WO2022169279A1 - 신장 질환의 예방 또는 치료용 조성물 - Google Patents
신장 질환의 예방 또는 치료용 조성물 Download PDFInfo
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Definitions
- the present invention relates to a composition for preventing or treating kidney disease.
- Kidney is an important organ that maintains homeostasis. It regulates body fluid volume, ion concentration and pH in the blood, excretes waste products such as metabolic wastes, toxins, and drugs, and controls blood pressure and other metabolic and endocrine functions. carry out In addition, it activates vitamin D to help calcium absorption in the small intestine and is also involved in the synthesis of various hormones.
- the kidney does not normally perform excretory, regulatory, metabolic and endocrine functions, and the overall function is reduced or abnormal is referred to as kidney disease.
- Decreased function due to kidney damage results in enlargement of the kidneys and related structures, atrophy of the kidneys, changes in body fluid volume, electrolyte imbalance, metabolic acidosis, gas exchange disorders, impaired anti-infective function, and accumulation of uremic toxins.
- acute renal failure is an intractable disease with a high mortality rate due to a decrease in renal function due to various causes. Even if renal function is restored, depending on the cause and severity of the damage, it may recur or progress to chronic or end-stage renal failure if not treated.
- stem cells such as mesenchymal stem cells, adipose-derived stem cells, amniotic fluid stem cells, and renal progenitor cells
- mesenchymal stem cells adipose-derived stem cells
- amniotic fluid stem cells adipose-derived stem cells
- renal progenitor cells are being studied in various ways to restore the kidney.
- a cell therapy agent using adult stem cells derived from an adult kidney has a potential advantage of improved kidney engraftment and differentiation, and there are advantages such as being very useful for self-treatment.
- One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of kidney disease comprising kidney tissue-derived stem cells as an active ingredient.
- Another object of the present invention is a method for preparing renal organoids; kidney organoids prepared through this; And to provide a pharmaceutical composition for the prevention or treatment of kidney disease comprising a kidney organoid as an active ingredient.
- Another object of the present invention is a composition for detecting kidney tissue-derived stem cells; And to provide a kit for detecting kidney stem cells comprising the same.
- Another object of the present invention is a kidney tissue-derived stem cell detection method; And to provide a method for isolating renal stem cells.
- Another object of the present invention is to provide a method for culturing kidney tissue-derived stem cells.
- composition comprising kidney tissue-derived stem cells as an active ingredient
- kidney tissue-derived stem cells as an active ingredient.
- the renal stem cells of the present invention express Lrig1 (Leucine Rich Repeats And Immunoglobulin Like Domains 1) protein or a gene encoding the same.
- Renal epithelial cells expressing Lrig1 or a gene encoding the same of the present invention are kidney tissue-derived stem cells having stem cell ability, unlike other cells, and are particularly involved in tubulogenesis, a later developmental stage of the kidney. It has the ability to split into nephrons. Therefore, in the case of using the renal epithelial cells expressing Lrig1 having the stem cell ability or a gene encoding the same for the purpose of the present invention, kidney disease can be very effectively prevented or treated.
- 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 humans and primates such as monkeys, rodents such as mice and rats, and the like, for example, human Lrig1 (accession number: a polypeptide encoded by NM_015541 or NP_056356, The polypeptide represented by SEQ ID NO: 1), but is not limited thereto.
- kidney tissue-derived stem cells may further express Klf6 (Krueppel-like factor 6) protein or a gene encoding the same.
- the "Klf6" of the present invention is a protein encoded by the KLF6 gene, which corresponds to a tumor suppressor gene.
- the Klf6 may be derived from mammals including humans and primates such as monkeys, rodents such as mice and rats, and the like, for example, human Lrig1 (accession number: a polypeptide encoded by NP_001153596.1 or NM_001160124 .1; polypeptide encoded by NP_001153597.1 or NM_001160125.1 [Q99612-3]; polypeptide encoded by NP_001291.3 or NM_001300.5 [Q99612-1], polypeptide represented by SEQ ID NO: 2) may be, but is not limited thereto.
- kidney tissue-derived stem cell refers to a stem cell existing in the kidney tissue, and refers to a self-renewing and pluripotent stem cell that can be differentiated into all cell types of the kidney. Such stem cells may be involved in the regeneration and homeostasis of kidney damage.
- kidney tissue-derived stem cells of the present invention may be used as cell therapy agents.
- the "cell therapy agent" of the present invention is a living cell used in a treatment method directly injected into a patient, and is manipulated by physical, chemical or biological methods such as culturing, proliferating, or selecting living autologous, allogeneic or xenogeneic cells in vitro. medicines manufactured by For the purpose of the present invention, the kidney tissue-derived stem cells have very excellent stem cell ability in which Lrig1 protein or a gene encoding the Lrig1 protein present in the patient's kidney is expressed. do.
- kidney disease of the present invention is a disease that causes weakening of renal function, and depending on the rate at which the deterioration of renal function progresses, acute kidney injury (AKI) and chronic kidney disease (CKD) Included, for example, may be acute kidney injury, but is not limited thereto.
- AKI acute kidney injury
- CKD chronic kidney disease
- the kidney disease of the present invention may be, for example, at least one selected from the group consisting of glomerulonephritis, chronic renal failure, acute renal failure, nephrotic syndrome, pyelitis, kidney stones and kidney cancer, but is not limited thereto.
- prevention of the present invention means a reduction in the degree of the occurrence of pathological cells or damage or loss of cells in an animal. Prevention may be complete or partial. In this case, it may mean a phenomenon in which the generation of pathological cells or abnormal immune action in the subject is reduced compared to the case where the composition for preventing and treating kidney disease is not used.
- the "treatment" of the present invention refers to any action that clinically intervenes to change the natural process of a subject or cell to be treated, and may be performed while a clinical pathology is in progress or to prevent it.
- the desired therapeutic effect is to prevent the occurrence or recurrence of a disease, alleviate symptoms, reduce any direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, or reduce the rate of disease progression. alleviating or temporarily ameliorating the condition, or improving the prognosis. That is, the treatment may be interpreted as encompassing all actions in which the symptoms of kidney disease are improved or cured by the composition.
