Induced pluripotent stem cell-derived mesenchymal stem cell precursor cells and preparation method thereof

Abstract

The present invention relates to a precursor cell of an induced pluripotent stem cell-derived mesenchymal stem cell and a preparation method thereof. The precursor cell of the induced pluripotent stem cell-derived mesenchymal stem cell of the present invention has enhanced functionality and better proliferation ability than general mesenchymal stem cells or induced pluripotent stem cell-derived mesenchymal stem cells.

Description

[Technical field]

[0001] This patent application claims priority to Korean Patent Application No. 10-2019-0091269 filed with the Korean Intellectual Property Office on July 26, 2019 and Korean Patent Application No. 10-2019-0173078 filed with the Korean Intellectual Property Office on December 23, 2019, and the disclosures of the above patent applications are incorporated into this specification by reference.

[0002] The present invention relates to induced pluripotent stem cell-derived mesenchymal stem cell precursor cells and a preparation method thereof. [Background technology]

[0003] Mesenchymal stem cells are a type of matrix cell with multi-differentiation ability, which refers to a type of cell that can differentiate into a variety of cells such as osteocytes, chondrocytes, muscle cells, and adipocytes. Mesenchymal cells can differentiate into various connective tissues such as cartilage, bone tissue, ligaments, and bone marrow matrix. Therefore, they are often used to study their use in treating various diseases (such as treating soft tissue defects caused by arthritis, trauma, burns, etc.). In addition to this structural support function, mesenchymal stem cells also have immunomodulatory or immunosuppressive functions, such as expressing anti-inflammatory cytokines and converting macrophages into M2 macrophages, so they can inhibit inflammatory responses. Many current studies aim to use them as therapeutic agents for treating immune diseases caused by excessive activation of the immune system (such as arthritis, inflammatory bowel disease, atopic dermatitis, and autoimmune diseases).

[0004] Recently, in the study of the therapeutic efficacy of mesenchymal stem cells for various diseases, most people choose to use exosomes secreted by mesenchymal stem cells rather than mesenchymal stem cells themselves. In order to realize the commercial applicability of such exosomes, a large amount of high-quality exosomes is required. However, the number of exosomes that can be obtained from mesenchymal stem cells is extremely small, and the function and proliferation ability of mesenchymal stem cells will also decrease with repeated passages. For this reason, it is necessary to develop a technology to establish cells that are equal to or better than mesenchymal stem cells in terms of function and have excellent proliferation ability.

[0005]

Prior technical literature

[0006] [Patent Literature]

[0007] Korean Patent Publication No. 10-2019-0063453 (published on January 15, 2019) [Summary of the invention]

[0008]

Technical issues

[0009] The present inventors have devoted themselves to research and aimed to develop a stem cell with equal or superior functions to mesenchymal stem cells and excellent proliferation ability. The results confirmed that by establishing mesenchymal cells in the differentiation stage, replacing mesenchymal stem cells derived from induced pluripotent stem cells, it is possible to show more excellent functionality and proliferation ability, thereby completing the present invention.

[0010] Therefore, an object of the present invention is to provide an induced pluripotent stem cell-derived mesenchymal stem cell precursor cell (precurosr cell of iPSC-derived mesenchymal stem cell), a cell population composed of the same, and a preparation method thereof.

[0011] Another object of the present invention is to provide a method for producing exosomes from the above-mentioned induced pluripotent stem cell-derived mesenchymal stem cell precursor cells.

[0012]

Technical solution

[0013] According to one aspect of the present invention, the present invention provides a cell population comprising mesenchymal stem cell (MSC) precursor cells.

[0014] In this specification, the term "stem cell" as an undifferentiated cell refers to a cell that has the ability to self-replicate and differentiate into two or more different types of cells. The stem cells of the present invention can be autologous or allogeneic tissue-derived stem cells.

[0015] In this specification, the term "mesenchymal stem cells" as a cell with multi-differentiation ability refers to stem cells that can differentiate into various cells including bone cells, chondrocytes, muscle cells, fat cells, etc. The most commonly used mesenchymal stem cells are bone marrow-derived mesenchymal stem cells, but in addition to bone marrow, they can also be derived from umbilical cord or umbilical cord blood, adipose tissue, amniotic fluid, and molar tooth buds. Mesenchymal stem cells are also called stromal cells.

[0016] In the present specification, the precursor cells of mesenchymal stem cells developed by the present inventors are not general mesenchymal stem cells, but precursor cells of mesenchymal stem cells derived from induced pluripotent stem cells (iPSCs).

[0017] In this specification, "induced pluripotent stem cells (iPSC)" refer to cells with pluripotent differentiation ability induced from differentiated cells through an artificial reverse differentiation process, also known as reverse differentiated stem cells. The artificial reverse differentiation process is completed by using viral mediation (which uses retrovirus, lentivirus, Sendai virus) or non-viral vectors, and by introducing non-viral mediated reverse differentiation factors (which use proteins or cell extracts, etc.); or includes a reverse differentiation process completed by stem cell extracts, compounds, etc. The above-mentioned induced pluripotent stem cells have almost the same characteristics as embryonic stem cells. Specifically, the two have similar cell shapes, similar gene and protein expressions, are omnipotent in vitro and in vivo, and form teratomas. When inserted into the blastocyst of a mouse, a chimera mouse is formed, and germline transmission of genes can be performed.

