Method for isolating and culturing tissue-resident uPAR+ / Nestin+ stem cells and its uses

Abstract

The present invention relates to a method for isolating and culturing tissue-resident uPAR+ / Nestin+ stem cells and its use, and provides a method for isolating tissue-resident stem cells present in solid tissue based on the uPAR-plasmin activity. The uPAR-plasmin activity of stem cells is closely related to the growth, migration ability, physiological activity, and differentiation ability of stem cells, and can be used as a method for isolating high-potency stem cells. The uPAR+ stem cells isolated from solid tissue of the present invention can be applied to the production of cell therapy agents, tissue engineering therapy agents, and new biopharmaceuticals using secretions containing exosomes, and have great industrial potential. In addition, the present invention can be applied to basic cell biological and molecular biological research and new drug development research related to cell division, migration, growth, and differentiation of tissue-resident stem cells.

Description

[Technical field]

[0001] The present invention relates to a method for isolating and culturing uPAR+ / Nestin+ tissue-resident stem cells, which play a key role in tissue regeneration and tissue homeostasis in solid tissues, and uses thereof. [Background technology]

[0002] A typical method for isolating and culturing stem cells present in solid tissue requires a process of dissociating the tissue by treating it with a protease, removing the cells that compose the tissue one by one, and then preparing a single cell suspension. In the tissue dissociation process, the yield of single cells obtained varies greatly depending on the type, potency, reaction time, reaction temperature, and type of tissue used of the protease used, and cell damage during the tissue dissociation process is unavoidable. As a result, the tissue dissociation process has a disadvantage that it is difficult to standardize, since the cell yield and the degree of damage vary greatly depending on the tissue type. In particular, the frequency of stem cells in solid tissue is less than 0.1%, and it is widely known that the yield at which a useful amount of stem cells can be isolated through the protease is extremely low.

[0003] A subsequent process is required to separate and purify tissue-resident stem cells from the single cell suspension dissociated from the tissue. The process of separating and purifying stem cells uses markers such as c-Kit or Sca-1 to separate cells that are positive or negative for the marker. However, these markers are expressed not only in tissue-resident stem cells but also in hematopoietic cells, so that unwanted cells are mixed in and separated, and there are also stem cells that are negative for these markers, so there are limitations to the method of separating and purifying tissue-resident stem cells using specific markers. Summary of the Invention [Problem to be solved by the invention]

[0004] The object of the present invention is to provide a method for activating, inducing the migratory growth and isolating tissue-resident uPAR+ / nestin+ stem cells located within solid tissues such as adipose tissue, bone marrow tissue, cardiac muscle tissue, peripheral nerve tissue, skeletal muscle tissue or synovial tissue.

[0005] Another object of the present invention is to provide tissue-resident uPAR+ and nestin+ stem cells isolated and cultured by the above method, or a culture medium thereof.

[0006] It is yet another object of the present invention to provide a pharmaceutical composition for preventing or treating inflammatory diseases or autoimmune diseases, comprising the tissue-resident uPAR+ and nestin+ stem cells or a culture medium thereof as an active ingredient.

[0007] It is yet another object of the present invention to provide a pharmaceutical composition for treating wounds or promoting vascular regeneration, comprising the tissue-resident uPAR+ and nestin+ stem cells or a culture medium thereof as an active ingredient. [Means for solving the problem]

[0008] To achieve the above objective, the present invention provides a method for activating tissue-resident uPAR+ and nestin+ stem cells, comprising: (1) preparing a wound repair matrix-mimicking hydrogel; (2) embedding an isolated tissue slice into the wound repair matrix-mimicking hydrogel; and (3) 3D culturing the wound repair matrix-mimicking hydrogel with the tissue slice embedded therein in a culture medium supplemented with a plasminogen activator inhibitor (PAI).

[0009] The present invention also provides a method for isolating and culturing tissue-resident uPAR+ and nestin+ stem cells, comprising: (1) preparing a wound repair matrix-mimicking hydrogel; (2) embedding an isolated tissue slice into the wound repair matrix-mimicking hydrogel; (3) 3D culturing the wound repair matrix-mimicking hydrogel with the tissue slice embedded therein in a culture medium containing PAI; (4) removing the 3D culture medium and washing to remove the PAI; (5) re-culturing the culture from which the PAI has been removed in a culture medium not containing PAI to degrade the wound repair matrix-mimicking hydrogel; and (6) isolating the stem cells released into the re-culture medium.

[0010] The present invention also provides tissue-resident uPAR+ and nestin+ stem cells isolated and cultured by the above method, or a culture medium thereof.

[0011] The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease, comprising the tissue-resident uPAR+ and nestin+ stem cells or a culture medium thereof as an active ingredient.

[0012] The present invention also provides a pharmaceutical composition for preventing or treating an autoimmune disease, comprising the tissue-resident uPAR+ and nestin+ stem cells or a culture medium thereof as an active ingredient.

[0013] The present invention also provides a pharmaceutical composition for wound treatment, comprising the tissue-resident uPAR+ and nestin+ stem cells or a culture medium thereof as an active ingredient.

[0014] The present invention also provides a pharmaceutical composition for promoting vascular regeneration, comprising the tissue-resident uPAR+ and nestin+ stem cells or a culture medium thereof as an active ingredient. Effect of the Invention

[0015] The present invention relates to a method for isolating and culturing tissue-resident uPAR+ / Nestin+ stem cells and its use, and provides a method for isolating tissue-resident stem cells present in solid tissue based on the uPAR-plasmin activity. The uPAR-plasmin activity of stem cells is closely related to the growth, migration ability, physiological activity, and differentiation ability of stem cells, and can be used as a method for isolating high-potency stem cells. The uPAR+ stem cells isolated from solid tissue of the present invention can be applied to the production of cell therapy agents, tissue engineering therapy agents, and new biopharmaceuticals using secretions containing exosomes, and have great industrial potential. In addition, the present invention can be applied to basic cell biological and molecular biological research and new drug development research related to cell division, migration, growth, and differentiation of tissue-resident stem cells. [Brief description of the drawings]

