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WO2022031008A1 - Procédé de production d'un modèle d'ostéoarthrite dérivé d'une cellule souche pluripotente induite à l'aide de la surexpression de l'aquaporine 1 - Google Patents

Procédé de production d'un modèle d'ostéoarthrite dérivé d'une cellule souche pluripotente induite à l'aide de la surexpression de l'aquaporine 1 Download PDF

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WO2022031008A1
WO2022031008A1 PCT/KR2021/010234 KR2021010234W WO2022031008A1 WO 2022031008 A1 WO2022031008 A1 WO 2022031008A1 KR 2021010234 W KR2021010234 W KR 2021010234W WO 2022031008 A1 WO2022031008 A1 WO 2022031008A1
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osteoarthritis
pluripotent stem
cells
induced pluripotent
derived
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Korean (ko)
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주지현
남유준
임예리
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Industry Academic Cooperation Foundation of Catholic University of Korea
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present invention relates to a method for producing an induced pluripotent stem cell-derived osteoarthritis model using aquaporin 1 overexpression, and more particularly, to an osteoarthritis model comprising the step of overexpressing aquaporin 1 (AQP 1) in induced pluripotent stem cells. It relates to a manufacturing method and a method for screening an osteoarthritis therapeutic agent using the osteoarthritis model manufactured by the method.
  • Osteoarthritis is a degenerative joint disease that occurs frequently in cartilage, and is an important disease that deteriorates the quality of life related to participation in social activities and health in the elderly. Osteoarthritis is a phenomenon in which articular cartilage cells normally remain in the intermediate stage of differentiation, but due to some cause, they differentiate and die like growth plate chondrocytes, and lose their properties. Articular cartilage cell death (apoptosis) occurs more frequently in osteoarthritis than in normal articular cartilage.
  • the vector overexpressing the AQP1 gene was transformed into induced pluripotent stem cell-derived embryonic outgrowth cells, and then the transformed cells were The osteoarthritis disease model was established by differentiation into chondrocytes.
  • the established osteoarthritis disease model observed cell hypertrophy, and the expression of SOX9, an early cartilage differentiation marker, ACAN, and COL2A1, a representative free cartilage marker, decreased, while IL-1 ⁇ and IL-6, important cytokines in osteoarthritis, and It was confirmed that osteoarthritis characteristics such as an increase in the expression of VEGFA, a marker of cell hypertrophy, were confirmed, and the present invention was completed.
  • AQP1 aquaporin 1
  • Another object of the present invention is to provide a method for screening an osteoarthritis therapeutic agent using the osteoarthritis model prepared by the above method.
  • the present invention comprises the steps of: (a) transforming a recombinant vector into which aquaporin 1 (AQP 1) gene is inserted into embryonic outgrowth cells derived from human induced pluripotent stem cells; and
  • the human induced pluripotent stem cells may be prepared by introducing Oct4, Sox2, Klf4 and c-Myc into fibroblasts derived from ⁇ .
  • the step (a) comprises the steps of (1) culturing human induced pluripotent stem cells to form and obtain an embryoid body; (2) inducing and separating the embryonic body obtained in step (1) into outgrowth cells; and (3) transforming the dendritic cells with a recombinant vector into which the aquaporin 1 (AQP 1) gene is inserted.
  • the culture in step (b) may be performed in a serum-free medium containing human bone morphogenetic protein (BMP) and transforming growth factor-beta (TGF- ⁇ ).
  • BMP bone morphogenetic protein
  • TGF- ⁇ transforming growth factor-beta
  • the present invention also provides a human induced pluripotent stem cell-derived aquaporin 1 overexpression osteoarthritis disease model prepared by the above production method.
  • the present invention also provides a method for selecting an osteoarthritis therapeutic agent comprising the step of treating a therapeutic agent candidate substance to the osteoarthritis disease model prepared by the above preparation method.
  • the present invention from a point of view, (a) transforming the recombinant vector into which the aquaporin 1 (AQP 1) gene is inserted into human induced pluripotent stem cell-derived embryonic outgrowth cells; and
  • Human induced pluripotent stem cells refer to stem cells with multifunctionality that can be differentiated into all three germ layers constituting a living body and can be differentiated into all cells or organ tissues of the human body.
  • Various technologies for producing chondrocytes from such human induced pluripotent stem cells are being studied (Korean Patent Publication No. 10-2016-0068982), and in order to obtain fully differentiated chondrocytes, an additional suspension culture period of 28 days or more is required. There is a disadvantage in that a long period of time is required to produce chondrocytes from induced pluripotent stem cells.