- the kidney tissue-derived stem cells of the present invention are 1 ⁇ 10 7 to 1 ⁇ 10 8 , 1 ⁇ 10 8 to 2 ⁇ 10 8 , 2 ⁇ 10 8 to 4 ⁇ 10 8 , 4 ⁇ 10 8 to 6 ⁇ 10 8 , 6 ⁇ 10 8 to 8 ⁇ 10 8 , 8 ⁇ 10 8 to 1 ⁇ 10 9 , 1 ⁇ 10 9 to 2 ⁇ 10 9 , 2 ⁇ 10 9 to 4 ⁇ 10 9 , 4 ⁇ 10 9 to 1 ⁇ 10 10 , 2 ⁇ 10 8 to 6 ⁇ 10 8 , 6 ⁇ 10 8 to 1 ⁇ 10 9 , 1 ⁇ 10 8 to 2 ⁇ 10 8 , 2 ⁇ 10 8 to 2 ⁇ 10 9 , 1 ⁇ 10 7 to 1 ⁇ 10 8 , 1 ⁇ 10 8 to 1 ⁇ 10 8 , 1 ⁇ 10 8 1 ⁇ 10 9 to 1 ⁇ 10 10 or 1 ⁇ 10 7 to 1 ⁇ 10 9 may be administered at a dose of any one cell/kg, but is not limited thereto.
- the pharmaceutical composition of the present invention may be characterized in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be characterized in that it is targeted to humans.
- the pharmaceutical composition of the present invention is not limited thereto, but each is formulated in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods to be used.
- the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.
- Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc., for oral administration, and in the case of injections, buffers, preservatives, and pain relief Agents, solubilizers, isotonic agents, stabilizers, etc.
- the dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above.
- a pharmaceutically acceptable carrier as described above.
- it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in the form of unit dose ampoules or multiple doses. have.
- it can be formulated as a solution, suspension, tablet, capsule, sustained release formulation, and the like.
- suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used.
- fillers, anti-agglomeration agents, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like may be further included.
- the route of administration of the pharmaceutical composition of the present invention is, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Sublingual or rectal is included and may be, for example, oral or parenteral administration.
- the "parenteral" of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
- the pharmaceutical composition may be administered by direct injection into the kidney, but is not limited thereto.
- the pharmaceutical composition of the present invention depends on several factors including the activity of the specific compound used, age, weight, general health, sex, formula, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated.
- the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg/day per day kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.
- the pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.
- Renal organoids to be described later in the present invention are easy to operate for treatment, can be supplied in large quantities with high self-proliferative ability, have excellent differentiation ability into renal cells, have low tumor formation potential, and are injected directly into the lesion. In this case, the ability to regenerate damaged tissue is very excellent. Therefore, it can be very suitably used for adult stem cell-based regenerative therapy.
- kidney organoids have an advantage in that they have a very excellent direct regeneration effect compared to cell therapeutics using conventional mesenchymal stem cells, embryonic stem cells, or pluripotent stem cells.
- renal organoids a method for preparing renal organoids, and uses thereof will be described in detail.
- Another embodiment of the present invention provides renal organoids.
- kidney organoids of the present invention include kidney tissue-derived stem cells expressing Lrig1 protein or a gene encoding the same.
- Renal epithelial cells expressing Lrig1 or a gene encoding the same of the present invention are kidney tissue-derived stem cells having stem cell ability, unlike other cells, and are particularly involved in tubulogenesis, a later developmental stage of the kidney. It has the ability to split into nephrons. Therefore, for the purpose of the present invention, kidney epithelial cells expressing Lrig1 having the stem cell ability or a gene encoding the same can form organoids very effectively.
- kidney tissue-derived stem cells may further express a Klf6 protein or a gene encoding the same.
- kidney organoid of the present invention the contents related to Lrig1 or Klf6 protein or a gene encoded by it, kidney tissue-derived stem cells, etc. are as described above in "1.
- Pharmaceutical composition comprising kidney tissue-derived stem cells as an active ingredient” In the same way, the description thereof is omitted.
- the "organoid” of the present invention refers to a cell having a 3D three-dimensional structure, and refers to a model similar to a tissue prepared through an artificial culture process that is not collected or acquired from animals. Unlike 2D culture, 3D cell culture allows cells to grow in all directions in vitro.
- Another embodiment of the present invention provides a method for preparing renal organoids.
- the production method of the present invention comprises the steps of (a) isolating a cell expressing a Leucine Rich Repeats And Immunoglobulin Like Domains 1 (Lrig1) protein or a gene encoding it from renal epithelial cells isolated from a target individual; (b) culturing the cells in which the Lrig1 protein or a gene encoding it is expressed; and (c) placing the cultured cells in matrigel to form organoids.
- Lrig1 Leucine Rich Repeats And Immunoglobulin Like Domains 1
- Renal epithelial cells expressing Lrig1 or a gene encoding the same of the present invention are kidney tissue-derived stem cells having stem cell ability, unlike other cells, and are particularly involved in tubulogenesis, a later developmental stage of the kidney. It has the ability to split into nephrons. 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 prepared in a very high yield.
- the cell separated in step (a) of the present invention may be one in which Klf6 (Krueppel-like factor 6) protein or a gene encoding it is expressed.
- the contents related to Lrig1 or Klf6 protein or a gene encoded by it, kidney tissue-derived stem cells, organoids, etc. are described above in “1.
- Pharmaceutical composition comprising renal stem cells as an active ingredient. It is the same as described in “ and “(1) renal organoid", and the description thereof is omitted.
- the step of isolating cells includes dissociating a sample isolated from a target subject, and then performing magnetic activated cell sorting (MACS) or flow cytometry according to a conventional method. flow cytometry analysis), but is not limited thereto.
- MCS magnetic activated cell sorting
- the step of dissociating the sample of the present invention may be using collagenase, but is not limited thereto.
- the step of culturing the gene-expressed cells may be using a cell culture medium containing fetal bovine serum, growth factors and antibiotics.
- the step of forming the organoid is a cell culture containing B27 supplement, conditioned medium, growth factor, N-acetylcysteine and ALK 5 (TGF ⁇ kinase/activin receptor-like kinase) inhibitor. It may be possible to use a medium.
- the "growth factor” of the present invention is a substance necessary for cell growth, for example, VEGF (Vascular endothelial growth factor), HGF (Hepatocyte growth factor), IGF (Insulin-like growth factor), EGF (Epidermal growth factor) ), may be Fibroblast growth factor (FGF), etc., but is not limited thereto.
- VEGF Vascular endothelial growth factor
- HGF Hepatocyte growth factor
- IGF Insulin-like growth factor
- EGF Extracellular growth factor
- FGF Fibroblast growth factor
- 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, Wnt3a conditioned medium, noggin conditioned medium and Rspo1 conditioned medium, and for example, may include, but is not limited to, 40% Wnt3a conditioned medium, 10% Noggin conditioned medium, and 10% Rspo1 conditioned medium.
- the "cell culture medium” of the present invention includes basic components for growth and maintenance of cell lines in vitro, for example, DMEM (Dulbeco's Modified Eagle's Medium), MEM (Minimal essential Medium), It may be 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., and the cell culture of the step (b)
- the medium may be RPMI-1640
- the cell culture medium of step (c) may be ADMEM/F12, but is not limited thereto.