[0016] [Figure 1] The expression rates of pFAK, uPAR, nestin, and Ki-67 in the cells that compose the organ after in vitro organ culture are shown. 2D, monolayer organ culture; 3D, 3D organ culture; AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, synovium. **, p<0.01 compared to 2D. [Diagram 2] The figures show that 3D myocardial organ culture increases intramyocardial pFAK+, uPAR+ and Nestin+ expressing cells. [Diagram 3] This shows the characteristic of increasing Ki-67 positive cells undergoing cell division within the myocardium in 3D organ culture. [Figure 4] The number of pFAK, uPAR, nestin, and ki-67 positive cells in the myocardium increases proportionally with the duration of 3D myocardial organ culture. **, p<0.01 compared to 0d (before culture). [Diagram 5] The figure shows the expression of uPAR, nestin, and BrdU in cells migrated and grown in the hydrogel after 3D myocardial organ culture. [Figure 6]The expression rates of uPAR and nestin in cells migrated and grown in the hydrogel after 2 weeks of 3D organ culture. AT, adipose tissue; BM, bone marrow; Myocar, cardiac muscle; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. [Figure 7] uPAR mRNA (A) and plasmin activity (B) in organs before and after 3 days of organ culture. AT, adipose tissue; BM, bone marrow; Myocar, cardiac muscle; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. *, p<0.05 compared to pre-culture; **, p<0.01 compared to pre-culture. [Figure 8] After organ culture, the hydrogel is decomposed by removing the PAI from the culture medium and washing the culture medium, and the cells that migrated and grew within the hydrogel are released into the culture medium. [Figure 9] After removing the PAI from the culture medium and washing the culture medium, the cells that had migrated and grown within the hydrogel detached from the hydrogel and the released cells contracted and aggregated (phase contrast micrographs). [Figure 10] After removal of PAI and washing of the culture, uPAR+ cells within the hydrogel were released and separated (HE staining of paraffin tissue sections and uPAR immunohistochemical staining). [Figure 11] After PAI removal, culture washing, and addition of exogenous urokinase, the cell yield and uPAR expression rate of the isolated and recovered cells after isolation from the hydrogel are shown. AT, adipose tissue; BM, bone marrow; Myocar, cardiac muscle; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. **, p<0.01 compared to urokinase. [Figure 12] After PAI removal and culture washing, tissue sections were harvested, showing the cell yields recovered after repeated isolation from the hydrogel through repeated organ culture. AT, adipose tissue; BM, bone marrow; Myocar, cardiac muscle; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. [Figure 13]After removing the PAI and washing the culture medium, the recovered cells were seeded in a culture vessel, and the cells attached and grew in a monolayer culture environment. A, Cell aggregates released from the hydrogel after removing the PAI and washing with water. B, Cell aggregates recovered from the hydrogel. C-E, Cell attachment and growth 30 min (C), 1 h (D), and 2 h (E) after seeding the recovered cells in a culture vessel (phase contrast micrographs). [Figure 14] After culturing adipose tissue, bone marrow, cardiac muscle, nerve, skeletal muscle, and synovial membrane organs, the PAI was removed and the cultures were washed. The cells that had been released into the hydrogel (top) and the collected cells were then seeded. After the cells attached to the culture vessel and their growth shape was observed, the results of the cells attaching and growing (bottom) are shown (phase contrast micrographs). [Figure 15] After culturing neural and cardiac organs, removal of the PAI and washing of the culture medium were followed, and the cells released from the hydrogel were shown to attach and grow in a monolayer culture environment. [Figure 16] Immunophenotypic characterization of cells withdrawn from hydrogels. A, Immunophenotypic characterization of cells released after withdrawal from hydrogels after PAI removal and culture washing. B, Immunophenotypic characterization of cells released from hydrogels after treatment with exogenous urokinase. AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. **, p<0.01 compared to urokinase (B). [Figure 17] Expression of hematopoietic and endothelial cell markers in cells recovered from hydrogels. A, Expression of cells isolated and recovered after PAI removal and culture washing. B, Expression of cells isolated and recovered after exogenous urokinase treatment. AT, adipose tissue; BM, bone marrow; Myocar, cardiac muscle; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. **, p<0.01 compared to urokinase (B). [Figure 18]The self-renewal ability of cells isolated and recovered from hydrogels is shown. A: Representative photographs of colony forming units (CFU). B: CFU frequency of cells isolated and recovered from hydrogels according to tissue origin. Urokinase, cells isolated and recovered after exogenous urokinase treatment; PAI withdrawal, cells isolated and recovered after PAI removal and culture washing; AT, adipose tissue; BM, bone marrow; Myocar, cardiac muscle; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. **, p<0.01 compared to urokinase. [Figure 19] The in vitro growth potential of cells isolated and recovered from hydrogels is shown. A; Population Doubling Time (PDT, cell doubling time). B; Population Doubling Level (PDL, cell doubling level). Urokinase, cells isolated and recovered after exogenous urokinase treatment; PAI withdrawal, cells isolated and recovered after PAI removal and culture washing; AT, adipose tissue; BM, bone marrow; Myocar, cardiac muscle; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. *, p<0.05 compared to urokinase; **, p<0.01 compared to urokinase. [Figure 20] The Ki-67 expression rate of cells isolated and recovered from the hydrogel is shown. Urokinase, cells isolated and recovered after exogenous urokinase treatment; PAI withdrawal, cells isolated and recovered after PAI removal and culture washing; AT, adipose tissue; BM, bone marrow; Myocar, cardiac muscle; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. **, p<0.01 compared to urokinase. [Figure 21] The differentiation potential of cells isolated and recovered from the hydrogel into osteoblasts (alizarin red) and adipocytes (Oil Red O). Urokinase, cells isolated and recovered after exogenous urokinase treatment; PAI withdrawal, cells isolated and recovered after PAI removal and culture washing. [Figure 22]Comparison of differentiation ability of cells isolated and recovered from hydrogel into osteoblasts (alizarin red) and adipocytes (Oil Red O). Urokinase, cells isolated and recovered after exogenous urokinase treatment; PAI withdrawal, cells isolated and recovered after PAI removal and culture washing; AT, adipose tissue; BM, bone marrow; Myocar, cardiac muscle; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. **, p<0.01 compared to urokinase. [Figure 23] Tissue regeneration mRNA expression profile of cells isolated and recovered from hydrogel. Urokinase, cells isolated and recovered after exogenous urokinase treatment; PAI withdrawal, cells isolated and recovered after PAI removal and culture washing; CB-MSC, cord blood-derived mesenchymal stem cells; AT, adipose tissue; BM, bone marrow; Myocar, cardiac muscle; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. *, p<0.05 compared to urokinase; **, p<0.01 compared to urokinase. [Figure 24] The plasmin activity and nestin expression rate of cells isolated and recovered from the hydrogel are shown. uPAR-, uPAR-negative cells isolated and purified after exogenous urokinase treatment; uPAR+, uPAR+ cells isolated and purified after exogenous urokinase treatment; PAI withdrawal, cells isolated and recovered after PAI removal and culture washing; AT, adipose tissue; BM, bone marrow; Myocar, cardiac muscle; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. *, p<0.05 compared to uPAR-; **, p<0.01 compared to uPAR-. *, p<0.05 compared to uPAR-; **, p<0.01 compared to uPAR-. [Diagram 25]The self-renewal (CFU) and in vitro growth (Ki-67) potential of cells isolated and recovered from hydrogels are shown. uPAR-, uPAR-negative cells isolated and purified after exogenous urokinase treatment; uPAR+, uPAR+ cells isolated and purified after exogenous urokinase treatment; PAI withdrawal, cells isolated and recovered after PAI removal and culture washing; AT, adipose tissue; BM, bone marrow; Myocar, cardiac muscle; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. *, p<0.05 compared to uPAR-; **, p<0.01 compared to uPAR-. *, p<0.05 compared to uPAR-; **, p<0.01 compared to uPAR-. [Figure 26] Anti-inflammatory ability of cells isolated and recovered from hydrogel. Inhibitory effect of TNFα and IL-1β secretion in RAW 264.7 cells depleted with LPS in the conditioned medium. uPAR-, uPAR-negative cells isolated and purified after exogenous urokinase treatment; uPAR+, uPAR+ cells isolated and purified after exogenous urokinase treatment; PAI withdrawal, cells isolated and recovered after PAI removal and culture washing; AT, adipose tissue; BM, bone marrow; Myocar, cardiac muscle; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. *, p<0.05 compared to uPAR-; **, p<0.01 compared to uPAR-. *, p<0.05 compared to uPAR-; **, p<0.01 compared to uPAR-. [Figure 27] The efficacy of cells isolated and recovered from hydrogels in inducing the growth of vascular endothelial cells (HUVEC) and fibroblasts (DF) is shown. uPAR-, uPAR-negative cells isolated and purified after exogenous urokinase treatment; uPAR+, uPAR+ cells isolated and purified after exogenous urokinase treatment; PAI withdrawal, cells isolated and recovered after PAI removal and culture washing; AT, adipose tissue; BM, bone marrow; Myocar, cardiac muscle; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. *, p<0.05 compared to uPAR-; **, p<0.01 compared to uPAR-. *, p<0.05 compared to uPAR-; **, p<0.01 compared to uPAR-. [Figure 28]The efficacy of cells isolated and recovered from hydrogels in protecting vascular endothelial cells (HUVEC) and fibroblasts (DF) is shown. uPAR-, isolated and purified uPAR-negative cells after exogenous urokinase treatment; uPAR+, isolated and purified uPAR+ cells after exogenous urokinase treatment; PAI withdrawal, isolated and recovered cells after PAI removal and culture washing; AT, adipose tissue; BM, bone marrow; Myocar, cardiac muscle; PN, peripheral nerve; SM, skeletal muscle; Syn, synovial membrane. *, p<0.05 compared to uPAR-; **, p<0.01 compared to uPAR-. *, p<0.05 compared to uPAR-; **, p<0.01 compared to uPAR-. [Figure 29] 1 shows a schematic diagram of the present invention compared to existing technology. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention provides a method for activating tissue-resident uPAR+ and nestin+ stem cells, comprising: (1) preparing a wound repair matrix-mimicking hydrogel; (2) embedding an isolated tissue slice into the wound repair matrix-mimicking hydrogel; and (3) 3D culturing the wound repair matrix-mimicking hydrogel with the tissue slice embedded in it in a culture medium containing a plasminogen activator inhibitor (PAI).