  • an embryoid body is formed from human induced pluripotent stem cells, and from this, outgrowth cells similar to mesenchymal cells are formed.
  • cell, OG octiocytes
  • aquaporin 1 AQP 1
  • Induction of differentiation into chondrocytes was performed using pellet culture. In the pellet culture, a relatively small number of cells were centrifuged to achieve cell aggregation and artificially developed an ultra-high-density culture system from the start of 3D culture. way to make
  • the method for producing osteoarthritis of the present invention is more specifically,
  • step (2) inducing and separating the embryonic body obtained in step (1) into outgrowth cells
  • (4) culturing the transformed dendritic cells in the form of pellets to differentiate them into chondrocytes can be prepared by a method comprising.
  • the culture of step (b) or step (4) may be performed in a serum-free medium containing human bone morphogenetic protein (BMP) and transforming growth factor-beta (TGF- ⁇ ).
  • BMP bone morphogenetic protein
  • TGF- ⁇ transforming growth factor-beta
  • SOX9, ACAN and COL2A1 expression may be decreased, and IL-1 ⁇ , IL-6 and VEGFA expression may be increased.
  • human induced pluripotent stem cell-derived dendritic cells were transformed using a vector into which the AQP1 gene was inserted.
  • AQP1 expression was observed using GFP ( FIGS. 2A and 2B ) and AQP1 antibody ( FIG. 3 ), and as a result, it was confirmed that AQP1 was expressed in the transformed dendritic cells.
  • cartilage pellets derived from induced pluripotent stem cells overexpressed with AQP1 by the method of the present invention exhibited osteoarthritis characteristics as well as enlarged cells, it can be usefully used as a model for osteoarthritis disease, and can also be used to select osteoarthritis therapeutics. can be used for
  • the present invention relates to a human induced pluripotent stem cell-derived aquaporin 1 overexpressing osteoarthritis disease model prepared by the above production method.
  • the present invention relates to a method for screening an osteoarthritis therapeutic agent comprising the step of treating a therapeutic agent candidate to the osteoarthritis disease model prepared by the above preparation method.
  • an osteoarthritis disease model can be prepared using human pluripotent stem cells
  • induced pluripotent stem cells are prepared from somatic cells isolated from each osteoarthritis patient, and then a patient-specific osteoarthritis model is prepared using the method of the present invention can do.
  • the prepared patient-specific osteoarthritis model can be used to select a therapeutic agent suitable for each patient.
  • the present invention relates to a method for selecting a patient-specific osteoarthritis therapeutic agent comprising the step of treating a candidate therapeutic agent to the patient-specific osteoarthritis disease model prepared by the above method.
  • AQP1 overexpressed induced pluripotent stem cell-derived cartilage pellets were prepared. And it can be easily used in a method for selecting an osteoarthritis therapeutic agent using the same.
  • FIG. 1 is a schematic diagram of a plasmid vector into which the AQP1 gene is inserted.
  • Figure 2a is a photograph of observing the cell shape of human induced pluripotent stem cell-derived embryonic body dendritic cells (EBOGC) and bone marrow-derived mesenchymal stem cells (BMSC) transformed with the vector into which the AQP1 gene is inserted.
  • EBOGC human induced pluripotent stem cell-derived embryonic body dendritic cells
  • BMSC bone marrow-derived mesenchymal stem cells
  • Figure 2b is a photograph of observing the expression of AQP1 in human induced pluripotent stem cell-derived embryonic body dendritic cells (EBOGC) and bone marrow-derived mesenchymal stem cells (BMSC) transformed with a vector into which the AQP1 gene is inserted using GFP expression.
  • EBOGC human induced pluripotent stem cell-derived embryonic body dendritic cells
  • BMSC bone marrow-derived mesenchymal stem cells
  • EBOGC + AQP1 human induced pluripotent stem cell-derived dendritic cell
  • EBOGC + Mock empty vector transformed human induced pluripotent stem cell-derived embryonic dendritic cell
  • Figure 4 is a human induced pluripotent stem cell-derived embryonic body dendritic cells (EBOGC + AQP1) transformed with the vector into which the AQP1 gene is inserted, and human induced pluripotent stem cell-derived embryonic body dendritic cells transformed with an empty vector (EBOGC + Mock) It is a photograph of observing the cartilage pellets formed when the cartilage was differentiated.
  • FIG. 5 is a result confirming the expression levels of SOX9, ACAN, COL2A1, IL-1 ⁇ , IL-6 and VEGFA genes in the cartilage pellet formed in FIG. 4 .
  • hiPSCs Human induced pluripotent stem cells
  • CBMC cord blood mononuclear cells
  • PBS phosphate buffered saline
  • STMCELL StemSpan medium