- the cell culture medium may include 10% fetal bovine serum, 20 ng/ml of EGF (Epidermal growth factor) and 1% penicillin-streptomycin. , but is not limited thereto.
- the cell culture medium in step (c), includes 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 ⁇ M A8301 (CAS no. CAS Number 909910-43-6) may be included, but is not limited thereto.
- the Matrigel of the present invention may be a growth factor reduced Matrigel, but is not limited thereto.
- Another embodiment of the present invention provides a kidney organoid prepared by the method for producing the kidney organoid according to the present invention.
- the kidney organoid of the present invention is prepared using kidney tissue-derived stem cells expressing Lrig1 or a gene encoding the same, preferably Lrig1 and Klf6 proteins or a gene encoding them, having very excellent stem cell ability. It can be implemented very effectively to effectively screen a therapeutic agent for kidney damage, and it can be used as a cell therapy directly used for kidney damage.
- kidney organoid of the present invention the Lrig1 or Klf6 protein or the gene encoded by it, the kidney organoid and the content related to the production method, etc. are described above in "1.
- Pharmaceutical composition comprising kidney stem cells as an active ingredient” and "(1 ) kidney organoid", and the description thereof is omitted.
- a pharmaceutical composition for preventing or treating kidney disease comprising a renal organoid as an active ingredient.
- kidney organoid of the present invention may be used as a cell therapy agent.
- kidney tissue-derived stem cells As it is the same as described in “Pharmaceutical composition comprising as an active ingredient” and “(1) Renal organoid", the description thereof is omitted.
- the kidney organoid of the present invention is 1 ⁇ 10 7 to 1 ⁇ 10 8 , 1 ⁇ 10 8 to 2 ⁇ 10 8 , 2 ⁇ 10 8 to 4 ⁇ 10 8 , 4 ⁇ 10 8 to 6 ⁇ 10 8 , 6 ⁇ 10 8 to 8 ⁇ 10 8 , 8 ⁇ 10 8 to 1 ⁇ 10 9 , 1 ⁇ 10 9 to 2 ⁇ 10 9 , 2 ⁇ 10 9 , 2 ⁇ 10 9 to 4 ⁇ 10 9 , 4 ⁇ 10 9 to 1 ⁇ 10 10 , 2 ⁇ 10 8 to 6 ⁇ 10 8 , 6 ⁇ 10 8 to 1 ⁇ 10 9 , 1 ⁇ 10 8 to 2 ⁇ 10 8 , 2 ⁇ 10 8 to 2 ⁇ 10 9 , 1 ⁇ 10 7 to 1 ⁇ 10 8 , 1 ⁇ 10 8 to 1 ⁇ 10 8 to to 1 ⁇ 10 10 or 1 ⁇ 10 7 to 1 ⁇ 10 9 may be administered at a dose of any one cell/kg, but is not limited thereto.
- Another embodiment of the present invention provides a composition for detecting kidney tissue-derived stem cells.
- composition for detection of the present invention includes an agent for measuring the expression level of Lrig1 protein or a gene encoding the same.
- the detection composition of the present invention may further include an agent for measuring the expression level of the Klf6 protein or a gene encoding the same.
- the contents related to Lrig1 or Klf6 protein and kidney tissue-derived stem cells are the same as those described above in "Pharmaceutical composition comprising one kidney tissue-derived stem cell as an active ingredient", so the description thereof is omitted. .
- the agent for measuring the expression level of the gene of the present invention may be included as long as it can measure the expression level of the DNA present in the biological sample or the mRNA transcribed by it, for example, it specifically binds to the gene. It may be at least one selected from the group consisting of primers, probes, and antisense nucleotides, but is not limited thereto.
- the "primer” of the present invention is a fragment recognizing a target gene sequence, including a pair of forward and reverse primers, but preferably, a primer pair that provides analysis results having specificity and sensitivity.
- the nucleic acid sequence of the primer is a sequence that is inconsistent with a non-target sequence present in the sample, and thus amplifies only the target gene sequence containing the complementary primer binding site and does not cause non-specific amplification, high specificity can be conferred. .
- the "probe” of the present invention means a substance capable of specifically binding to a target substance to be detected in a sample, and means a substance capable of specifically confirming the presence of a target substance in the sample through the binding .
- the type of probe is not limited as a material commonly used in the art, but preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, and most preferably It is PNA.
- the probe is a biomaterial derived from or similar thereto, or manufactured in vitro, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, neurons, DNA, and It may be RNA, DNA includes cDNA, genomic DNA, and oligonucleotides, RNA includes genomic RNA, mRNA, and oligonucleotides, and examples of proteins include antibodies, antigens, enzymes, peptides, and the like.
- LNA Locked nucleic acids
- the "LNA (Locked nucleic acids)" of the present invention means a nucleic acid analog comprising a 2'-O, 4'-C methylene bridge.
- LNA nucleosides include general nucleic acid bases of DNA and RNA, and can form base pairs according to the Watson-Crick base pairing rule. However, due to the 'locking' of the molecule due to the methylene bridge, the LNA does not form an ideal shape in the Watson-Crick bond.
- LNAs When LNAs are incorporated into DNA or RNA oligonucleotides, LNAs can pair with complementary nucleotide chains more rapidly, increasing the stability of the double helix.
- the "antisense” of the present invention means that the antisense oligomer is hybridized with a target sequence in RNA by Watson-Crick base pairing, and typically mRNA and RNA: oligomeric heteroduplex formation in the target sequence, allowing the formation of a sequence of nucleotide bases and oligomers having an inter-subunit backbone.
- An oligomer may have exact sequence complementarity or approximate complementarity to a target sequence.
- the agent for measuring the expression level of the protein of the present invention may be included as long as it can measure the amount of protein present in a biological sample, for example, an antibody, oligopeptide, ligand, which specifically binds to the protein; It may be at least one selected from the group consisting of peptide nucleic acid (PNA) and aptamer, but is not limited thereto.
- PNA peptide nucleic acid
- the "protein” of the present invention is not only the protein itself, but also a protein isomer (Isoform) or protein variant (Variant) that can be generated by splicing and variable promoters, or can be generated due to genetic changes such as mutation or polymorphism includes
- the "antibody” of the present invention refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction.
- an antibody refers to an antibody that specifically binds to the biomarker protein.
- Antibodies of the present invention include polyclonal antibodies, monoclonal antibodies and recombinant antibodies.
- the antibody can be readily prepared using techniques well known in the art.
- the polyclonal antibody can be produced by a method well known in the art, including the process of injecting an antigen of the protein into an animal and collecting blood from the animal to obtain a serum containing the antibody.
- Such polyclonal antibodies can be prepared from any animal such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, and the like.
- monoclonal antibodies can be prepared using hybridoma methods well known in the art, or phage antibody library techniques.
