shown in Fig. 25, it was confirmed that the KLF6 + - expressing PT tubules were significantly increased in the kidney on day 365. From this, it was found that the increase in PTQPs distribution in the kidney on day 365 could be confirmed using Klf6.

[0145] To confirm whether the progeny cells derived from Lrig1-positive cells form a PTQPs population, tdTomato-expressing cells were identified in kidney sections on day 1 and day 365. As a result, as shown in Fig. 26, on day 1, tdTomato+ cells accounted for 54% of PTS1 cells and 27% of PTS3 in the kidney section, but decreased to 50% and 11% respectively in the kidney section on day 365. In contrast, on day 1, they accounted for 12% of PTS2 cells and 17.8% of PTQPs cells in the kidney section, but increased to 20% and 18% respectively on day 365. From this, it was found that the progeny cells derived from Lrig1-positive cells form a PTQPs cell population. Also, the trajectory of cells indicating the progression to differentiation was analyzed based on single-cell information. At the initial stage of the trajectory, it corresponded to PTS3, known as the PT stem cell niche, and PTQPs, identified as a new stem cell niche. Such cell clusters are divided into two distinguishable trajectories to PTS1 (Fig. 27). In the kidney on day 1, it showed a pattern of differentiating into the PTS1 cluster after PTS3 became dominant, and on day 365, it was found that the PTQPs cluster mainly maintains the homeostasis of PTS1 cluster cells. As a result of aligning PTS3 and PTQPs with tdTomato-expressing cells on day 1 and day 365, as shown in Fig. 28, it was confirmed that tdTomato-expressing cells differentiated into PTS1 in the PTS3 cluster on day 1, and on day 365, it was found that tdTomato-expressing cells in the PTQPs cluster mainly maintained PTS1. When Klf6 and tdTomato+ cells were stained, as shown in Figs. 29 and 30, it was confirmed that Klf6+tdTomato+ tubules increased significantly on day 365 compared to day 1. From this, it was found that Lrig1-positive cells and their progeny cells form a PTQPs cluster. Although the kidney on day 1 was taken from 6-week-old mice under the experimental conditions, the kidney on day 365 was taken from 13-month-old mice. From the above experimental results, it was found that kidney homeostasis is regulated by PTS3 in the young kidney (kidney on day 1), and PTQPs represents a new stem cell niche in the aged kidney (kidney on day 365).

[0146] Although the present invention has been described in detail above, the scope of the rights of the present invention is not limited thereto, and it will be apparent to those skilled in the art that various modifications and variations are possible without departing from the technical idea of the present invention described in the claims.

Industrial Applicability

[0147] The present invention is for overcoming the limitations of the treatment of kidney diseases such as conventional acute renal failure and developing a more effective therapeutic agent. At present, research on identifying kidney stem cells, identifying stem cells for application as therapeutic agents, and the applicability for clinical use is currently insufficient, but the need for development is required. The present invention uses kidney tissue-derived stem cells; or organoids, which are easy to administer for treatment, have excellent differentiation ability into kidney cells, have a low possibility of tumor formation, and when directly injected into lesions, have an extremely excellent ability to regenerate damaged tissues, and thus are expected to be very effectively used for the prevention or treatment of kidney diseases. [Free text of sequence listing] SEQ ID NO: 1 MARPVRGGLG APRRSPCLLL LWLVLVRLEP VTAAAGPRAP CAAACTCAGD SLDCGGRGLA ALPGDLPSWT RSLNLSYNKL SEIDPAGFED LPNLQEVNLS YNKLSEIDPA GFEDLPNLQE VYLNNNELTA VPSLGAASSH VVSLFLQHNK IRSVEGSQLK AYLSLEVLDL SLNNITEVRN TCFPHGPPIK ELNLAGNRIG TLELGAFDGL SRSLLTLRLS KNRITQLPVR AFKLPRLTQL DLNRNRIRLI EGLTFQGLNS LEVLKLQRNN ISKLTDGAFW GLSKMHVLHL EYNSLVEVNS GSLYGLTALH QLHLSNNSIA RIHRKGWSFC QKLHELVLSF NNLTRLDEES LAELSSLSVL RLSHNSISHI AEGAFKGLRS LRVLDLDHNE ISGTIEDTSG AFSGLDSLSK LNLGGNAIRS VQFDAFVKMK NLKELHISSD SFLCDCQLKW LPPWLIGRML QAFVTATCAH PESLKGQSIF SVPPESFVCD DFLKPQIITQ PETTMAMVGK DIRFTCSAAS SSSSPMTFAW KKDNEVLTNA DMENFVHVHA QDGEVMEYTT ILHLRQVTFG HEGRYQCVIT NHFGSTYSHK ARLTVNVLPS FTKTPHDITI RTTTVARLEC AATGHPNPQI AWQKDGGTDF PAARERRMHV MPDDDVFFIT DVKIDDAGVY SCTAQNSAGS ISANATLTVL ETPSLVVPLE DRVVSVGETV ALQCKATGNP PPRITWFKGD RPLSLTERHH LTPDNQLLVV QNVVAEDAGR YTCEMSNTLG TERAHSQLSV LPAAGCRKDG TTVGIFTIAV VSSIVLTSLV WVCIIYQTRK KSEEYSVTNT DETVVPPDVP SYLSSQGTLS DRQETVVRTE GGPQANGHIE SNGVCPRDAS HFPEPDTHSV ACRQPKLCAG SAYHKEPWKA MEKAEGTPGP HKMEHGGRVV CSDCNTEVDC YSRGQAFHPQ PVSRDSAQPS APNGPEPGGS DQEHSPHHQC SRTAAGSCPE CQGSLYPSNH DRMLTAVKKK PMASLDGKGD SSWTLARLYH PDSTELQPAS SLTSGSPERA EAQYLLVSNG HLPKACDASP ESTPLTGQLP GKQRVPLLLA PKS Sequence number 2: MDVLPMCSIF QELQIVHETG YFSALPSLEE YWQQTCLELE RYLQSEPCYV SASEIKFDSQ EDLWTKIILA REKKEESELK ISSSPPEDTL ISPSFCYNLE TNSLNSDVSS ESSDSSEELS PTAKFTSDPI GEVLVSSGKL SSSVTSTPPS SPELSREPSQ LWGCVPGELP SPGKVRSGTS GKPGDKGNGD ASPDGRRRVH RCHFNGCRKV YTKSSHLKAH QRTHTGEKPY RCSWEGCEWR FARSDELTRH FRKHTGAKPF KCSHCDRCFS RSDHLALHMK RHL SEQ ID NO: 3: GGTGAGCCTGGCCTTATGTGAATA SEQ ID NO: 4: CACCACCATCCTGCACCTCC SEQ ID NO: 5: TACTCTTTCCTTCAGGCAGTGA SEQ ID NO: 6: GATCGAGGCAAGTGCATGG SEQ ID NO: 7: CACCTCCACAAGAATGAAAGCG SEQ ID NO: 8: CTCCGCCTCGATGTAGTGC SEQ ID NO: 9: AACCTTGGAGTGAAGGATCGC SEQ ID NO: 10: GTAGGAGAGCCTATTGGAGATGT SEQ ID NO: 11: CTCAACATTTCCAATCCGACCC SEQ ID NO: 12: GGCATCCTTGCTCTTAGTGGG SEQ ID NO: 13: GAGAGCCAGCCTACCATCC SEQ ID NO: 14: GGGTCCTCGTGTTTGAAGGAA SEQ ID NO: 15: AAGTTCGGTTTTCGCTCCAGG SEQ ID NO: 16: ACACCCCGGAGAGGTTCTG SEQ ID NO: 17: CTCGGCCATTCGTACATGGAA SEQ ID NO: 18: GGATACCTCTGCACCGTAGC SEQ ID NO: 19: GCGTCAGGGAGATGGTAAAG SEQ ID NO: 20: CATCAGGGAAACAGTTGCAG SEQ ID NO: 21: CGCTAAGAATCCGCTGGTGAAG SEQ ID NO: 22: GGATCTTGACGAAGCAGTCGTT SEQ ID NO: 23: CCTCGGGTCAGTTTGAGCTG SEQ ID NO: 24: CCTTGAGGCACACTTTGAAGTA