[0018] The induced pluripotent stem cells of the present invention may include induced pluripotent stem cells from all species (such as humans, monkeys, pigs, horses, cattle, sheep, dogs, cats, mice, rats, and rabbits), but preferably, they are induced pluripotent stem cells of human origin.

[0019] In addition, the somatic cells before reverse differentiation of the induced pluripotent stem cells of the present invention may be precursor cells derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion, amniotic fluid or placenta, but are not limited thereto. Specifically, the somatic cells include: fibroblasts, hepatocytes, adipose cells, epithelial cells, epidermal cells, chondrocytes, muscle cells, cardiac muscle cells, melanocytes, neural cells, glial cells, astrocytes, monocytes, macrophages, etc., but are not limited thereto.

[0020] In one embodiment of the present invention, the cell population is characterized in that, in at least 80% of the cells, the marker protein expressed on the cell surface is CD73 + 、CD90 + 、CD105+ In a specific implementation example of the present invention, the cell population is characterized in that, in at least 80%, 85%, 90% or more than 95% of the cells, the marker protein expressed on the cell surface is CD73 + 、CD90 + 、CD105 + or a combination thereof.

[0021] In another embodiment of the present invention, the cell population is characterized in that, in at most 5% or less of the cells, the marker protein expressed on the cell surface is CD34. + and / or CD45 + In a specific implementation example of the present invention, the cell population is characterized in that, in at most 5%, 4%, or less than 3% of the cells, the marker protein expressed on the cell surface is CD34. + and / or CD45 + .

[0022] In this specification, CD refers to cell surface antigen cluster of differentiation, "+" or "(+)" refers to the presence of a corresponding cell surface marker on the cell surface (positive), and "-" or "(-)" refers to the absence of a corresponding cell surface marker on the cell surface (negative). The presence (positive) or absence (negative) of the above-mentioned marker does not mean absolute physical presence or absence, but means the presence or absence determined according to a relative standard, specifically, if it is higher than the experimental detectable value of the technical level in the art, it is determined to be present (positive), and if it is lower than this value, it is determined to be absent (negative).

[0023] In another embodiment of the present invention, the precursor cells of the mesenchymal stem cells of the present invention express one or more genes selected from the group consisting of ANKRD1, CPE, NKAIN4, LCP1, CCDC3, MAMDC2, CLSTN2, SFTA1P, EPB41L3, PDE1C, EMILIN2, SULT1C4, TRIM58, DENND2A, CADM4, AIF1L, NTM, SHISA2, RASSF4, and ACKR3 at a higher level than the same amount of mesenchymal stem cells.

[0024] The gene expression level expressed at a higher level by the precursor cells of the mesenchymal stem cells of the present invention means that the expression level is at least 3 times, 5 times, 7 times or 10 times higher than the gene expression level expressed by the same amount of mesenchymal stem cells.

[0025] In addition, in another embodiment of the present invention, the precursor cells of the mesenchymal stem cells of the present invention express one or more genes selected from the group consisting of DHRS3, BMPER, IFI6, PRSS12, RDH10, and KCNE4 at a lower level than the same amount of mesenchymal stem cells.

[0026] The gene expression level expressed at a lower level by the precursor cells of the mesenchymal stem cells of the present invention means that the expression level is at least 3, 5, 7 or 10 times lower than the gene expression level expressed by the same amount of mesenchymal stem cells.

[0027] In a specific implementation of the present invention, the precursor cells of the mesenchymal stem cells and the same amount of mesenchymal stem cells are from the same tissue source. More specifically, the precursor cells of the mesenchymal stem cells are precursor cells of mesenchymal stem cells derived from induced pluripotent stem cells of the same tissue source.

[0028] In one embodiment of the present invention, the precursor cells of the mesenchymal stem cells are precursor cells of mesenchymal stem cells derived from umbilical cord tissue-derived induced pluripotent stem cells, and the equal amount of mesenchymal stem cells compared thereto are umbilical cord tissue-derived mesenchymal stem cells.

[0029] The inventors named the precursor cells of the above-mentioned induced pluripotent stem cell-derived mesenchymal stem cells as BxC (brexogen stem cell). In this specification, the above-mentioned "precursor cells of induced pluripotent stem cell-derived mesenchymal stem cells (BxC)" can also be expressed as "induced pluripotent stem cell-derived mesenchymal precursor cells" or "induced pluripotent stem cell-derived mesenchymal precursor cells with enhanced functions".

[0030] In the present specification, the above-mentioned "induced pluripotent stem cell-derived mesenchymal stem cell precursor cells (BxC)" are deposited in the "Korean Collection for Type Culture (KCTC)" with the accession number "KCTC14019BP".

[0031] As demonstrated in the examples of the present invention, the precursor cells (BxC) of mesenchymal stem cells of induced pluripotent stem cells of the same tissue source (e.g., umbilical cord tissue) of the present invention have no abnormal chromosome karyotype and have a more excellent proliferation capacity than mesenchymal stem cells (MSC) of the same tissue source (e.g., umbilical cord tissue). Specifically, when BxC completes more than 9 subcultures, it shows a proliferation capacity difference of more than 10 times compared to mesenchymal stem cells (MSC) of the same tissue source, and even after more than 12 subcultures, no signs of decreased proliferation capacity are observed. In addition, BxC shows more than 2 times higher expression of Ki67, a marker related to cell proliferation capacity, than MSC.