- the antibody prepared by the above method may be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.
- the antibodies of the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains.
- a functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and includes all of Fab, F(ab'), F(ab')2 and Fv.
- PNA 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, Denmark. Whereas DNA has a phosphate-ribose sugar backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases binding strength and stability to DNA or RNA, resulting in molecular biology , diagnostic assays and antisense therapy.
- the PNA can be further specified with reference to the background well known in the art.
- the "aptamer” of the present invention is an oligonucleic acid or a peptide molecule, and the general contents of the aptamer are described in Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine”. J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R. "Anartificial cell-cycle inhibitor isolated from a combinatorial library”. Proc Natl Acad Sci USA. 95(24): 142727(1998)].
- the antibody, oligopeptide, ligand, PNA, aptamer, etc. of the present invention can be easily produced by those skilled in the art based on the amino acid sequence identifiable by the site identifiable through the NCBI accession number.
- Another embodiment of the present invention provides a kit for detecting kidney tissue-derived stem cells comprising the composition for detecting according to the present invention.
- kidney tissue-derived stem cells information related to Lrig1 protein or a gene encoding the same, kidney tissue-derived stem cells, agents for measuring protein expression levels, agents for measuring gene expression levels, etc. are described above in "1.
- Kidney tissue-derived stems It is the same as described in "Pharmaceutical Composition Containing Cells as an Active Ingredient” and "(1) Composition for Detection of Renal Stem Cells", and thus description thereof will be omitted.
- 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 a multiple reaction monitoring (MRM) kit, but is not limited thereto.
- MRM multiple reaction monitoring
- the kit of the present invention may further include one or more other component compositions, solutions or devices suitable for the assay method.
- the kit may further include essential elements necessary for performing the reverse transcription polymerase reaction.
- the reverse transcription polymerase reaction kit includes a pair of primers specific for the gene.
- the primer is a nucleotide having a sequence specific to the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include primers specific for the nucleic acid sequence of a control gene (eg, a housekeeping gene).
- reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffers (varying pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors.
- DEPC-water DEPC-water
- the diagnostic kit of the present invention may include essential elements necessary for performing a DNA chip.
- the DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for preparing a fluorescently-labeled probe.
- the substrate may include a cDNA or oligonucleotide corresponding to a control gene (eg, a housekeeping gene) or a fragment thereof, but is not limited thereto.
- the diagnostic kit of the present invention may include essential elements necessary for performing ELISA.
- the ELISA kit contains an antibody specific for this protein.
- Antibodies are antibodies with high specificity and affinity for a marker protein and little cross-reactivity with other proteins, and are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies.
- the ELISA kit may include an antibody specific for a control (eg, housekeeping protein) protein.
- Other ELISA kits include reagents capable of detecting bound antibody, for example, labeled secondary antibodies, chromophores, enzymes (eg, conjugated with an antibody) and substrates thereof or capable of binding the antibody. other materials and the like.
- Another embodiment of the present invention provides a method for detecting kidney tissue-derived stem cells.
- the detection method of the present invention includes the step of measuring the expression level of the Lrig1 protein or a gene encoding the same from a biological sample isolated from a subject of interest.
- the expression level of the Klf6 protein or a gene encoding the same from the biological sample may be further measured.
- kidney tissue-derived stem cells Lrig1 or Klf6 protein or genes encoding them, etc. are described above in "1.
- Pharmaceutical composition comprising kidney tissue-derived stem cells as an active ingredient” and “(1) Composition for the detection of renal stem cells", and the description thereof is omitted.
- the "biological sample” of the present invention refers to any material, biological fluid, tissue or cell obtained from or derived from an individual, for example, whole blood, leukocytes, peripheral blood mononuclear peripheral blood mononuclear cells, buffy coat, blood including plasma and serum, urine, semen, organ secretions, cells Or it may include a cell extract (cell extract), for example, may be kidney tissue or kidney cells, but is not limited thereto.
- a cell extract may be kidney tissue or kidney cells, but is not limited thereto.
- RT-PCR reverse transcription Polymerase reaction
- RPA RNase protection assay
- Northern blotting Northern blotting
- DNA chip a DNA chip
- the step of measuring the expression level of the protein of the present invention is at least one selected from the group consisting of an antibody, oligopeptide, ligand, PNA (peptide nucleic acid) and aptamer that specifically binds to the protein.
- kidney tissue-derived stem cells when the expression level of the Lrig1 protein or a gene encoding the same is increased compared to the control, detecting with kidney tissue-derived stem cells may be further included.
- Another embodiment of the present invention provides a method for isolating kidney tissue-derived stem cells.
- the isolation method of the present invention includes a step of isolating cells expressing Lrig1 protein or a gene encoding the same from a biological sample isolated from a target individual.
- the cell may be one in which the Klf6 protein or a gene encoding the same is additionally expressed.
- kidney tissue-derived stem cells the contents related to Lrig1 or Klf6 protein or a gene encoding it, kidney tissue-derived stem cells, biological samples, etc. are described above in "1.
- Pharmaceutical composition comprising kidney stem cells as an active ingredient
- (1 ) composition for detecting renal stem cells and “(3) method for detecting kidney tissue-derived stem cells”, and the description thereof will be omitted.
- the separation of the present invention may be performed through magnetic activated cell sorting (MACS) or flow cytometry analysis.
- MCS magnetic activated cell sorting
- flow cytometry analysis may be performed through magnetic activated cell sorting (MACS) or flow cytometry analysis.
- Another embodiment of the present invention provides a method for culturing kidney tissue-derived stem cells.
- the culturing method of the present invention comprises the steps of isolating a kidney tissue-derived stem cell expressing the Lrig1 protein or a gene encoding it; and culturing the isolated kidney tissue-derived stem cells.
- the kidney tissue-derived stem cells may be those in which Klf6 protein or a gene encoding the same is additionally expressed.
- kidney stem cells In the isolation method of the present invention, the contents related to Lrig1 or Klf6 protein or a gene encoding it, kidney tissue-derived stem cells, etc. are the same as described above in "1.
- Pharmaceutical composition comprising kidney stem cells as an active ingredient" omit
- the culturing method of the present invention is performed after isolating kidney tissue-derived stem cells according to the method described in 5. Method for isolating kidney tissue-derived stem cells of the present invention and then culturing them, for example, in vitro. It may be cultured in, but is not limited thereto.
- culture means any in vitro culture of cells.
- the culture of the present invention may use a cell culture medium, and the cell culture medium refers to any type of medium used in an environment for culturing cells, for example, amino acids, at least one carbohydrate as an energy source, trace elements, vitamins , salts and possibly additional ingredients (eg, to affect cell growth, productivity, or product quality).