[0018] Preferably, the wound repair matrix-mimicking hydrogel is a fibrin hydrogel obtained by mixing a fibrinogen solution having a concentration of 0.25 to 2.5% and a thrombin solution having a concentration of 0.5 to 5 I.U. / mL, a fibrin / collagen mixed hydrogel obtained by mixing the fibrin hydrogel with a collagen solution having a concentration of 0.1 to 0.5%, or a fibrin / gelatin mixed hydrogel obtained by mixing the fibrin hydrogel with a gelatin solution having a concentration of 0.1 to 0.5%, but is not limited to these.

[0019] Preferably, the tissue is, but is not limited to, adipose tissue, bone marrow tissue, cardiac muscle tissue, peripheral nerve tissue, skeletal muscle tissue or synovial tissue.

[0020] Preferably, the PAI is tranexamic acid or aminomethyl benzoic acid, but is not limited thereto.

[0021] Preferably, the method can activate integrin-FAK cell signaling in cells within the tissue to induce cell division and cell growth of tissue-resident uPAR+ and nestin+ stem cells, but is not limited thereto.

[0022] Preferably, but not limited to, the method can induce cell migration and growth of tissue-resident uPAR+ and nestin+ stem cells within the wound repair matrix-mimicking hydrogel.

[0023] In order to induce the migration of uPAR+ and nestin+ tissue-resident stem cells of the present invention to the wound repair matrix, a tissue-matched wound repair matrix is ​​required. The wound repair matrix can be prepared using a biopolymer that mimics the wound repair matrix components formed by coagulation of plasma released from blood vessels after tissue injury. The wound repair matrix of the present invention can be prepared by using fibrin, collagen, and gelatin alone or in combination. The engineered wound repair matrix can be prepared by preparing a wound repair matrix that is resistant to uPA-plasmin activity, since the activity of uPA-plasmin varies depending on the organ. The wound repair matrix can be prepared by adjusting the content or composition of the biopolymer. That is, a wound repair matrix that is a mixture of two or more components, such as fibrin-collagen, fibrin-gelatin, and collagen-gelatin, can be used. The wound repair matrix of the present invention can be composed of the constituent polymers fibrinogen, collagen, and gelatin in a content of 1.0 to 20.0 mg / ml. To enhance the structural properties of the wound repair matrix, the degree of cross-linking can be adjusted, and cross-linkers such as Ca++ or Factor XIIIa can be used to adjust the degree of cross-linking of the wound repair matrix to control the structural properties.

[0024] In order to induce cell division and growth of tissue-resident stem cells through in vitro culture, activation of cell signaling pathways is required. Signals capable of inducing cell growth and migration by binding to integrin ligands of cells can be transmitted and activated through the wound repair matrix. As tissue is supported with a wound repair matrix capable of directly binding to integrins of cells to transmit signals into cells, cell division, growth and migration of tissue-resident stem cells can be induced. The integrin-β1-FAK signaling pathway transmits signals to tissue-resident stem cells through integrins through the support of the wound repair matrix, inducing activation, and the tissue-resident stem cells can increase uPAR and nestin expression by the transmitted signals, thereby inducing the division, growth and migration of tissue-resident regenerative stem cells. The present invention can achieve the above object by preparing a wound repair matrix-mimicking hydrogel composed of a polymer having an RGD motif such as fibrin, collagen, and gelatin as a wound repair matrix capable of activating the integrin-FAK cell signaling pathway.

[0025] The present invention provides a method for inducing cell division, growth and migration of tissue-resident stem cells by activating the integrin-β1-FAK-uPAR signaling pathway. The efficiency of signaling to tissue-resident stem cells is proportional to the density of receptors that can bind to integrin ligands. The density of receptors that can bind to integrin ligands can be increased by increasing the contact area with tissue. By providing a three-dimensional environment rather than a two-dimensional one, the area that can be bonded to cells can be increased, thereby increasing the binding between integrin ligands and receptors. As a result, the efficiency of signaling to tissue-resident stem cells can be increased. The present invention provides a method for activating the integrin-β1-FAK-uPAR signaling pathway by providing three-dimensional support to tissue using an artificial wound repair matrix. To this end, a method is provided for enhancing the signaling pathway by providing three-dimensional binding to tissue slices using a wound repair matrix that is a hydrogel capable of sol-gel phase transition.

[0026] uPA-plasmin activity may vary depending on the tissue, the degree of injury, and the cause. Compared to fat, placenta, and umbilical cord, nervous system tissue has a higher uPA-plasmin activity, and as a result, the degree of decomposition of the wound repair matrix is ​​higher in nervous system tissue. The decomposition of the wound repair matrix can be controlled by adjusting the content, molecular weight, and crosslinking degree of the components constituting the wound repair matrix. The present invention provides a method for adjusting the resistance to uPA-plasmin activity by increasing the content, molecular weight, and crosslinking degree of the polymers constituting the wound repair matrix. In addition, a method for continuously inducing the activity, growth, and migration of stem cells in tissue in the wound repair matrix by adjusting the uPA-plasmin activity through the addition of PAI is provided. The PAI that can be applied to the present invention can be selected from the group consisting of aminocaproic acid, tranexamic acid, aprotinin, and aminomethylbenzoic acid. The PAI that can be applied to the present invention can be added at a concentration of 10 μg to 10 mg per ml of hydrogel volume. As a result, the present invention provides a method to support the induction of tissue-resident stem cell growth and migration during organ culture by incorporating PAIs and maintaining the structural stability of the wound repair matrix.

[0027] Excessive uPAR-uPA-plasmin activity of stem cells may result in rapid degradation of the wound repair matrix mimicking hydrogel during in vitro culture, resulting in the degradation and disappearance of the wound repair matrix around the tissue slice and cells, resulting in the disappearance of the wound repair matrix to which stem cells can attach and migrate, and resulting in the disappearance, reduction, or disappearance of cell migration from the tissue into the hydrogel. In order to control the excessive degradation and disappearance of the wound repair matrix mimicking hydrogel, the present invention provides a method for controlling the excessive plasmin activity of stem cells and tissue slices by adding PAI, thereby allowing the wound repair matrix mimicking hydrogel to maintain its role as a physically supporting matrix for the tissue slice during the organ culture period, and at the same time maintain its structural and functional role as a matrix to which activated tissue-resident stem cells can migrate and grow.

[0028] The present invention also provides a method for isolating and culturing tissue-resident uPAR+ and nestin+ stem cells, comprising: (1) preparing a wound repair matrix-mimicking hydrogel; (2) embedding an isolated tissue slice into the wound repair matrix-mimicking hydrogel; (3) 3D culturing the wound repair matrix-mimicking hydrogel with the tissue slice embedded therein in a culture medium containing PAI; (4) removing the 3D culture medium and washing to remove the PAI; (5) re-culturing the culture from which the PAI has been removed in a culture medium not containing PAI to degrade the wound repair matrix-mimicking hydrogel; and (6) isolating the stem cells released into the re-culture medium.