[0032] In one embodiment of the present invention, the precursor cells (BxC) of mesenchymal stem cells derived from induced pluripotent stem cells of the present invention secrete a large amount of functional proteins such as endostatin, endothelin-1, vascular endothelial growth factor A (VEGF-A), thrombospondin-2, placental growth factor (PlGF), platelet-derived growth factor (PDGF-AA), β-nerve growth factor (β-NGF), and heparin-binding epidermal growth factor (HB-EGF) compared to the same amount of mesenchymal stem cells.

[0033] In the present specification, the above-mentioned endostatin is a 20 kDa C-terminal fragment derived from naturally occurring type XVIII collagen, which is reported to be an anti-angiogenesis inhibitor.

[0034] In the present specification, the above-mentioned endothelin-1 (endothelin-1), also called preproendothelin-1 (PPET1), is a protein encoded by the EDN1 gene and produced in vascular endothelial cells. Endothelin-1 is known to be a potent vasoconstrictor.

[0035] In the present specification, the vascular endothelial growth factor A (VEGF-A) is a protein encoded by the VEGFA gene, and is known to induce blood vessel growth by interacting with VEGFR1 and VEGFR2 of vascular endothelial cells.

[0036] In this specification, the above-mentioned thrombospondin-2, as a protein encoded by the THBS2 gene, is known to mediate cell-cell interaction or cell-matrix interaction. The role of thrombospondin-2 in cancer is still controversial, but it is reported that it can regulate the cell surface properties of mesenchymal stem cells and participate in cell adhesion and cell migration.

[0037] In the present specification, the placental growth factor (PlGF) is a protein encoded by the PFG gene, and is known to be a member of the vascular endothelial growth factor subfamily (VEGF sub-family), and is a protein that plays a major role in angiogenesis during embryonic development.

[0038] In the present specification, the platelet-derived growth factor (PDGF-AA) is a growth factor that regulates cell growth and division, and is known to play an important role in the formation and growth of blood vessels, and the proliferation, chemotaxis and migration of mesenchymal stem cells.

[0039] In this specification, nerve growth factor (NGF) is a nerve affinity factor and neuropeptide, which is mainly involved in the growth, maintenance, proliferation and survival of nerves. NFG is a complex of three proteins, among which the expression ratio of α-, β-, and γ-NGF is about 2:1:2. It is known that γ-NGF plays the role of serine protease and activates NGF by cutting the N-terminus of beta-NGF.

[0040] In the present specification, the above-mentioned heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family protein encoded by the HBEGF gene. It is known that HB-EGF plays an important role in cardiac development and vascular distribution, and is also an essential protein in the epithelialization process in terms of epidermal wound healing.

[0041] According to another aspect of the present invention, the present invention provides a method for preparing precursor cells of mesenchymal stem cells, comprising the following steps:

[0042] Culturing the induced pluripotent stem cells in a medium containing FBS and bFGF for 1 to 10 days; and

[0043] SSEA-4(-) cells were isolated and cultured in a medium containing FBS and bFGF for 1 to 10 days.

[0044] The precursor cells of the mesenchymal stem cells of the present invention belong to the same cell type as the precursor cells of the mesenchymal stem cells described above, and therefore, the common contents between the two are omitted.

[0045] According to an implementation example of the present invention, the induced pluripotent stem cells are prepared by culturing somatic cells derived from human umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion, amniotic fluid or placenta in a reverse differentiation medium.

[0046] Specifically, the above-mentioned somatic cells include: fibroblasts, hepatocytes, adipose cells, epithelial cells, epidermal cells, chondrocytes, muscle cells, cardiac muscle cells, melanocytes, neural cells, glial cells, astrocytes, monocytes, macrophages, or mesenchymal stem cells derived from bone marrow, umbilical cord, umbilical cord blood, adipose tissue, amniotic fluid, tooth buds, etc., but are not limited to these.

[0047] In the present specification, stage-specific embryonic antigen-4 (SSEA-4) is one of the characteristic cell surface markers of human induced pluripotent stem cells and embryonic stem cells. In one embodiment of the present invention, in the process of differentiating into the mesenchymal stem cell precursor cell (BxC) of the present invention, the above SSEA-4 can be used as a screening factor to distinguish cells that retain the characteristics of induced pluripotent stem cells from cells that lose the characteristics of induced pluripotent stem cells.

[0048] In one embodiment of the present invention, the inventors cultured induced pluripotent stem cells and screened them based on whether they expressed SSEA-4, separated SSEA-4(-) cells, and then further cultured them.

[0049] In one embodiment of the present invention, the induced pluripotent stem cells and SSEA-4(-) cells can be cultured for 1 to 10 days, 3 to 10 days, 5 to 10 days, and more specifically, 5 to 7 days or 7 to 10 days.

[0050] In one implementation example of the present invention, after the above-mentioned induced pluripotent stem cells are cultured for 1 to 10 days, the separation of SSEA-4(-) cells can use fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) methods. Preferably, the FACS method can be used, but it is not limited thereto, and cell separation methods known in the art can be used without limitation.

[0051] In another embodiment of the present invention, the culture medium for culturing cells contains 5-20%, 5-15%, 10-15% or 10-20% FBS, and most specifically, contains 10% FBS, but is not limited thereto.

[0052] In another implementation example of the present invention, the culture medium for culturing the cells contains 1-20 ng / ml, 2-20 ng / ml, 3-20 ng / ml, 5-20 ng / ml, 7-20 ng / ml, 10-20 ng / ml, 1-15 ng / ml, 2-15 ng / ml, 3-15 ng / ml, 5-15 ng / ml, 7-15 ng / ml, 10-15 ng / ml, 1-10 ng / ml, 2-10 ng / ml, 3-10 ng / ml, 5-10 ng / ml, 7-10 ng / mld bFGF, most specifically, contains 10 ng / ml of bFGF.