- Another embodiment of the present invention provides a method for preparing an animal model for screening a cell therapy agent for the prevention or treatment of kidney disease.
- the method for producing the animal model of the present invention comprises the steps of inducing kidney damage in an animal in which a desired gene is conditionally expressed by the CreERT2-LoxP system; and treating the animal with an estrogen antagonist to induce expression of a desired gene.
- kidney tissue-derived stem cells contents of the kidney tissue-derived stem cells, kidney disease, and cell therapy are the same as described above in "1.
- Pharmaceutical composition comprising kidney tissue-derived stem cells as an active ingredient”. omit the description.
- the promoter-specifically expressed Cre-ERT2 polypeptide recognizes two LoxP nucleotide sequences and catalyzes site-specific recombination between the two LoxP nucleotide sequences, so that the LoxP nucleo It refers to a system that specifically expresses a gene downstream of the tide. How to build such a system is described in Lab Anim Res. 2018 Dec; 34(4): 147-159. A person skilled in the art can easily construct it according to the contents described in others.
- the "gene of interest" of the present invention is a gene that is specifically expressed or expected to be expressed in a cell with potential to be used as a cell therapy agent, and for the purpose of the present invention, a gene encoding Lrig1, preferably It may be a gene encoding Lrig1 and a gene encoding Klf6, but is not limited thereto.
- the "animal model” of the present invention means an animal capable of exhibiting a form very similar to a human disease.
- the causes and pathogenesis of various diseases can be studied, and basic data for judging the feasibility, such as treatment selection and toxicity testing, can be obtained.
- the animal of the present invention means any mammal other than humans, and includes animals of all ages including embryos, fetuses, newborns, and adults.
- Such animals include rabbits, rodents (mice, rats, hamsters, gerbils or guinea pigs), cattle, sheep, pigs, goats, horses, dogs, cats, birds (chickens, turkeys, ducks, geese) and primates (chimpanzees, monkey, rhesus monkey) may be any one selected from the group consisting of, but is not limited thereto.
- the step of inducing kidney damage of the present invention includes intraperitoneal administration of folic acid; induce ischemia/reperfusion injury; And it may be to perform any one of the methods consisting of induction of unilateral ureteral obstruction, but is not limited thereto.
- the folic acid of the present invention may be administered intraperitoneally at 100 mg/kg to 300 mg/kg, for example, 150 mg/kg or 250 mg/kg, but is not limited thereto. If the dose of folic acid is less than 100 mg/kg, acute kidney damage may not be induced, and if it exceeds 300 mg/kg, toxicity exists in animal models, affecting the interpretation of the desired experimental results. or the animal model may die.
- the estrogen antagonist of the present invention may be tamoxifen, 4-hydroxytamoxifen, clomiphene, raloxifene, or a combination thereof, but is not limited thereto.
- Another embodiment of the present invention provides an animal model for screening a cell therapy agent for the prevention or treatment of kidney disease produced by the production method of the present invention.
- Kidney tissue-derived stem cells according to the present invention.
- organoids are easy to operate for treatment, can be supplied in large quantities due to their high self-proliferative capacity, have excellent differentiation capacity into kidney cells, have a low probability of tumor formation, and have the ability to regenerate damaged tissue when directly injected into the lesion. This is very excellent. Therefore, among these stem cells, in particular, it can be used very suitably for adult stem cell-based regenerative therapy.
- composition according to the present invention can specifically select only renal stem cells from among renal cells.
- kidney stem cells expressing Lrig1 protein or a gene encoding the same have excellent regenerative ability and pluripotency, and have the ability to divide into nephrons, so they can be used very effectively for the prevention or treatment of kidney disease.
- FIG. 1 shows a schematic diagram of an experimental process using a mouse animal model used in an in vivo lineage tracking study according to an embodiment of the present invention.
- FIG. 2 is a schematic diagram of an experimental process using an embryonic mouse animal model used in an in vivo lineage tracking study according to an embodiment of the present invention.
- FIG. 3 is a schematic diagram illustrating a method for preparing a high-dose (250 mg/kg) folate-induced acute kidney injury animal model according to an embodiment of the present invention.
- FIG. 4 is a schematic diagram of an analysis process after transplantation of the renal organoid according to the present invention into a high-dose (150 mg/kg) folate-induced acute kidney injury animal model according to an embodiment of the present invention.
- FIG. 5 is a schematic diagram showing a method for preparing an animal model of ischemia/reperfusion injury-induced acute kidney injury according to an embodiment of the present invention.
- FIG. 6 is a schematic diagram showing a method of manufacturing an animal model of unilateral ureteral obstruction-induced acute kidney injury according to an embodiment of the present invention.
- FIG. 7 shows the results of confirming the presence of tdTomato+ cells (Lrig1tdT+) in the kidneys of mice according to an embodiment of the present invention through tissue staining.
- FIG. 8 shows the results of quantifying the number of tdTomato+ cells (Lrig1tdT+) in the kidney of a mouse according to an embodiment of the present invention.
- FIG. 10 shows the results of quantifying the number of tdTomato+ cells (Lrig1tdT+) in mice in the developmental stage according to an embodiment of the present invention.
- FIG. 11 shows the results of confirming the kidney organoid according to an embodiment of the present invention using a fluorescence microscope.
- FIG. 12 shows the result of confirming the expression level of the gene expressed in the kidney organoid according to an embodiment of the present invention through real-time polymerase chain reaction.
- FIG. 13 shows the results confirming that the number of kidney organoids is increased when the kidney organoids according to an embodiment of the present invention are cultured for a long period from 1 day to 19 days.
- FIG. 14 is a high-dose (150 mg/kr) folic acid-induced acute kidney injury animal model according to an embodiment of the present invention transplanted with PBS or a renal organoid according to the present invention and tissue staining and It shows the result of confirming the expression level of KIM1.
- 15 is a high-dose (150 mg/kr) folic acid-induced acute kidney injury animal model according to an embodiment of the present invention transplantation of PBS or a renal organoid according to the present invention and confirming the presence of kidney repair, blood BUN and Shows the results of checking the creatine concentration.
- 16 shows the results of confirming the expression of Ki-67 in kidney organoids according to an embodiment of the present invention by fluorescence microscopy.
- FIG. 17 shows the classification of the proximal tubule (PT) cluster into four sub-clusters of PTS1, PTS2, PTS3 and PTQPs in renal organoids according to an embodiment of the present invention.
- 18 and 19 are bubble-plots showing the results of confirming the expression levels of genes for each cluster of PTS1, PTS2, PTS3 and PTQPs in kidney organoids according to an embodiment of the present invention.
- FIG. 21 shows the results of measuring changes in the number of PTS1, PTS2, PTS3 and PTQPs cluster cells at 365 days compared to the 1st day after Cre-loxp induction after transplanting the renal organoid according to an embodiment of the present invention.