[0029] Preferably, the wound repair matrix-mimicking hydrogel is a fibrin hydrogel obtained by mixing a fibrinogen solution having a concentration of 0.25 to 2.5% and a thrombin solution having a concentration of 0.5 to 5 I.U. / mL, a fibrin / collagen mixed hydrogel obtained by mixing the fibrin hydrogel with a collagen solution having a concentration of 0.1 to 0.5%, or a fibrin / gelatin mixed hydrogel obtained by mixing the fibrin hydrogel with a gelatin solution having a concentration of 0.1 to 0.5%, but is not limited to these.

[0030] Preferably, the tissue is, but is not limited to, adipose tissue, bone marrow tissue, cardiac muscle tissue, peripheral nerve tissue, skeletal muscle tissue or synovial tissue.

[0031] Preferably, the PAI is tranexamic acid or aminomethylbenzoic acid, but is not limited thereto.

[0032] Preferably, the step (5) can induce an increase in uPAR expression in the tissue and degrade the wound repair matrix-mimicking hydrogel through an increase in plasmin activity, but is not limited thereto.

[0033] Preferably, the tissue-resident uPAR+ and nestin+ stem cells have enhanced self-renewal ability, in vitro growth ability, differentiation ability, or tissue regeneration induction ability, but are not limited thereto.

[0034] Preferably, the tissue slices recovered from the reculture medium in step (5) may further include, but are not limited to, a process of repeating steps (2) to (5) 1 to 10 times.

[0035] The present invention provides a method for selectively isolating uPAR+ stem cells that have migrated and grown within a wound repair matrix. The addition of PAI can prevent the wound repair matrix from being degraded by excessive uPAR-plasmin activity, and a method for recovering cells from the wound repair matrix after the targeted stem cell migration and growth has been achieved is provided. As the PAI is washed and removed, the wound repair matrix is ​​degraded using the uPAR-plasmin activity characteristic inherent in the stem cells, and the stem cells that have migrated and grown within the matrix are released and recovered. The present invention provides a method for isolating uPAR+ stem cells from solid tissues without the use of a specific exogenous protease and without a subsequent purification process, in which the wound repair matrix is ​​degraded by uPAR-plasmin depending on the uPAR expression rate and plasmin activity in the stem cells.

[0036] The present invention provides a method for recovering tissue slices used in organ culture in a state where the structure and function are preserved by not using a specific exogenous tissue degrading enzyme, and a method for recovering uPAR+ tissue-resident stem cells by inducing their migration into a wound repair matrix through repeated organ culture, while preserving the structure of the tissue slices recovered after organ culture.

[0037] The present invention provides a composition of a wound repair matrix that is not degraded and lost due to the uPAR-uPA-plasmin activity of tissue-resident stem cells, which varies depending on the tissue, and provides a tissue-specific PAI type and concentration that can control excessive degradation.

[0038] By subjecting uPAR+ / Nestin+ tissue-resident stem cells that have migrated and grown in the final wound repair matrix mimicking hydrogel to PAI washing and withdrawal, a method for isolating highly pure stem cells without a subsequent purification process is provided. uPAR+ / Nestin+ tissue-resident stem cells are used to manufacture stem cell therapeutics and stem cell-derived biopharmaceuticals with high self-renewal, in vitro growth, differentiation potential, tissue regeneration-inducing gene expression, vascular regeneration, and wound repair efficacy.

[0039] The present invention also provides tissue-resident uPAR+ and nestin+ stem cells isolated and cultured by the above method, or a culture medium thereof.

[0040] In the present invention, the term "culture medium" includes a medium capable of supporting the growth and survival of stem cells in vitro, and secretions of cultured stem cells contained in the medium. The medium used for the culture includes any medium suitable for culturing stem cells and commonly used in the art. The medium and culture conditions can be selected according to the type of cells. The medium used for the culture is preferably a cell culture minimum medium (CCMM), which generally contains a carbon source, a nitrogen source, and trace element components. Examples of such cell culture minimum medium include, but are not limited to, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM (α Minimal essential Medium), GMEM (Glasgow's Minimal essential Medium), and Iscove's Modified Dulbecco's Medium.

[0041] Meanwhile, the present invention can be used in any of the following forms: a form containing all of the stem cells, their secretions, and medium components; a form containing only the secretions and medium components; a form in which only the secretions are isolated and used alone or together with stem cells; or a form in which only stem cells are administered to produce secretions in the body.

[0042] The stem cells can be obtained using routine methods known to those of ordinary skill in the art.

[0043] The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease, comprising the tissue-resident uPAR+ and nestin+ stem cells or a culture medium thereof as an active ingredient.

[0044] The present invention also provides a pharmaceutical composition for preventing or treating an autoimmune disease, comprising the tissue-resident uPAR+ and nestin+ stem cells or a culture medium thereof as an active ingredient.

[0045] The present invention also provides a pharmaceutical composition for wound treatment, comprising the tissue-resident uPAR+ and nestin+ stem cells or a culture medium thereof as an active ingredient.

[0046] The present invention also provides a pharmaceutical composition for promoting vascular regeneration, comprising the tissue-resident uPAR+ and nestin+ stem cells or a culture medium thereof as an active ingredient.

[0047] The tissue-resident uPAR+ and nestin+ stem cells of the present invention are used as a cell therapy agent for the treatment of a particular disease, and the treatment is a direct treatment or a pretreatment with the molecule.

[0048] The term "cell therapy" refers to a pharmaceutical product used for therapeutic, diagnostic, or preventive purposes through a series of actions such as expanding or selecting living autologous, allogenic, or xenogenic cells in vitro, or changing the biological properties of cells in other ways, in order to restore the functions of cells and tissues.

[0049] The cell therapy agent is administered to the human body via a common route as long as it can reach the target tissue.

[0050] The pharmaceutical composition of the present invention can be prepared using pharmaceutical suitable and physiologically acceptable auxiliary agents in addition to the active ingredient, and the auxiliary agents can be solubilizers such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants, or flavoring agents. The pharmaceutical composition of the present invention can be preferably formulated as a pharmaceutical composition by further including one or more pharmaceutical acceptable carriers in addition to the active ingredient for administration. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are those suitable for sterilization and living organisms, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components, and other conventional additives such as antioxidants, buffers, bacteriostatic agents, etc. can be added as necessary. In addition, the composition can be formulated as an injectable dosage form such as an aqueous solution, suspension, or emulsion, or as a pill, capsule, granule, or tablet by additionally adding a diluent, dispersant, surfactant, binder, and lubricant.

[0051] The dosage form of the drug of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops, or injectable liquids and sustained release formulations of active compounds. The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. The effective amount of the active ingredient of the pharmaceutical composition of the present invention means the amount required for the prevention or treatment of a disease. Therefore, it is adjusted according to various factors including the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of dosage form, and the patient's age, weight, general health condition, sex and diet, administration time, administration route, and excretion rate of the composition, treatment period, and drugs used simultaneously.

[0052] The present invention provides a method for isolating stem cells without using a specific exogenous tissue decomposition enzyme during the process of isolating solid tissue-resident stem cells and without a tissue dissociation process. Since no exogenous tissue decomposition enzyme is used, the tissue can be used as a tissue source that can repeatedly isolate stem cells from the tissue since the function and structure of the tissue can be preserved after isolating the stem cells.