[0053] According to another aspect of the present invention, the present invention provides a method for preparing exosomes, comprising the following steps:

[0054] (a) culturing a cell population comprising precursor cells of the mesenchymal stem cells in a cell culture medium; and

[0055] (b) Exosomes were isolated from the above cell culture medium.

[0056] In one implementation example of the present invention, the cell culture medium is characterized in that it contains fetal bovine serum from which exosomes have been removed.

[0057] In a specific implementation example of the present invention, the reason why the above-mentioned cell culture medium uses FBS without exosomes is that commonly used FBS contains a large amount of exosomes derived from bovine serum. Therefore, the purpose of doing so is to avoid mixing with exosomes derived from FBS other than exosomes secreted by stem cells.

[0058] In this specification, the term "exosome" is a nanoscale membrane vesicle, which is released by the fusion of multivesicular bodies and plasma membranes in the cell organelles of the endocytic pathway, and is present in the body fluids of almost all eukaryotic organisms. The diameter of the exosome is about 30 to 150 nm, which is larger than LDL protein, but significantly smaller than red blood cells. Exosomes are released from cells when multivesicular bodies fuse with cell membranes, or directly from cell membranes. Exosomes are known to play important specialized functions, such as: mediation of cellular immunity and intercellular signal transduction. Exosome marker proteins are known to include CD9, CD63, CD81, etc. The above-mentioned exosomes of the present invention are not strictly limited to a size of 30 to 150 nm, and the concept includes: multivesicular bodies falling within the above size range, microvesicles composed of lipid bilayers greater than or equal to the above size, and mixtures thereof.

[0059] In an implementation example of the present invention, the above-mentioned culture can be cultured for 12 hours to 120 hours, 24 hours to 96 hours, 48 ​​hours to 96 hours, or 60 hours to 84 hours, and most specifically, can be cultured for 72 hours, but is not limited thereto.

[0060] In one implementation of the present invention, the precursor cells of the mesenchymal stem cells may be pretreated with various substances before isolating exosomes. For example, before isolating exosomes, the precursor cells of the mesenchymal stem cells of the present invention can be pretreated with one or more substances selected from the group consisting of interferon-gamma, pioglitazone, metformin, 5-Aminoimidazole-4-carboxamideribonucleotide (AICAR), thrombin, resveratrol, substance P, hyaluronic acid, tetrandrine, phorbol 12-myristate 13-acetate (PMA), exendine-4, dexamethasone, insulin, ascorbate, insulin-like growth factor (IGF), and transforming growth factor β1 (TGF-β1), but are not limited thereto.

[0061] In addition, in another embodiment of the present invention, the step of isolating exosomes from the above-mentioned cell culture medium is a process of isolating exosomes secreted into the cell culture medium from the precursor cells of the cultured mesenchymal stem cells.

[0062] In one implementation example of the present invention, the method for isolating the exosomes is as follows.

[0063] Centrifuge the culture medium at 200-400×g for 5-20 minutes to remove the remaining cells and cell residues, then take the supernatant and centrifuge at 9,000-12,000×g for 60-80 minutes. At this time, take the supernatant again and centrifuge at 90,000-120,000×g for 80-100 minutes, remove the supernatant, and you can get the exosomes remaining in the lower layer.

[0064] According to a specific implementation example of the present invention, the mesenchymal stem cell culture medium is collected, centrifuged at 300×g for 10 minutes, the remaining cells and cell residues are removed, and the culture medium is centrifuged at 100×g and 4°C for 70 minutes using a high speed centrifuge. The supernatant after centrifugation is then taken and centrifuged at 100,000×g and 4°C for 90 minutes using an ultracentrifuge, and the supernatant is removed to separate the exosomes remaining in the lower layer.

[0065] The method for preparing the exosomes is to prepare the exosomes by culturing the precursor cells of the mesenchymal stem cells, which is similar to the precursor cells of the mesenchymal stem cells. The exosome separation method in this specification is only used as an example, and the exosome separation method used in the art can also be used without limitation.

[0066] The exosomes prepared by the preparation method of the present invention may or may not contain a therapeutic agent.

[0067] In one embodiment of the present invention, the above-mentioned therapeutic agent is a therapeutic protein, an antibody (for example, a full-length antibody, a monoclonal antibody, a single-chain antibody (scFv), a Fab fragment, F(ab')2, a diabody, a triabody or a minibody), an inhibitory RNA or a small molecule drug.

[0068] In one embodiment of the invention, the therapeutic protein is a known disease-associated protein (eg, a tumor suppressor, kinase, phosphatase, or transcription factor) that is lost or inactivated for therapeutic purposes.

[0069] In one embodiment of the present invention, the antibody binds to an intracellular antigen. This intracellular antigen can be a protein (e.g., a tumor gene), the activity of which is required for cell proliferation and / or survival. In some cases, the antibody blocks the function of the antigen. In some cases, the antibody also interferes with protein-protein interactions.

[0070] In one embodiment of the present invention, the inhibitory RNA is siRNA, shRNA, miRNA or pre-miRNA. In various embodiments, the inhibitory RNA blocks the expression of a protein (e.g., a tumor gene), the activity of which is required to maintain the disease state. When the tumor gene is a mutant form of the gene, the inhibitory RNA will preferentially block the expression of the mutant tumor gene rather than the wild-type protein.