- FIG. 23 shows the top 10 genes that are highly expressed in PTS3 and PTQPs clusters in kidney organoids according to an embodiment of the present invention.
- FIG. 24 shows immunofluorescence staining photographs of LTL (PT marker) and KLF6 staining in kidney sections on days 1 and 365 after Cre-loxp induction after transplanting kidney organoids according to an embodiment of the present invention.
- 26 shows the ratio of tdTomato+ cells for each cluster at the 1st day and 365 days after Cre-loxp induction after transplanting the renal organoid according to an embodiment of the present invention.
- FIG. 27 is a pseudotime system in the proximal tubule (PT) of a renal organoid according to an embodiment of the present invention divided into three different states, PTS1, PTS3 and PTQPs clusters are shown in different colors, respectively. . Black arrows indicate the direction of pseudotime.
- FIG. 29 shows immunofluorescence staining pictures of Lrig1tdTomato+ and KLF6 staining of kidney sections on days 1 and 365 after Cre-loxp induction after transplanting kidney organoids according to an embodiment of the present invention.
- the present invention is to overcome the limitations of the conventional treatment of kidney diseases such as acute renal failure, and to develop a more effective therapeutic agent.
- the present invention is for the development of an effective therapeutic agent for kidney disease using kidney tissue-derived stem cells or organoids.
- F high-concentration folic acid
- F+PBS high-concentration folic acid
- the levels of BUN and creatine in the blood were very high, whereas renal organoids were injected.
- the levels in which BUN and creatine were present were reduced to a degree similar to that of Normal.
- the animals were housed in a Specific pathogen-free (SPF) barrier facility, and PicoLab Lab Rodent Diet 20, LabDiet, St. Louis, MO, USA. ) The experimental animals were bred by feeding them.
- SPF Specific pathogen-free
- Lrig1CreERT2/+ was provided by Robert J. Coffey of Venterbilt University, and 2) B6.Cg-Gt(ROSA)26Sortm14 (CAG-tdTomato)/Hze/J(R26R-LSL-tdTomato; The Jackson Laboratory, 007914) was provided by Professor Bok Jin-woong, Yonsei University, 3) B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdToamto) -EGFP)Luo/J(ACTB-mT/mG; The Jackson Laboratory, 007676) was provided by Professor Ki Hyun-woong, Yonsei University.
- heterozygous mice were produced by crossing Lrig1CreERT2/+ mice and R26R-LSL-tdTomato mice, which are reporter mice of the same type as in Experimental Method 1. Cre-loxp recombination was induced by intraperitoneally injecting 2 mg of tamoxifen (Sigma-Aldrich) contained in corn oil to the mice for 3 consecutive days, 1 day, 3 days, 10 days, 30 days, 60 days, 90 days , analysis was performed on day 180 or day 365.
- tamoxifen Sigma-Aldrich
- E9.5, E10.5, E13.5 and E18.5 embryos were generated by crossing Lrig1CreERT2/+ mice and R26R-LSL-tdTomato mice, which are isoform reporter mice.
- 2 mg of 4-hydroxytamoxifen (Sigma-Aldrich) contained in corn oil was injected once, and the analysis was performed at 6 weeks postpartum.
- reporter mice of the same type produced by crossing Lrig1CreERT2/+ mice and R26R-ACTB-mT/mG mice were bred for 6 to 10 weeks, and then primary renal epithelial cells were collected. 2 mg/ml of type 1 collagenase was added to the collected primary renal epithelial cells, and cultured at 37° C. for 30 minutes with gentle agitation. Then, after filtering the primary renal epithelial cells using a filter, the isolated single cells were cultured.
- the cells expressing the Lrig1 protein were placed in RPMI 1640 (10% fetal bovine serum (FBS), 20 ng/ml of EGF, and 1% penicillin-streptomycin) medium, 5% CO 2 and 37 It was cultured for 7 to 8 days at a confluence of 80% under the condition of °C. Thereafter, 1 ⁇ 10 3 of the cultured cells were dispensed and cultured in the wells containing the growth factor-reduced matrigel and culture medium.
- FBS fetal bovine serum
- EGF EGF
- penicillin-streptomycin penicillin-streptomycin
- the culture medium is based on ADMEM/F12 culture medium containing 1% penicillin-streptomycin, HEPS and glutamax, 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 EGF, 100 ng/mL FGF-10, 1.25 mM N-acetylcysteine, and 5 ⁇ M A8301 (CAS) No. CAS Number 909910-43-6) was used.
- an organoid culture medium was added, and the organoid culture medium was replaced every 3 days.
- Lrig1 protein was expressed in adult (8 to 10 week old) Lrig1-CreERT2;LSL-tdTomato mice through the injection of tamoxifen. Thereafter, 250 mg/kg of folic acid (FA) was intraperitoneally injected to induce acute kidney injury.
- FA folic acid
- Lrig1 protein was expressed in adult (8 to 10 week old) Lrig1-CreERT2;LSL-tdTOmato mice through the injection of tamoxifen. Then, acute renal injury was induced through the process of closing the renal artery of the mouse with forceps for 20 minutes. After 3 days, as a result of collecting and confirming the kidney tissue of the mouse, it was confirmed that acute kidney damage was induced.
- Lrig1 protein was expressed in adult (8 to 10 week old) Lrig1-CreERT2;LSL-tdTomato mice through the injection of tamoxifen. Then, acute kidney injury was induced through the process of closing the ureter with forceps for 7 days. After 7 days, as a result of collecting and confirming the kidney tissue of the mouse, it was confirmed that acute kidney damage was induced.
- 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, the nephrogenesis stage, and most of the cells had expanded to form a tube structure.
- Lrig1 is a stem cell population that is involved in generating nephron division after developing from an early stage to a mature kidney.
- the gene expression level on the 17th day after culturing kidney organoids was confirmed by performing a real-time polymerase chain reaction (Real-time PCR) using the primers in Table 1 below according to a conventional method. It was confirmed that not only the expression level of Lrig1, but also the expression level of kidney stem cells such as Sall1, Six2, Foxo1, Cited, Osr1, Hoxp7, Jagged1 and Gata3 were increased together. Furthermore, as shown in FIG. 13 , in the case of Lrig1-positive kidney organoids, the number of generated organoids increased to about 40 or more from the 9th day to the 19th day.
- the organoids injected into the kidneys were confirmed through immunofluorescence staining, and it was found that proliferation markers such as Ki-67 were expressed. It was confirmed that organoids were proliferative.