Claims

1. A pharmaceutical composition for preventing or treating kidney diseases, comprising, as an active ingredient, kidney tissue-derived stem cells that express Lrig1 (Leucine Rich Repeats And Immunoglobulin Like Domains 1) protein or a gene encoding the same.

2. The pharmaceutical composition according to claim 1, wherein the kidney tissue-derived stem cells additionally express Klf6 (Kruppel-like factor 6) protein or a gene encoding the same.

3. The pharmaceutical composition according to claim 1, wherein the kidney disease is acute kidney injury (AKI) or chronic kidney disease (CKD).

4. A kidney organoid comprising kidney tissue-derived stem cells that express Lrig1 protein or a gene encoding the same.

5. The kidney organoid according to claim 4, wherein the kidney tissue-derived stem cells additionally express Klf6 (Kruppel-like factor 6) protein or a gene encoding the same.

6. A pharmaceutical composition for preventing or treating kidney diseases, comprising, as an active ingredient, the kidney organoid according to claim 4 or 5.

7. A method for detecting kidney tissue-derived stem cells, comprising the step of measuring the expression level of Lrig1 (Leucine Rich Repeats And Immunoglobulin Like Domains 1) protein or a gene encoding the same in a biological sample isolated from a target individual.

8. The detection method according to claim 7, wherein, when measuring the expression level, the expression level of Klf6 (Kruppel-like factor 6) protein or a gene encoding the same is additionally measured in the biological sample.

9. The detection method according to claim 7, wherein the expression level of the gene is measured by at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically binds to the gene.

10. The detection method according to claim 7, wherein the expression level of the protein is measured by at least one selected from the group consisting of an antibody, an oligopeptide, a ligand, a PNA, and an aptamer that specifically binds to the protein.

11. The detection method according to claim 7, further comprising the step of detecting as kidney tissue-derived stem cells when the expression level of the Lrig1 (Leucine Rich Repeats And Immunoglobulin Like Domains 1) protein or the gene encoding the same is increased as compared with a control group.

12. A method for isolating kidney tissue-derived stem cells, comprising the step of isolating cells in which the Lrig1 (Leucine Rich Repeats And Immunoglobulin Like Domains 1) protein or the gene encoding the same is expressed from a biological sample isolated from a target individual.

13. The isolation method according to claim 12, wherein the isolated cells additionally express the Klf6 (Kruppel-like factor 6) protein or the gene encoding the same.

14. The isolation method according to claim 12, wherein the isolating step is performed by magnetic activated cell sorting (MACS) or flow cytometry analysis.

15. A step of separating kidney tissue-derived stem cells that express the Lrig1 (Leucine Rich Repeats And Immunoglobulin Like Domains 1) protein or a gene encoding the same, and a step of culturing the separated kidney tissue-derived stem cells, a method for culturing kidney tissue-derived stem cells comprising the same.

16. The method for culturing according to claim 15, wherein the cells to be separated additionally further express the Klf6 (Kruppel-like factor 6) protein or a gene encoding the same.

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