[0071] In one implementation example of the present invention, the small molecule drug is a contrast agent. In another implementation example of the present invention, the small molecule drug is a chemotherapeutic agent.

[0072] In one embodiment of the present invention, the composition is formulated for oral administration, or is formulated for parenteral administration (eg, intravenous, intramuscular, subcutaneous or intraperitoneal injection).

[0073] In one implementation of the present invention, the therapeutic agent includes an antimicrobial agent. The antimicrobial agent may be benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide bromide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerol, ethidine, acylide urea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol or thimerosal, but is not limited thereto.

[0074] According to another aspect of the present invention, the present invention provides a precursor cell of mesenchymal stem cells derived from induced pluripotent stem cells. In one implementation example of the present invention, the induced pluripotent stem cells are derived from human umbilical cord. In another implementation example of the present invention, the precursor cell has been deposited with the deposit number KCTC14019BP.

[0075] [Effects of the Invention]

[0076] The present invention provides a precursor cell of mesenchymal stem cells derived from induced pluripotent stem cells and a preparation method thereof. The precursor cell of mesenchymal stem cells derived from induced pluripotent stem cells of the present invention has enhanced functionality and better proliferation ability than general mesenchymal stem cells or mesenchymal stem cells derived from induced pluripotent stem cells.

Brief Description of the Drawings

[0077] Figure 1a-1b The graphs show the expression ratios and microscopic photographs of the cell surface expression marker SSE-4 on day 0 and day 7 after culture of the induced pluripotent stem cells (iPSCs) of the present invention. Figure 1c The figure shows the surface expression marker SSEA-4 of cells (BxC) after culturing the induced pluripotent stem cells of the present invention for 7 days, separating the corresponding SSEA-4(-) cells therefrom and culturing them for 7 days. The figure shows the expression ratio of SSE-4 and micrographs.

[0078] Figure 2 It is a graph showing the types of cell surface markers of the induced pluripotent stem cells of the present invention on day 0 of culture (iPSC), day 7 of culture (pre-BxC), day 7 of culture after isolation of SSEA-4 (1) (BxC), and mesenchymal stem cells (MSC).

[0079] Figure 3 Shown are the results of karyotype analysis of precursor cells (BxC) of mesenchymal stem cells derived from induced pluripotent stem cells of the present invention and mesenchymal stem cells (MSC) derived from the same tissue.

[0080] Figure 4aThis is a graph showing the number of cells obtained by subculture of precursor cells (BxC) of mesenchymal stem cells derived from induced pluripotent stem cells of the present invention and mesenchymal stem cells (MSC) derived from the same tissue.

[0081] Figure 4b The graph shows a comparison of the expression ratio of the cell proliferation factor Ki67 between the precursor cells (BxC) of mesenchymal stem cells derived from induced pluripotent stem cells of the present invention and mesenchymal stem cells (MSC) derived from the same tissue.

[0082] Figures 5a to 5d Shown is a comparative graph of the secretion amounts of functional proteins expressed by precursor cells (BxC) of mesenchymal stem cells derived from induced pluripotent stem cells of the present invention (5a: endostatin, endothelin-1; 5b: VEGF-A, thrombospondin-2; 5c: PlGF, PDGF-AA; 5d: β-NGF, HB-EGF).

[0083] Figure 6a and Figure 6b The graph shows the results of a CFU-F assay for comparing the proliferation capacity of the precursor cells (BxC) of mesenchymal stem cells derived from induced pluripotent stem cells of the present invention and mesenchymal stem cells (MSC) derived from the same tissue.

[0084] Figure 7a Shown is a schematic diagram of RNA-seq analysis results, aiming to identify genes with differential expression levels between the precursor cells (BxC) of mesenchymal stem cells derived from induced pluripotent stem cells of the present invention and mesenchymal stem cells (MSC) derived from the same tissue. Figure 7b Shown is a graph of genes whose expression levels in BxC are higher than those in MSC. Figure 7c Shown is a graph of genes that are less expressed in BxC than in MSC.

[0085] Figure 8a is a graph confirming the number and size distribution of exosomes separated from BxC of the present invention, Figure 8b The diagram confirms the expression of exosome-specific surface antigens TSG101 and CD63. [Specific implementation method]

[0086] Hereinafter, the present invention will be described in more detail with reference to the embodiments. These embodiments are only used for a more detailed description of the present invention. According to the gist of the present invention, the scope of the present invention is not limited by these embodiments, which is obvious to those skilled in the art.

[0087] [Example]

[0088] In this specification, "%" used to express the concentration of a specific substance, i.e., solid / solid is (weight / weight)%, solid / liquid is (weight / volume)%, liquid / liquid is (volume / volume)%, unless otherwise mentioned.

[0089] [Example 1: Isolation and culture of mesenchymal stem cell precursor cells (BxC) derived from induced pluripotent stem cells (iPSC)]

[0090] First, induced pluripotent stem cells (iPSCs) were cultured for 7 days in DMEM supplemented with 10% FBS and 10 ng / ml of bFGF. Then, SSEA-4(-) cells that do not express stage-specific embryonic antigen-4 (SSEA-4) protein on the cell surface were separated from the cultured induced pluripotent stem cells by FASC. In addition, the isolated SSEA-4(-) cells were subcultured and further cultured in the same culture medium for 7 days to prepare the precursor cells of the induced pluripotent stem cell-derived mesenchymal stem cells of the present invention. The inventors named the precursor cells of the above-mentioned induced pluripotent stem cell-derived mesenchymal stem cells as BxC (brexogen stem cell) and preserved them in the Korean Collection for Type Culture (KCTC) with the accession number "KCTC14019BP".