[0053] It is known that tissue-resident stem cells account for a very small proportion of the cells that make up tissue, usually less than 0.01%. After tissue injury, the number of somatic cells decreases, and stem cells to replace or regenerate these cells are activated, increasing the frequency of stem cells compared to before injury. After injury, tissue-resident stem cells self-renew through cell division and produce daughter progenitor cells, which then migrate to the damaged site. This patent provides a method for activating in vivo tissue homeostasis mechanisms through in vitro culture to induce self-renewal and cell division of stem cells in tissue slices, and for mobilizing the stem cells that have self-renewed and divided in vitro to the damaged site.

[0054] After tissue injury, a wound repair matrix (provisional matrix) is formed around the injured tissue, and the matrix-cellular integrin pathway is activated through these matrices, resulting in stem cells acting on the tissue injury migrating to the injured site after cell division, and tissue regeneration begins. The present invention provides a method for activating stem cells in tissue by maximizing matrix-cellular integrin interactions through three-dimensional support of a wound repair matrix-mimicking hydrogel around a tissue slice, inducing cell division, growth, and migration into the wound repair matrix-mimicking hydrogel, and isolating stem cells that have migrated into the hydrogel.

[0055] It has been reported that tissue-resident stem cells are activated in response to tissue damage, and the activated stem cells have the characteristic of increasing uPAR or nestin expression. It has been revealed that uPAR and nestin expression plays an important role in stem cell growth and migration, and uPAR+ and nestin+ cells are known to play a central role in tissue regeneration. Since uPAR and nestin are increased in reparative stem cells, uPAR and nestin can be used as primary target markers in stem cell isolation. The present invention provides a method for selectively isolating and recovering uPAR+ stem cells by inducing cell division, growth, and migration of tissue-resident uPAR+ and nestin+ stem cells as a result of activation of the matrix-cellular integrin pathway through in vitro culture.

[0056] The present invention also provides a method for inducing migration and growth within a provided wound repair substrate mimicking hydrogel during 3D culture by uPAR-plasmin-MMP activity as a result of stem cell activation and physiological response to stimulation, and then isolating and recovering cells from the hydrogel without exogenous tissue degrading enzymes based on the characteristics of uPAR expression and plasmin activity of stem cells that have migrated and grown within the hydrogel.

[0057] The present invention provides a common method for isolating tissue-resident uPAR+ and nestin+ stem cells from representative solid tissues such as adipose tissue, bone marrow, cardiac muscle, nerve, skeletal muscle, synovial membrane, etc. Tissue-resident uPAR+ and nestin+ stem cells have self-renewal, high in vitro growth, multilineage differentiation properties, and high tissue regeneration ability, and can be used as a regenerative medicine therapeutic agent.

[0058] The present invention will be described in more detail with reference to the following examples. It will be apparent to those skilled in the art that these examples are merely intended to more specifically illustrate the present invention, and that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0059] <Example 1> Preparation of wound repair matrix mimicking hydrogel Wound repair mimicking hydrogels are made from fibrinogen, collagen, gelatin, or a mixture of these polymers. Plasma-derived fibrinogen is dissolved in phosphate-buffered saline (PBS) containing 10 to 50 mM CaCl2 at a concentration of 2.5 to 10 mg / ml to make a fibrinogen solution. Thrombin is dissolved in PBS at a concentration of 1 to 10 units / ml to make a thrombin solution.

[0060] Dermis-derived collagen (Matrix BioScience, Germany) or gelatin (befMatrix Collagen, Nitta Gelatin, Japan) is dissolved in 0.1% (wt. / vol.) acetic acid to prepare a collagen solution with a concentration of 1.0 to 20.0 mg / ml. A 10X reconstitution buffer for preparing a neutral gelatin or collagen solution is prepared with 50 mM NaHCO3, 40 mM HEPES, and 0.01 N NaOH, and mixed with the collagen or gelatin solution at a volume ratio of 9:1 to prepare a neutral collagen or gelatin solution.

[0061] Wound repair substrate-mimicking hydrogels are produced by preparing fibrin hydrogel from a 0.25-2.5% fibrinogen solution and a 0.5-5 I.U. / mL thrombin solution, and mixing these with a 0.1-0.5% collagen or gelatin solution during the production of the fibrin hydrogel to produce fibrin / collagen or fibrin / gelatin mixed hydrogels.

[0062] Example 2: Monolayer (2-dimension, 2D) and 3-dimensional (3D) organ culture The organs used for the culture were adipose tissue (AT), bone marrow (BM), myocardium (Myocar), peripheral nerve (PN), skeletal muscle (SM), and synovium (Syn). Approval for the research using tissues from brain-dead subjects was obtained from the institutional ethics committee, and tissues donated by brain-dead subjects were used. Hematoma and fibrous tissues attached around the tissue were removed with scissors, and the donated tissue was cut into 0.2-2 mm pieces using a surgical scalpel. 3 The tissue slices were then cut into small pieces according to size, suspended in PBS, centrifuged at 1,000 rpm, and the supernatant was removed, and this washing process was repeated three times.

[0063] After washing, the tissue slices are suspended in culture medium and then cultured using two types of organ culture methods. The first method is defined as monolayer (2D) organ culture, in which tissue slices suspended in culture medium are seeded in a culture vessel and cultured on the surface of the vessel.

[0064] The second method was defined as 3D organ culture, in which tissue slices were invaginated into fibrin, fibrin / collagen, or fibrin / gelatin wound repair matrix-mimicking hydrogels and then cultured in a three-dimensional (3D) environment. In the 3D organ culture method, tissue slices were mixed with 0.25-2.5% fibrinogen solution, then mixed with an equal amount of 0.5-5 unit / mL thrombin solution in a 1:1 ratio, and the tissue slices were invaginated into the fibrin hydrogel after polymerization at 37°C for 1 hour. For fat, bone marrow, heart, and muscle, tissue slices were invaginated into the wound repair matrix-mimicking fibrin hydrogel with a final fibrinogen solution of 0.25-1.25% and thrombin solution of 0.25-2.5 unit / mL. Nerve and synovial tissue sections were invaginated into a wound repair matrix-mimicking fibrin / collagen or gelatin hydrogel consisting of 0.25-2.5% fibrinogen, 0.1-0.5% collagen or gelatin neutral solution, and 0.25-2.5 unit / mL thrombin solution.

[0065] The tissue slices mixed with the wound repair matrix mimicking hydrogel solution were transferred to a 100-150 mm culture vessel and then left in a 37°C incubator for 1 hour for polymerization, followed by conversion to a gel. The organ culture medium consisted of 45% v / v DMEM, 45% v / v Ham's F12, 10% fetal bovine serum (FBS, Invitrogen), 20 ng / ml EGF, 2 ng / ml bFGF, 10 ng / ml IGF, and 10 μg / ml gentamicin (Invitrogen). The culture medium was added in a volume equivalent to twice the gel volume, and after adding the culture medium, the culture vessel was placed on an orbital shaker and cultured for 14 days while stirring at 30 rpm. The culture medium was replaced twice a week.

[0066] To suppress hydrogel degradation, the PAI tranexamic acid or aminomethylbenzoic acid was added to the culture medium every day during the culture period at a concentration of 10 to 500 μg / mL. For adipose, bone marrow, heart, and skeletal muscle organ cultures, tranexamic acid or aminomethylbenzoic acid was added to the culture medium at a concentration of 100 to 250 μg / mL to suppress the degradation of the wound repair matrix mimicking hydrogel. For peripheral nerve and synovial membrane, tranexamic acid or aminomethylbenzoic acid was added to the culture medium at a concentration of 250 to 500 μg / mL.

[0067] <Example 3> Expression of pFAK, uPAR, nestin, and Ki-67 in organs by organ culture After 7 days of 2D and 3D organ culture, tissue sections of cultured adipose tissue, bone marrow, cardiac muscle, nerve, skeletal muscle, and synovial membrane were harvested from the culture vessel or hydrogel. After fixation for 2 hours in 4% neutral formalin solution, paraffin blocks were prepared. Sections 4 μm thick were cut from the paraffin blocks and immunochemical staining was performed.