- Lrig1-positive cells survive for a long time in the proximal tubule (PT) and correspond to potential renal stem/progenitor cells, and the Lrig1-positive cell-derived descendants maintain PT homeostasis. was found to have made a substantial contribution to the In order to confirm the cellular heterogeneity of Lrig1+ cells and their derived cells in PT, as shown in FIG. 17 , the PT cluster was classified into four sub-clusters of PTS1, PTS2, PTS3 and PTQPs.
- Slc5a12 and Slc5a2 markers were used for PTS1 classification, Slc13a3 and Ddah1 markers for PTS2 classification, and Slc16a9 and Slc7a13 markers for PTS3 classification.
- pyruvate dehydrogenase kidney 4 Pyruvate dehydrogenase kidney 4; Pdk4
- cysteine-rich protein Cystein-rich protein 61; Cyr61
- PTQPs were compared with other PT sub-clusters.
- PT cells increase with time, and in particular, the number of PTQPs and PTS2 cluster cells increases.
- clusters of PTQPs in the kidney were defined using the adult stem cell gene module. As a result, a total of 650 genes were detected in DEGs. 22, stem-related genes were highly expressed in the PTQPs cluster, self-renewal-related genes (except for 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 confirmed to be up-regulated. Through this, it was found that the PTQPs cluster expresses stem function-related genes, including kidney injury-recovery-related genes and markers of mature PT cells. will be named as
- PTQPs marker distinct from PTS3 genes known as stem cell niche highly expressed in DEGs could be identified.
- PT segment 3 specific genes were highly expressed, whereas in PTQPs, they were not highly expressed.
- kidney sections were IF-stained using Jun, Klf6 and Cyr61. Jun and Cyr61 were highly expressed in various nephron fragments as well as PT, and these were excluded.
- KLF6 and LTL were stained on day 1 and day 365 kidney sections to confirm whether they were expressed in PT. As shown in FIG.
- 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.
- FIG. 25 it was confirmed that the KLF6+ expression PT tubule significantly increased in the kidney on day 365. From this, it can be seen that the increase in the distribution of PTQPs in the kidney on day 365 can be confirmed using Klf6.
- tdTomato-expressing cells were identified in kidney sections at day 1 and day 365. As a result, as shown in FIG. 26 , tdTomato+ cells constituted 54% of PTS1 cells and 27% of PTS3 cells in the kidney section on day 1, but decreased to 50% and 11% in the kidney section on day 365, respectively. On the other hand, 12% of PTS2 cells and 17.8% of PTQPs cells in the kidney section on day 1 were increased to 20% and 18%, respectively, on day 365. From this, it was found that progeny cells derived from Lrig1-positive cells constitute a PTQPs cell population.
- PTS3 known as a PT stem cell niche
- PTQPs a novel stem cell niche
- the present invention is to overcome the limitations of the conventional treatment of kidney diseases such as acute renal failure, and to develop a more effective therapeutic agent.
- kidney diseases such as acute renal failure
- the present invention is kidney tissue-derived stem cells;
- organoids are easy to operate for treatment, have excellent ability to differentiate into renal cells, have a low probability of tumor formation, and have excellent ability to regenerate damaged tissues when directly injected into a lesion. It is expected to be used very effectively for treatment.
- SEQ ID NO: 3 GGTGAGCCTGGCCTTATGTGAATA
- SEQ ID NO: 4 CACCACCATCCTGCACCTCC
- SEQ ID NO: 5 TACTCTTTCCTTCAGGCAGTGA
- SEQ ID NO: 7 CACCTCCACAAGAATGAAAGCG
- SEQ ID NO: 8 CTCCGCCTCGATGTAGTGC
- SEQ ID NO: 9 AACCTTGGAGTGGAAGGATCGC
- 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
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Abstract
Description
서열번호 | 유전자 | 특징 | 염기 서열 |
서열번호 3 | mLrig1 | 정방향 | GGTGAGCCTGGCCTTATGTGAATA |
서열번호 4 | 역방향 | CACCACCATCCTGCACCTCC | |
서열번호 5 | Osr1 | 정방향 | TACTCTTTCCTTCAGGCAGTGA |
서열번호 6 | 역방향 | GATCGAGGCAAGTGCATGG | |
서열번호 7 | Six2 | 정방향 | CACCTCCACAAGAATGAAAGCG |
서열번호 8 | 역방향 | CTCCGCCTCGATGTAGTGC | |
서열번호 9 | 1 | 정방향 | AACCTTGGAGTGAAGGATCGC |
서열번호 10 | 역방향 | GTAGGAGAGCCTATTGGAGATGT | |
서열번호 11 | Sall1 | 정방향 | CTCAACATTTCCAATCCGACCC |
서열번호 12 | 역방향 | GGCATCCTTGCTCTTAGTGGG | |
서열번호 13 | WT1 | 정방향 | GAGAGCCAGCCTACCATCC |
서열번호 14 | 역방향 | GGGTCCTCGTGTTTGAAGGAA | |
서열번호 15 | Hoxb1 | 정방향 | AAGTTCGGTTTTCGCTCCAGG |
서열번호 16 | 역방향 | ACACCCCGGAGAGGTTCTG | |
서열번호 17 | Gata3 | 정방향 | CTCGGCCATTCGTACATGGAA |
서열번호 18 | 역방향 | GGATACCTCTGCACCGTAGC | |
서열번호 19 | cRet | 정방향 | GCGTCAGGGAGATGGTAAAG |
서열번호 20 | 역방향 | CATCAGGGAAACAGTTGCAG | |
서열번호 21 | Foxd1 | 정방향 | CGCTAAGAATCCGCTGGTGAAG |
서열번호 22 | 역방향 | GGATCTTGACGAAGCAGTCGTT | |
서열번호 23 | Jagged1 | 정방향 | CCTCGGGTCAGTTTGAGCTG |
서열번호 24 | 역방향 | CCTTGAGGCACACTTTGAAGTA |
Claims (30)
- Lrig1(Leucine Rich Repeats And Immunoglobulin Like Domains 1) 단백질 또는 이를 암호화하는 유전자를 발현하는 신장 조직유래 줄기세포를 유효성분으로 포함하는 신장 질환의 예방 또는 치료용 약학 조성물.
- 제1항에 있어서,상기 신장 조직유래 줄기세포는 Klf6(Krueppel-like factor 6) 단백질 또는 이를 암호화하는 유전자를 추가로 더 발현하는, 약학 조성물.
- 제1항에 있어서,상기 신장 질환은 급성 신손상(Acute kidney injury; AKI) 또는 만성 신손상(Chronic kidney disease; CKD)인 것인, 약학 조성물.
- Lrig1 단백질 또는 이를 암호화하는 유전자를 발현하는 신장 조직유래 줄기세포를 포함하는 신장 오가노이드.
- 제4항에 있어서,상기 신장 조직유래 줄기세포는 Klf6(Krueppel-like factor 6) 단백질 또는 이를 암호화하는 유전자를 추가로 더 발현하는, 신장 오가노이드.