[0091] The precursor cells of induced pluripotent stem cell-derived mesenchymal stem cells named BxC were further cultured in a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100×) (Gibco, Cat no. 11140-050)].

[0092] The cell surface marker SSEA-4 ratio of each cell in the above-mentioned induced pluripotent stem cells (iPSC) on day 0 (D0), induced pluripotent stem cells after 7 days of culture (Pre-BxC) (D7), and precursor cells of mesenchymal stem cells derived from induced pluripotent stem cells of the present invention (BxC) is as shown in Tables 1 and Figure 1a-1c , Figure 2 shown.

[0093] 96.6% of the initial induced pluripotent stem cell population expressed SSEA-4 protein on the cell surface ( Figure 1a ), but after 7 days of culture, the population of SSEA04(+) cells expressing SSEA-4 protein was reduced to 35.8% of the total cells ( Figure 1bAfter isolating the SSEA-4(-) protein, which accounts for 64.2% of the total cells, and subculturing and culturing for 7 days (BxC cells), it was confirmed that the SSEA-4 protein-expressing cells accounted for less than 3% of the total cell population ( Figure 1c ).

[0094] This indicates that the SSEA-4 protein expression rate in the cell population of the induced pluripotent stem cell (iPSC)-derived mesenchymal progenitor cells (BxC) with enhanced functions is 5% or less.

[0095] [Example 2: Expression of cell surface markers of mesenchymal precursor cells (BxC) with enhanced functions derived from induced pluripotent stem cells (iPSC)]

[0096] The following analysis was performed on the cell surface markers (SSEA-4, CD34, CD45, CD73, CD90, CD105) of each cell of induced pluripotent stem cells (iPSC) on day 0 (D0), induced pluripotent stem cells after 7 days of culture (Pre-BxC) (D7), and precursor cells of mesenchymal stem cells derived from induced pluripotent stem cells of the present invention (BxC).

[0097] Using rypLE TM Express (Gibco, 12604-021), cells at each stage were processed into single cells and washed with PBS. Then they were divided into tubes for separation to avoid wavelength overlap of antibodies specifically binding to SSEA-4 (BD560128), CD73 (BD550257), CD90 (BD5555950), CD105 (BD560839), CD45 (BD555483) and CD34 (BD348053). The cell mass was resuspended in 100 μL of FACS suspension, and 6 μL of antibodies were added 2 times at a time to avoid wavelength overlap. The cell mass was allowed to stand for 30 minutes at room temperature in the dark for 30 minutes, then washed with PBS, and then suspended in 300 μL FACS buffer and analyzed using a FACS device.

[0098] The expression ratio of each cell surface marker (SSEA-4, CD34, CD45, CD73, CD90, CD105) of stem cells is shown in Table 1 and Figure 2 shown.

[0099] From the above results, it can be seen that the expression rate of CD73, CD90, and CD105 proteins in the cell population of the mesenchymal precursor cells (BxC) with enhanced functions derived from induced pluripotent stem cells (iPSC) of the present invention is 90% or more.

[0100]

Table 1

[0101] Differentiation stage SSEA-4 (%) CD34 (%) CD45 (%) CD73 (%) CD90(%) CD105(%) iPSC 96.7 0.1 0.7 0.3 0.6 0.9 Pre-BxC 38.8 0.4 0.9 1.3 1.7 3.8 BxF 2.7 1.4 1.2 99.0 96.9 98.6 MSC 0.2 0.3 0.5 99.7 99.2 98.6

[0102] [Example 3: Chromosome Analysis of Functionally Enhanced Mesenchymal Precursor Cells (BxC) Derived from Induced Pluripotent Stem Cells (iPSCs)]

[0103] In order to confirm whether the induced pluripotent stem cell-derived function-enhanced mesenchymal progenitor cells (BxC) and induced pluripotent stem cell-derived mesenchymal stem cells (MSC) of the present invention have chromosomal abnormalities, the karyotypes of BxC and MSC passaged 6 times were analyzed.

[0104] like Figure 3 As shown, it was confirmed that the BxC and MSC of the present invention had no abnormalities in chromosome number and structure.

[0105] [Example 4: Comparison of proliferation characteristics of mesenchymal precursor cells (BxC) with enhanced functions derived from induced pluripotent stem cells (iPSC)]

[0106] In order to compare the proliferation capacity of the induced pluripotent stem cell-derived mesenchymal precursor cells (BxC) with enhanced functions and the induced pluripotent stem cell-derived mesenchymal stem cells (MSC) of the present invention, the following experiment was performed.

[0107] In a 6-well culture plate, 1×10 cells were seeded per well of induced pluripotent stem cell-derived mesenchymal precursor cells (BxC) and mesenchymal stem cells (MSC) derived from the same tissue (umbilical cord) at the same passage number (P4). 5 After 10 cells were grown, the medium was removed and the cells were washed with PBS every 3.5 days. TM Express treatment was performed to separate the cells. After centrifugation at 300×g for 5 minutes, the cells were resuspended in 1 mL of culture medium and the cell number was measured. The cells whose cell number had been measured were inoculated again, with 1×10 5 After the cells were cultured, the culture was repeated every 3.5 days, and the increase in cell number at each passage was confirmed.