[0068] To evaluate the activity of the integrin-FAK cell signaling pathway in the cultured tissue, expression was evaluated using primary antibodies such as anti-phospho FAK (3283, Cell Signaling Technology, USA; pFAK), anti-uPAR (MAB807, R&D Systems, USA), and Ki-67 (M7240, DAKO, USA). After reacting with the primary antibodies, the cells were washed three times with water and reacted with HRP-conjugated secondary antibodies (ImmPRESS One-Step Polymer Systems, Vector Laboratories, USA), and then colored using DAB as a substrate. After counterstaining with hematoxylin (H-3401-500, Vector Laboratories), the expression rate and the location of positive cells were evaluated.

[0069] In vitro organ culture activates the constituent cells in the tissue slice, and the activated cells proliferate after cell division, thereby replacing or regenerating damaged or lost cells, activating the tissue homeostasis mechanism. The integrin cell signaling pathway is one of the main mechanisms that induce tissue regeneration, and integrin cell signaling is activated by the binding of cell ligands, extracellular matrix, and receptors. In this example, the tissue slice was directly seeded in a culture vessel and then cultured on the surface of the culture vessel in a monolayer culture (2-dimension, 2D), and in a 3D (3-dimension, 3D) environment after invaginating into a hydrogel. The expression of pFAK, uPAR, and nestin, which are target factors activated as a result of integrin cell signaling, was analyzed in the constituent cells inside the tissue slice.

[0070] After 3D organ culture, the pFAK expression rate in tissue slices was confirmed to be significantly higher than that in 2D organ culture. Differences in pFAK expression rate were confirmed after organ culture according to tissue type, with pFAK expression being significantly higher in cardiac, nervous, and skeletal muscles after 3D organ culture (Figure 1A).

[0071] Expression of uPAR, a downstream target of the integrin signaling pathway, was also significantly elevated in the 3D organ culture environment, with high expression confirmed in cardiac, nervous, and skeletal muscles, in a similar manner to that of pFAK (Figure 1B).

[0072] Integrin signaling can induce cell division and growth of constituent cells within the organ. Cell regeneration and growth were evaluated through the expression of nestin and Ki-67, which are characteristically expressed in cells undergoing regeneration or cell division. After 3D organ culture, nestin expression in constituent cells in tissue slices was significantly higher than that in tissues after 2D organ culture (Figure 1C). Ki-67 expression, a cell division marker, was also significantly higher in tissue slices after 3D organ culture (Figure 1D).

[0073] In vitro organ culture can induce cell division and growth of cells that make up an organ, and integrin signaling can be maximized through the 3D culture environment using ligands that can bind to integrins, thereby enhancing the expression of downstream target factors pFAK and uPAR. As a result, it was confirmed that 3D organ culture effectively induces cell division and growth of cells that make up the tissue slices more effectively than 2D organ culture.

[0074] 3D physical stimulation of wound repair matrix-mimicking hydrogels can provide a way to activate signaling pathways in constituent cells within organs, meaningfully increasing cell division and growth.

[0075] Example 4: Location and characteristics of pFAK+, uPAR+, and nestin+ cells in tissues after 3D organ culture Cultured myocardial tissue slices were harvested from the hydrogel before 3D organ culture (0 d) or after 3, 5, 7, and 14 days of culture. After fixation for 2 hours in 4% neutral formalin solution, paraffin blocks were prepared. Sections with a thickness of 4 μm were cut from the paraffin blocks and immunochemical staining was performed. After culture, expression was evaluated using primary antibodies such as anti-phospho FAK (3283, Cell Signaling Technology, USA; pFAK), anti-uPAR (MAB807, R&D Systems, USA), nestin (MAB5326, Millipore, USA), and Ki-67 (M7240, DAKO, USA) to evaluate the activity of the integrin-FAK cell signaling pathway in the tissue. After reacting with the primary antibody, the cells were washed three times with water and then reacted with an HRP-conjugated secondary antibody (ImmPRESS One-Step Polymer Systems, Vector Laboratories, USA). Color was developed using DAB as a substrate, and the cells were counter-stained with hematoxylin (H-3401-500, Vector Laboratories) to evaluate the expression rate and the location of positive cells.

[0076] After invaginating myocardium into a wound repair matrix mimicking hydrogel and carrying out 3D organ culture, the characteristics of pFAK+, uPAR+ and nestin+ cells in myocardium were investigated. Before culture, the expression rates of pFAK, uPAR and nestin were less than 6%. However, after 3D organ culture, the number of pFAK, uPAR and nestin positive cells increased significantly in proportion to the culture period. After 2 weeks of culture, the expression rates of pFAK, uPAR and nestin in tissue sections were more than 30% (Figures 2 and 3). pFAK, uPAR and nestin were expressed by cells in the epilepsy between mature somatic cells, and pFAK, uPAR and nestin were expressed in perivascular cells surrounding capillaries rather than in endothelial cells of capillaries.

[0077] Ki-67, a cell division marker, is not expressed in mature somatic cells and was expressed at less than 1% before culture. However, after in vitro 3D organ culture, the Ki-67 expression rate increased in proportion to the culture period, and after 2 weeks of organ culture, the intramyocardial Ki-67 expression rate was 45.7%, confirming that 3D organ culture can induce the division and growth of intramyocardial cells, and Ki-67 was also mainly expressed in perivascular cells (Figure 4).

[0078] This example confirmed that intramyocardial cell division and growth could be induced through 3D wound repair matrix-mimicking hydrogel, and that the division and growth of pFAK+, uPAR+, and nestin+ perivascular cells could be induced through organ culture.

[0079] Example 5: Induction of migration and growth of tissue uPAR+ and nestin+ cells into a wound repair matrix-mimicking hydrogel through 3D organ culture Tissue slices of fat, bone marrow, cardiac muscle, nerve, skeletal muscle, and synovial membrane were invaginated into fibrin or fibrin / collagen hydrogels, and then 3D organ culture was performed. Organ culture medium was added in an amount twice the volume of the hydrogel, and the culture vessel was placed on an orbital shaker and cultured for 14 days with stirring at 30 rpm. PAI was added to the culture medium to suppress hydrogel degradation. During in vitro culture, 5 μM bromodeoxyuridine (B5002, Sigma-Aldrich, USA) was added to the culture medium every day for 7 days to label cells that had undergone cell division. The culture medium was replaced twice a week and cultured for 2 weeks.

[0080] After 3, 7, and 14 days of 3D organ culture, the cultured tissues and hydrogels were simultaneously harvested. After fixation for 2 hours in 4% neutral formalin solution, paraffin blocks were prepared. Sections with a thickness of 4 μm were cut from the paraffin blocks and immunochemical staining was performed.

[0081] To evaluate the expression of uPAR, nestin, and BrdU in cells migrated and grown in the hydrogel, anti-uPAR (MAB807, R&D Systems, USA), -nestin (MAB5326, Millipore, USA), and -BrdU (347580, BD Biosciences, USA) primary antibodies were used. After reacting with the primary antibodies, the cells were washed three times with water and reacted with HRP-conjugated secondary antibodies (ImmPRESS One-Step Polymer Systems, Vector Laboratories, USA), and then colored using DAB as a substrate. After counterstaining with hematoxylin (H-3401-500, Vector Laboratories), the expression rates were evaluated.

[0082] Wound repair matrix mimicking hydrogel plays a role of an extracellular matrix that can support cell migration and growth, and can achieve this by providing cell adhesion factors essential for cell migration, such as fibronectin, collagen, and fibrin. The 3D wound repair matrix hydrogel can activate the integrin-pFAK cell signaling pathway through receptor-ligand binding, inducing cell division and growth of uPAR+ and nestin+ cells in the organ, and recruiting uPAR+ and nestin+ cells to the wound repair matrix mimicking hydrogel, inducing their migration and growth.