- 제4항 또는 제5항의 신장 오가노이드를 유효성분으로 포함하는, 신장 질환의 예방 또는 치료용 약학 조성물.
- (a) 목적하는 개체로부터 분리된 신장 상피세포에서 Lrig1(Leucine Rich Repeats And Immunoglobulin Like Domains 1) 단백질 또는 이를 암호화하는 유전자가 발현된 세포를 분리하는 단계;(b) 상기 Lrig1 단백질 또는 이를 암호화하는 유전자가 발현된 세포를 배양하는 단계; 및(c) 상기 배양된 세포를 마트리겔에 넣고 오가노이드를 형성하는 단계;를 포함하는 신장 오가노이드의 제조 방법.
- 제7항에 있어서,상기 (a) 단계에서 분리된 세포는 Klf6(Krueppel-like factor 6) 단백질 또는 이를 암호화하는 유전자를 추가로 더 발현하는 것인, 제조 방법.
- 제7항에 있어서,상기 (b) 단계에서, 상기 유전자가 발현된 세포를 배양하는 단계는 우태아혈청, 성장인자 및 항생제가 포함된 세포 배양 배지를 이용하는 것인, 제조 방법.
- 제7항에 있어서,상기 (c) 단계에서, 상기 오가노이드를 형성하는 단계는 B27 보충제, 조건 배지, 성장인자, N-아세틸시스테인 및 ALK 5(TGFβ kinase/activin receptor-like kinase) 억제제가 포함된 세포 배양 배지를 이용하는 것인, 제조 방법.
- 제10항에 있어서,상기 조건 배지는 Wnt3a 조건 배지, 노긴(noggin) 조건 배지 및 Rspo1 조건 배지로 이루어진 군으로부터 선택되는 적어도 하나 이상인 것인, 제조 방법.
- 제7항에 있어서,상기 마트리겔은 성장인자 감소 마트리겔인 것인, 제조 방법.
- Lrig1(Leucine Rich Repeats And Immunoglobulin Like Domains 1) 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정하는 제제를 포함하는 신장 조직유래 줄기세포 검출용 조성물.
- 제13항에 있어서,상기 검출용 조성물은 Klf6(Krueppel-like factor 6) 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정하는 제제를 더 포함하는, 검출용 조성물.
- 제13항에 있어서,상기 유전자의 발현 수준을 측정하는 제제는 상기 유전자에 특이적으로 결합하는 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군으로부터 선택되는 적어도 어느 하나인 것인, 검출용 조성물.
- 제13항에 있어서,상기 단백질의 발현 수준을 측정하는 제제는 상기 단백질에 특이적으로 결합하는 항체, 올리고펩타이드, 리간드, PNA(peptide nucleic acid) 및 압타머(aptamer)로 이루어진 군으로부터 선택되는 적어도 어느 하나인 것인, 검출용 조성물.
- 제13항 내지 제16항 중 어느 한 항의 검출용 조성물을 포함하는 신장 조직유래 줄기세포 검출용 키트.
- 목적하는 개체로부터 분리된 생물학적 시료로부터 Lrig1(Leucine Rich Repeats And Immunoglobulin Like Domains 1) 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정하는 단계;를 포함하는 신장 조직유래 줄기세포 검출 방법.
- 제18항에 있어서,상기 발현 수준을 측정하는 단계 시 상기 생물학적 시료로부터 Klf6(Krueppel-like factor 6) 단백질 또는 이를 암호화하는 유전자의 발현 수준을 추가로 측정하는, 검출 방법.
- 제18항에 있어서,상기 유전자의 발현 수준은 상기 유전자에 특이적으로 결합하는 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군으로부터 선택되는 적어도 어느 하나 이상을 통해 측정되는 것인, 검출 방법.
- 제18항에 있어서,상기 단백질의 발현 수준은 상기 단백질에 특이적으로 결합하는 항체, 올리고펩타이드, 리간드, PNA 및 압타머로 이루어진 군에서 선택되는 적어도 하나 이상을 통해 측정되는 것인, 검출 방법.
- 제18항에 있어서,상기 Lrig1(Leucine Rich Repeats And Immunoglobulin Like Domains 1) 단백질 또는 이를 암호화하는 유전자의 발현 수준이 대조군과 비교하여 증가된 경우, 신장 조직유래 줄기세포로 검출하는 단계를 더 포함하는, 검출 방법.
- 목적하는 개체로부터 분리된 생물학적 시료에서, Lrig1(Leucine Rich Repeats And Immunoglobulin Like Domains 1) 단백질 또는 이를 암호화하는 유전자가 발현되는 세포를 분리하는 단계;를 포함하는 신장 조직유래 줄기세포의 분리 방법.
- 제23항에 있어서,상기 분리되는 세포는 Klf6(Krueppel-like factor 6) 단백질 또는 이를 암호화하는 유전자를 추가로 더 발현하는 것인, 분리 방법.
- 제23항에 있어서,상기 분리하는 단계는 자기 활성 세포 분류법(Magnetic activated cell sorting; MACS) 또는 유세포 분석(Flow cytometry Analysis)을 통해 수행되는 것인, 분리 방법.
- Lrig1(Leucine Rich Repeats And Immunoglobulin Like Domains 1) 단백질 또는 이를 암호화하는 유전자를 발현하는 신장 조직유래 줄기세포를 분리하는 단계; 및상기 분리된 신장 조직유래 줄기세포를 배양하는 단계;를 포함하는 신장 조직 유래 줄기세포를 배양하는 방법.
- 제26항에 있어서,상기 분리되는 세포는 Klf6(Krueppel-like factor 6) 단백질 또는 이를 암호화하는 유전자를 추가로 더 발현하는 것인, 배양하는 방법.
- 신장 질환의 예방 또는 치료를 위한 세포치료제 스크리닝용 동물 모델의 제조 방법에 있어서,Lrig1(Leucine Rich Repeats And Immunoglobulin Like Domains 1)을 암호화하는 유전자가 CreERT2-LoxP 시스템에 의해 조건적으로 발현되는 동물에 신장 손상을 유도하는 단계; 및상기 동물에 에스트로겐 길항제를 처리하여, 목적하는 유전자의 발현을 유도하는 단계;를 포함하는, 동물 모델의 제조 방법.
- 제28항에 있어서,상기 신장 손상을 유도하는 단계는 엽산의 복강내 투여; 허혈/재관류 손상을 유도; 및 일측성 요관 폐쇄 유도로 구성된 방법 중 어느 하나를 수행하는 것인, 동물 모델의 제조 방법.
- 제28항의 제조 방법에 따라 제조된 신장 질환의 예방 또는 치료를 위한 세포치료제 스크리닝용 동물 모델.
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