[0108] In addition, in order to confirm the expression ratio of Ki67, a cell proliferation marker, per cell, the following experiment was performed.

[0109] By TrypLE TMExpress (Gibco, 12604-021) was used to treat the same passage number (P4) of induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) and the same tissue (umbilical cord)-derived mesenchymal stem cells (MSC) into single cells and washed with PBS. After washing, the cells were placed in cold 80% ethanol (ethanol), fixed at -20°C for more than 2 hours, and washed 3 times with PBS. 20 μL of Ki-67-specific antibody in each 100 μL buffer contained in PE Mouse Anti-Ki-67kit (BD556027) was transferred to a new tube, left at room temperature for 30 minutes, then washed with PBS, reacted with the labeled secondary antibody, and left to stand at room temperature for 30 minutes in the dark, and then analyzed using a FASC device.

[0110] like Figure 4a As shown, compared with mesenchymal stem cells (MSC), the mesenchymal precursor cells (BxC) derived from induced pluripotent stem cells of the present invention with enhanced functions have shown a proliferation capacity difference of more than 10 times since the 9th passage. In addition, the proliferation capacity of mesenchymal stem cells began to decline since the 12th passage, while the mesenchymal precursor cells (BxC) derived from induced pluripotent stem cells of the present invention with enhanced functions showed no signs of decreased proliferation capacity after more than 12 passages.

[0111] In addition, if Figure 4b As shown, the expression level of Ki67, a cell proliferation marker, in the induced pluripotent stem cell-derived mesenchymal precursor cells (BxC) with enhanced functions of the present invention is more than two times higher than that in mesenchymal stem cells (MSC).

[0112] This indicates that the mesenchymal progenitor cells (BxC) with enhanced functions derived from induced pluripotent stem cells of the present invention have a much superior proliferation effect compared to mesenchymal stem cells (MSC).

[0113] [Example 5: Comparison of protein secretion ability of mesenchymal precursor cells (BxC) with enhanced functions derived from induced pluripotent stem cells (iPSC)]

[0114] The following experiment was conducted to compare the secretion capacity of proteins related to the functionality of the induced pluripotent stem cell-derived mesenchymal precursor cells (BxC) with enhanced functions and the induced pluripotent stem cell-derived mesenchymal stem cells (MSC) of the present invention.

[0115] In a 6-well culture plate, induced pluripotent stem cell-derived mesenchymal precursor cells (BxC) and mesenchymal stem cells (MSC) with enhanced functions were seeded at the same passage number, with 1×10 per well. 5After 24 hours, the supernatant was removed. After washing with DPBS, 1 mL of serum free medium was added and the medium was removed after 48 hours. Dead cells and debris were precipitated by centrifugation at 300 × g for 5 minutes, and only the supernatant was taken and stored at -80°C. The array kit was made using the custom service of Luminex High performance assay of R&D systems, and Luminex assay was performed.

[0116]

Table 2

[0117] protein MSC BxF Endostatin 3516.6pg / ml 6682.0pg / ml Endothelin-1 17.0pg / ml 79.4pg / ml VEGF-A 32.2pg / ml 78.5pg / ml Thrombospondin-2 21802.9pg / ml 36912.7pg / ml PlGF 5.8pg / ml 9.4pg / ml PDGF-AA 29.2pg / ml 328.4pg / ml β-NGF 10.5pg / ml 17.3pg / ml HB-EGF 3.9pg / ml 15.7pg / ml

[0118] like Figures 5a to 5d As shown, compared with induced pluripotent stem cell-derived mesenchymal stem cells (MSC), the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) of the present invention secrete a large amount of proteins related to the functionality of mesenchymal stem cells (MSC). It can be seen that compared with mesenchymal stem cells (MSC), the functionality of the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) of the present invention is more enhanced.

[0119] [Example 6: Investigation of stem cell characteristics (Stemness) of mesenchymal precursor cells (BxC) with enhanced functions derived from induced pluripotent stem cells (iPSC)]

[0120] In order to compare the stem cell characteristics (Stemness) of the induced pluripotent stem cell-derived mesenchymal precursor cells (BxC) with enhanced functions and mesenchymal stem cells (MSC) of the present invention, the following experiment was conducted.

[0121] Mesenchymal stem cells and induced pluripotent stem cell-derived mesenchymal stem cell precursor cells (BxC) with enhanced functions were inoculated in a 100 mm cell culture dish containing 10 mL of culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100×) (Gibco, Cat no. 11140-050)]. 1,000 cells were inoculated in each dish and cultured for 21 days. The dish with cultured cells attached was washed twice with PBS and fixed with 95% methanol at room temperature for about 2 minutes. After washing the fixed cells three times with PBS, 0.5% crystal violet solution [5 g of crystal violet (Sigma, C-3886, USA), 100 ml of methanol] was added and stained for 5 minutes. After washing with water, it was left to dry at room temperature. The number of colonies consisting of more than 50 cells in each dish was counted and compared.

[0122] like Figure 6a-6b As shown in the results, the number of colony forming unit fibroblasts (CFU-F) of induced pluripotent stem cell-derived mesenchymal stem cells (MSC) is about 3 times higher than that of the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) of the present invention. It is thus confirmed that the stem cell characteristics (stemness) of the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) of the present invention are superior to those of mesenchymal stem cells (MSC).