[0083] It was confirmed that the number of cells migrating and growing within the hydrogel increased in proportion to the duration of 3D myocardial organ culture (Figure 5). It was confirmed that the cells migrating and growing within the hydrogel bound to the hydrogel in a spindle shape and had expanded cytoplasm.

[0084] It was confirmed that the cells that migrated and grew from the myocardium into the hydrogel had uPAR and nestin expression rates of 88.5% and 95.4% or more, respectively, and showed the same uPAR+ and nestin+ characteristics as the cells that divided and grew after organ culture in the tissue. More than 89.4% of the cells that migrated into the hydrogel were BrdU positive, which means that they were cells that had uptaken the added BrdU during organ culture and synthesized DNA, proving that most of the cells in the hydrogel were cells that had migrated after the tissue cells divided and grew after organ culture. Before organ culture, no BrdU+ cells were detected in the myocardium, and uPAR+ and nestin+ cells were less than 2%.

[0085] After two weeks of 3D organ culture of tissue slices from adipose tissue, bone marrow, cardiac muscle, nerve, skeletal muscle, and synovial membrane, approximately 90% of the cells in the wound repair substrate-mimicking hydrogel were uPAR positive and over 90% were BrdU positive, confirming that the tissue-resident cells had undergone cell division and growth through organ culture and had migrated into the hydrogel, with no significant differences observed depending on the tissue (Figure 6).

[0086] <Example 6> Isolation of cells that migrated and grew in the hydrogel after 3D organ culture Tissue slices of adipose tissue, bone marrow tissue, cardiac muscle tissue, nerve tissue, skeletal muscle tissue, and synovial membrane tissue were invaginated into fibrin or fibrin / collagen hydrogels, and then 3D organ culture was performed. Organ culture medium was added in an amount twice the volume of the hydrogel, and the culture vessel was placed on an orbital shaker and cultured for 14 days while stirring at 30 rpm. To suppress hydrogel degradation, PAI was added daily during the culture period, and the PA activity of cells that migrated and grew in the hydrogel suppressed the degradation of the wound repair matrix-mimicking hydrogel.

[0087] After 3D organ culture, the uPAR mRNA and plasmin activity of the collected tissue sections were evaluated. Tissue lysate was prepared from 10 mg of tissue section, and plasmin activity was analyzed using a Plasmin Activity Assay Kit (ab204728, Abcam). Fluorescent substrate was added to the tissue lysate, and the tissue was incubated at 37°C for 20 minutes, after which the fluorescence intensity was measured to determine plasmin activity. Plasmin activity was calculated using the following formula (△RFU360 / 450nm=(RFU2-RFU 2BG )-(RFU1-RFU 1BG The expression rate of uPAR mRNA in tissue sections was calculated by extracting total RNA from 10 mg tissue sections, generating cDNA through reverse transcriptase reaction, and analyzing it through real time qPCR.

[0088] After 14 days of 3D organ culture, the culture medium was removed. After adding DMEM, the cells were washed while stirring at 30 rpm for 30 minutes, and the washing solution was removed. This washing process was repeated three times. After washing, the cells that had migrated and grown in the hydrogel were separated and collected by the following two methods. In the first method, 1,000 unit / mL urokinase was added to the culture medium, and the hydrogel was decomposed for 2 hours after adding it to the culture vessel. The cells and tissue slices released from the decomposed hydrogel were transferred to a tube, centrifuged at 3,000 rpm for 10 minutes, the supernatant was removed, and the collected cells and tissue slice pellets were suspended in culture medium.

[0089] The PAI withdrawal method allows cells to be separated and collected from the hydrogel. After 14 days of organ culture, the culture medium was removed, and the hydrogel was washed three times with DMEM to remove the PAI remaining in the culture medium and in the hydrogel. Fresh culture medium was added, and the cells were cultured for 1 hour while stirring at 30 rpm, without adding PAI in the culture medium. The cells separated and released from the hydrogel were collected using a transfer pipette, transferred to tubes, and centrifuged at 3,000 rpm for 10 minutes, after which the supernatant was removed, and the collected cells and tissue slice pellets were suspended in culture medium.

[0090] The cells separated and recovered from the hydrogel were suspended in culture medium, and the total cell number was calculated using a hemocytometer. The recovered cells were analyzed for uPAR expression rate using flow cytometry.

[0091] This embodiment aims to provide a method for selectively isolating and recovering uPAR+ / nestin+ cells by inducing the growth of uPAR+ / nestin+ cells after 3D organ culture and cell division within the tissue to migrate and grow within the hydrogel, and then decomposing the hydrogel using the plasmin activity of the uPAR+ / nestin+ cells themselves without the use of exogenous proteases, thereby selectively isolating and recovering the uPAR+ / nestin+ cells.

[0092] The expression of uPAR mRNA and plasmin activity in tissue slices were evaluated before and after organ culture. Even before in vitro culture, the expression of uPAR mRNA and plasmin activity varied depending on the organ, with the expression of uPAR mRNA and plasmin activity being higher in cardiac muscle, nerve, and skeletal muscle than in other tissues (Figure 7). After organ culture, the expression of uPAR mRNA and plasmin activity significantly increased in all tissue slices evaluated compared to before culture. In particular, the highest levels of mRNA expression and plasmin activity were measured in cardiac muscle, nerve, and skeletal muscle. As plasmin activity is proportional to uPAR expression, it is possible to induce an increase in uPAR expression in tissues through organ culture, providing a method to increase plasmin activity in tissues through an increase in uPAR after organ culture.

[0093] During 3D organ culture, Plasminogen Activator Inhibitor (PAI) is added to the culture medium, and the added PAI can suppress hydrogel degradation by regulating excessive plasmin and protease activity of organs and cells. After inducing the migration and growth of cells that have divided and grown in tissues through organ culture into the hydrogel, the PAI can be removed from the culture medium and hydrogel through a water washing and cleaning process to induce hydrogel degradation only with the plasmin and protease activity in the organ and cells, and as a result, the cells that have migrated and grown into the hydrogel are released, and the released cells can be separated and collected.

[0094] For PAI withdrawal, the culture medium containing PAI was removed, and the PAI remaining in the hydrogel and organ was removed through the washing process by washing three times with DMEM, and the culture medium not containing PAI was added to twice the volume of the hydrogel and cultured. After 30 minutes of culture, the hydrogel began to decompose as a result of PAI withdrawal, and the cells that migrated and grew in the hydrogel as the hydrogel was decomposed were released from the hydrogel and formed cell-cell aggregates, and after 2 hours of culture, the hydrogel around the tissue slice and the cells that migrated and grew were completely decomposed, resulting in the dissociation of the cells in the hydrogel (Figure 8). In this example, the wound repair matrix mimicking hydrogel disappeared due to the plasmin and protease activity in the cells only due to PAI withdrawal, and the cells in the hydrogel were released into the culture medium, and as a result, the cells released into the culture medium could be separated and collected in an aggregated form with their cytoplasm contracted.

[0095] During organ culture, hydrogel degradation can be suppressed by adding PAI, and cells in the hydrogel show a spindle shape due to cytoplasmic expansion. However, with PAI withdrawal in the culture medium, the hydrogel around the cells begins to degrade, resulting in the contraction of the cytoplasm and the formation of cell-cell junctions, and the aggregated cells are released into the culture medium (Figure 9).