  • CBMCs were inoculated into a 24-well plate at a concentration of 3 ⁇ 10 5 , and reprogramming was induced according to the protocol provided by the manufacturer using the CytoTuneTM-iPS 20 Sendai reprogram kit (A16518, Life Technologies). Thus, hiPSCs derived from CBMC were obtained.
  • the obtained hiPSCs were cultured in a vitronectin-coated vessel (Thermo Fisher Scientific, Waltham, MA, USA), and the culture medium was cultured with Essential 8 media (Thermo Scientific) being replaced once a day.
  • the hiPSCs derived from CBMC prepared in ⁇ Example 1-1> were resuspended in Aggrewell medium (STEMCELL), and inoculated into a 100-mm culture plate at a concentration of 2 ⁇ 10 6 cells/well.
  • the inoculated hiPSCs were cultured in an incubator at 37° C. for 24 hours, and the next day, the culture medium was cultured in Essential 8 medium for 3 days.
  • the embryoid body (EB) formed and obtained in ⁇ Example 1-2> was placed in DMEM medium (Thermo Fisher Scientific) containing 20% Fetal Bovine Serum and 10% penicillin/streptomycin. Suspended and cultured in 5% CO 2 at 37 ° C. for 7 days on a gelatin-coated plate to induce the formation of outgrowth cells (OG). For this, the culture plate was used after coating the bottom surface with 0.1% gelatin for 30 minutes and removing the solution.
  • DMEM medium Thermo Fisher Scientific
  • the outgrowth cells (OG) formed and obtained in ⁇ Example 1-3> were transformed using the AQP1 CRISPR activation plasmid (h2) (Santa Cruz, FIG. 1).
  • BMSC bone marrow-derived mesenchymal stem cells
  • the shape of the transformed cells was observed through an optical microscope, and AQP1 expression was observed with a fluorescence microscope using GFP expression and AQP1 antibody.
  • the transformed cells were washed with PBS, fixed with 4% paraformaldehyde, and then permeabilized with 0.1% Triton X-100 for 10 minutes. After permeation, PBS (PBA) containing 2% bovine serum albumin was added, followed by blocking at room temperature for 30 minutes.
  • the primary antibody, AQP1 antibody was diluted 1:100 in PBA and added to the cells, and then reacted at room temperature for 2 hours, and then the cells were washed with PBS. Thereafter, the Alexa Fluor 488-conjugated secondary antibody was diluted 1:400 in PBA and added to the cells, and then reacted for 1 hour at room temperature while blocking light.
  • the cells were washed and fixed on a glass slide by adding ProLong Antifade, and the stained cells were observed using a fluorescence microscope.
  • the dendritic cells induced overexpression of AQP1 were differentiated into chondrocytes through pellet culture.
  • human induced pluripotent stem cell-derived embryonic body dendritic cells (EBOGC + AQP1) transformed with the vector into which the AQP1 gene prepared in ⁇ Example 3> is inserted and human induced pluripotent stem cell-derived embryos transformed with the empty vector
  • the body dendritic cells (EBOGC + Mock) were separated from the gelatin-coated plate and passed through a 40 ⁇ m-sized cell strainer (Thermo Fisher Scientific) to remove EB clumps and isolate only single-cell units of OG cells. and obtained.
  • Count the isolated and obtained single-cell unit outgrowth cells prepare 1 ⁇ 10 3 , 2 ⁇ 10 3 or 3 ⁇ 10 3 cells per pellet in a microwell, and prepare cells in the form of pellets Cells were precipitated by centrifugation at 1800 rpm for 5 minutes to facilitate aggregation of chondrocytes to prepare a chondrocyte pellet.
  • the prepared chondrocyte pellet was inoculated into a chondrogenic differentiation medium, and the differentiation into chondrocytes was induced for 21 days while replacing with a new medium once every 2-3 days.
  • a cartilage pellet induced to differentiate into chondrocytes was obtained, total RNA was isolated, and then PCR was performed using the primer sequences in Table 1 below according to a method known in the art.
  • AQP1 overexpressed induced pluripotent stem cell-derived cartilage pellets were prepared. And since it can be easily used for screening methods for treating osteoarthritis using the same, it has industrial applicability.
  • SEQ ID NO: 1 is a forward primer sequence of SOX9 as a chondrogenic marker.
  • SEQ ID NO: 2 is a reverse primer sequence of SOX9 as a chondrogenic marker.
  • SEQ ID NO: 3 is a forward primer sequence of ACAN as a chondrogenic marker.
  • SEQ ID NO: 4 is a reverse primer sequence of ACAN as a chondrogenic marker.
  • SEQ ID NO: 5 is a forward primer sequence of COL2A1 as a chondrogenic marker.
  • SEQ ID NO: 6 is a reverse primer sequence of COL2A1 as a chondrogenic marker.
  • SEQ ID NO: 7 is a forward primer sequence of VEGFA as a cell hypertrophy marker (Hypertrophic Marker).
  • SEQ ID NO: 8 is a reverse primer sequence of VEGFA as a hypertrophic marker.
  • SEQ ID NO: 9 is a forward primer sequence of IL-1 ⁇ as an inflammatory cytokine.
  • SEQ ID NO: 10 is a reverse primer sequence of IL-1 ⁇ as an inflammatory cytokine.
  • SEQ ID NO: 11 is a forward primer sequence of IL-6 as an inflammatory cytokine.
  • SEQ ID NO: 12 is a reverse primer sequence of IL-6 as an inflammatory cytokine.
  • SEQ ID NO: 13 is a forward primer sequence of GAPDH as a house keeping gene.
  • SEQ ID NO: 14 is a reverse primer sequence of GAPDH as a house keeping gene.