[0123] [Example 7: Investigation of characteristic gene expression patterns of mesenchymal precursor cells (BxC) with enhanced functions derived from induced pluripotent stem cells (iPSCs)]

[0124] In order to confirm the differences in gene expression patterns between the induced pluripotent stem cell-derived functionally enhanced mesenchymal precursor cells (BxC) and mesenchymal stem cells (MSC) of the present invention, mRNA was extracted from each cell population, and the gene expression levels of each cell were compared using RNA-seq analysis.

[0125] The gene types showing differences in gene expression and the differences in expression (gene expression of BxC / gene expression of MSC) are shown in Tables 2 and Figure 7a-7c shown.

[0126] like Figure 7a-7cAs shown, compared with the same amount of mesenchymal stem cells (MSC), the gene expression levels of ANKRD1, CPE, NKAIN4, LCP1, CCDC3, MAMDC2, CLSTN2, SFTA1P, EPB41L3, PDE1C, EMILIN2, SULT1C4, TRIM58, DENND2A, CADM4, AIF1L, NTM, SHISA2, RASSF4 and ACKR3 of the induced pluripotent stem cell-derived mesenchymal precursor cells (BxC) of the present invention are at least 10 times higher and at most 55 times higher.

[0127] In contrast, the expression of DHRS3, BMPER, IFI6, PRSS12, RDH10 and KCNE4 genes in the induced pluripotent stem cell-derived function-enhanced mesenchymal precursor cells (BxC) of the present invention is at least 10- to 20-fold lower.

[0128]

Table 3

[0129]

[0130]

[0131] [Example 8: Isolation and confirmation of exosomes from induced pluripotent stem cell (iPSC)-derived mesenchymal precursor cells (BxC) with enhanced functions]

[0132] The precursor cells (BxC) of the induced pluripotent stem cell-derived functionally enhanced mesenchymal stem cells prepared in Example 1 above were further cultured in a medium supplemented with 10% exosome-depleted FBS (exosome-free fetal bovine serum). After 72 hours of culture, the BxC cell culture medium was collected and centrifuged at 300×g for 10 minutes to remove the remaining cells and cell debris. Centrifuge at 1,000×g and 4°C for 70 minutes using a high speed centrifuge. The supernatant after centrifugation was taken and centrifuged at 100,000×g and 4°C for 90 minutes using an ultracentrifuge. The supernatant was removed and the exosomes remaining in the lower layer were diluted with phosphate buffered saline (PBS) for use. The number and size distribution of the isolated exosomes were confirmed by nanoparticle tracking assay (NanoSight NS300, Malvern) ( Figure 8a ). The expression of exosome-specific surface antigens TSG101 and CD63 was confirmed by Western blotting ( Figure 8b ).

[0133]

preservation

[0134]

Collection

[0135]

Accession number

[0136]

Collection date

[0137] Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure

[0138] International Forms

[0139] Receipt in original preservation condition

[0140] Rule 7.1 is issued to

[0141] To: KIM Sue

[0142] KIM Sue

[0143] 109 Jeongdae-ro, Songpa-gu, Seoul

[0144] South Korea

[0145]

[0146] BP / 4 Form (KCTC Form 17) (single page)

Claims

1. A cell population comprising precursor cells of mesenchymal stem cells (MSC) derived from induced pluripotent stem cells, wherein at least 80% of the cells in the cell population are CD73 + 、CD90 + or CD105 + , wherein no more than 5% of the cells in the cell population are SSEA-4 + 、CD34 + or CD45 + , The precursor cells of mesenchymal stem cells derived from induced pluripotent stem cells have been deposited under the accession number KCTC14019BP, and The mesenchymal stem cells derived from induced pluripotent stem cells are obtained by the following steps: (i) culturing induced pluripotent stem cells in a medium containing FBS and bFGF for 7 days; (ii) isolating SSEA-4(-) cells; and (iii) culturing the SSEA-4(-) cells in a medium containing FBS and bFGF for 7 days.

2. The cell population according to claim 1, wherein the precursor cells of the mesenchymal stem cells express one or more genes selected from the group consisting of ANKRD1, CPE, NKAIN4, LCP1, CCDC3, MAMDC2, CLSTN2, SFTA1P, EPB41L3, PDE1C, EMILIN2, SULT1C4, TRIM58, DENND2A, CADM4, AIF1L, NTM, SHISA2, RASSF4 and ACKR3 at a higher level than the mesenchymal stem cells of the same tissue origin.

3. The cell population according to claim 1, wherein the precursor cells of the mesenchymal stem cells express one or more genes selected from the group consisting of DHRS3, BMPER, IFI6, PRSS12, RDH10 and KCNE4 at a lower level than an equal amount of mesenchymal stem cells derived from the same tissue.

4. A method for preparing precursor cells of mesenchymal stem cells derived from induced pluripotent stem cells, wherein the precursor cells of mesenchymal stem cells derived from induced pluripotent stem cells have been deposited under the deposit number KCTC14019BP, wherein the method comprises the following steps: Induced pluripotent stem cells were cultured in a medium containing FBS and bFGF for 7 days; and SSEA-4(-) cells were isolated and cultured in a medium containing FBS and bFGF for 7 days.

5. A method for producing exosomes, comprising the following steps: Cultivating the cell population according to any one of claims 1 to 3 in a cell culture medium; and Exosomes are isolated from the cell culture medium. The method according to claim 5 , wherein the cell culture medium contains exosome-depleted fetal bovine serum.

7. A precursor cell of a mesenchymal stem cell derived from an induced pluripotent stem cell, The induced pluripotent stem cells are derived from human umbilical cord. The precursor cells have been deposited under the accession number KCTC14019BP.

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