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Abstract

La présente invention concerne un procédé de production d'un modèle d'ostéoarthrite dérivé d'une cellule souche pluripotente induite à l'aide de la surexpression de l'aquaporine 1, et plus spécifiquement : un procédé de production d'un modèle d'arthrose, le procédé comprenant une étape de surexpression de L'aquaporine 1 (AQP1) dans des cellules souches pluripotentes induites ; et un procédé de sélection d'un agent thérapeutique pour l'arthrose à l'aide du modèle d'arthrose préparé par ledit procédé. Dans la présente invention, des granulats de cartilage dérivés de cellules souches pluripotentes induites dans lesquelles l'AQP 1 est surexprimée ont été préparés afin d'établir le modèle d'ostéoarthrite, et il a été observé que les granulats de cartilage préparés avaient non seulement des cellules hypertrophiées, mais présentaient également des caractéristiques d'ostéoarthrite. Par conséquent, les granulats de cartilage peuvent être facilement utilisés dans un modèle de maladie d'ostéoarthrite et une méthode de sélection d'un agent thérapeutique de l'ostéoarthrite les utilisant.
PCT/KR2021/010234 2020-08-05 2021-08-04 Procédé de production d'un modèle d'ostéoarthrite dérivé d'une cellule souche pluripotente induite à l'aide de la surexpression de l'aquaporine 1 Ceased WO2022031008A1 (fr)

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CN118221761A (zh) * 2024-03-04 2024-06-21 广州汉密顿生物科技有限公司 一种用于治疗骨关节炎的干细胞组合物及其制备方法

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KR102622158B1 (ko) * 2020-08-05 2024-01-08 가톨릭대학교 산학협력단 골관절염 진단용 조성물 및 골관절염 진단을 위한 정보 제공방법

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118221761A (zh) * 2024-03-04 2024-06-21 广州汉密顿生物科技有限公司 一种用于治疗骨关节炎的干细胞组合物及其制备方法

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