[0096] Using histological methods, it was confirmed that before PAI withdrawal, cells in the hydrogel had expanded cytoplasm, but after PAI withdrawal, as the hydrogel began to decompose, the hydrogel to which the cells could attach disappeared, and as a result of cell-cell junctions, the cells aggregated and were released from the hydrogel (Figure 10). All of the cells released and aggregated from the hydrogel showed the characteristic of expressing uPAR, and the higher the uPAR expression, the higher the plasmin activity, confirming the basis for being able to separate uPAR+ cells in the hydrogel with PAI withdrawal alone (Figure 10).

[0097] The cells that migrated and grew in the hydrogel can be isolated and collected after PAI withdrawal or urokinase treatment. The present invention compared the usefulness of a method for isolating cells that migrated and grew in the hydrogel with PAI withdrawal without urokinase treatment.

[0098] Through the examples, it was confirmed that the PAI withdrawal method could separate and recover the same number of cells as those recovered through urokinase treatment (Figure 11A). After 2 weeks of organ culture per 100 mg of tissue, more than 2 million cells could be separated and recovered, which was lower than the number of cells recovered after urokinase treatment, but the difference was not significant.

[0099] However, the uPAR expression rate of cells collected after PAI withdrawal was significantly higher than that of cells collected after urokinase treatment (Figure 11B). In particular, the yield of selectively isolating uPAR+ cells was high in tissues with high uPAR expression rate and plasmin activity. This demonstrated that uPAR+ cells have high hydrogel decomposition activity, and as a result, PAI removal and washing are methods for selectively isolating uPAR+ cells with high efficiency.

[0100] <Example 7> Repeated organ culture and cell separation of tissue slices harvested after 3D organ culture Tissue slices of fat, bone marrow, cardiac muscle, nerve, skeletal muscle, and synovial membrane were invaginated into fibrin or fibrin / collagen hydrogels, and then 3D organ culture was performed. Organ culture medium was added in an amount twice the volume of the hydrogel, and the culture vessel was placed on an orbital shaker and cultured for 14 days with stirring at 30 rpm. To suppress hydrogel degradation, PAI was added daily during the culture period, and the plasmin activity of cells that migrated and grew in the hydrogel suppressed the degradation of the wound repair matrix-mimicking hydrogel.

[0101] After 14 days of 3D organ culture, the culture medium was removed. After adding DMEM, the cells were washed with stirring at 30 rpm for 30 minutes, and the washing solution was removed. The washing process was repeated three times to remove the PAI remaining in the culture medium and hydrogel. After adding fresh culture medium, the cells were cultured for 2 hours with stirring at 30 rpm, and no PAI was added to the culture medium. The cells and tissue slices separated and released from the hydrogel were collected using a transfer pipette, transferred to tubes, and centrifuged at 3,000 rpm for 10 minutes, and the supernatant was removed. Since the tissue slices precipitate faster than the cells due to their high specific gravity, the cells and tissue slice pellets were dispersed in DMEM, and then left for 30 seconds to induce precipitation of the tissue slices, and only the supernatant was transferred to a new tube. This process was repeated three times to separate the collected cells and tissue slices.

[0102] The tissue slices separated from the hydrogel by the PAI withdrawal method were again invaginated into the hydrogel, and then 3D organ culture was performed by the method described in Example 2. After two weeks, the cells that had migrated and grown in the hydrogel and the tissue slices were isolated and collected by the method described in Example 7. After the PAI withdrawal, the collected tissue slices were subjected to three more consecutive 3D organ culture processes to induce the migration and growth of the cells in the tissue, and the cells in the hydrogel were isolated and collected, and the total number of cells isolated and collected was measured using a hemocytometer.

[0103] This example provides a method for recovering tissue slices with preserved structure after PAI withdrawal, repeatedly culturing the recovered tissue slices, and then isolating and recovering the cells that have grown after moving into the hydrogel after organ culture. Treatment with an exogenous protease such as Urokinase degrades the tissue slices that are invaginated together with the hydrogel, resulting in the loss of the structural and functional microenvironment of the tissue slices.

[0104] In this example, the cells and tissue slices that migrated after growing in the tissue were isolated and collected without exogenous protease treatment through PAI withdrawal, and the collected tissue slices were also repeatedly cultured. After the organ culture, three consecutive organ cultures were performed using the collected tissue slices, and it was confirmed that cells could be collected at a yield similar to that of the first organ culture (FIG. 12). It was confirmed that the PAI withdrawal method is a method that can preserve the structural and functional microenvironment of the tissue slices that migrated and grew in the hydrogel after 3D organ culture, and that it is a method for isolating endogenous cells in the tissue efficiently and stably through repeated organ culture.

[0105] Example 8: Culture of cells separated from hydrogel after PAI withdrawal and urokinase treatment Tissue slices of adipose, bone marrow, cardiac muscle, nerve, skeletal muscle, and synovial membrane were invaginated into fibrin or fibrin / collagen hydrogels, and then 3D organ culture was performed. Organ culture medium was added in an amount twice the volume of the hydrogel, and the culture vessel was placed on an orbital shaker and cultured for 14 days with agitation at 30 rpm. To inhibit hydrogel degradation, tranexamic acid, a PAI, was added to the culture medium every day.

[0106] After 14 days of 3D organ culture, the culture medium was removed. After adding DMEM, the cells were washed for 30 minutes while stirring at 30 rpm, and this washing process was repeated three times. After washing, the cells that had migrated and grown in the hydrogel were separated and then collected using the PAI withdrawal or urokinase method presented in Example 6. After 14 days of organ culture, the culture medium was removed, and the PAI remaining in the culture medium and hydrogel was removed by washing three times with DMEM. After adding fresh culture medium, the cells were cultured for 1 hour while stirring at 30 rpm, and no PAI was added to the culture medium. The cells separated and released from the hydrogel were collected using a transfer pipette, transferred to tubes, and centrifuged at 3,000 rpm for 10 minutes, the supernatant was removed, and the collected cells and tissue slice pellets were suspended in culture medium.

[0107] For urokinase treatment, after washing three times, 1,000 unit / mL urokinase was added to the culture medium and the hydrogel was decomposed for 2 hours. The cells and tissue fragments released from the decomposed hydrogel were collected and transferred to a tube, and then centrifuged at 3,000 rpm for 10 minutes. The supernatant was removed, and the collected pellets of cells and tissue fragments were suspended in culture medium.

[0108] The cells isolated and recovered from the hydrogel were cultured in polystyrene culture vessels. 2 After seeding 5,000 cells per well, culture medium was added. The attachment and growth characteristics of cells detached and harvested from the hydrogel in a monolayer culture environment were evaluated using a microscope.

[0109] After PAI withdrawal, it was confirmed that the cells were released from the hydrogel and formed cell aggregates (Figure 13A). The collected cells were suspended in culture medium and formed small circular aggregates (Figure 13B), which were then seeded on a PS culture vessel and cultured in a monolayer environment. The collected cells in the intercellular aggregate state attached to the culture vessel 30 minutes after seeding (Figure 13C), and 1 hour after seeding, the cytoplasm of the seeded cells expanded (Figure 13D). After 2 hours of culture, the cells were stably attached, and it was confirmed that the cells around the cell aggregates grew (Figure 13E).

[0110] After organ culture of various tissue slices such as fat, bone marrow, cardiac muscle, nerve, skeletal muscle, synovial membrane, etc., cells can be stably isolated and recovered through PAI withdrawal. The recovered cells are mixed with culture medium and seeded in a culture vessel. It was confirmed that all the recovered cells attached to the culture vessel within 30 minutes and the cytoplasm stably expanded, with no significant difference depending on the tissue (Figure 14).

[0111] Cells seeded and cultured in a monolayer environment showed a high growth rate, and after two days of culture, they grew and expanded around the seeded cells. It was confirmed that cells isolated and recovered through PAI withdrawal maintained a high growth rate even when cultured in a monolayer environment (Figure 15).

[0112] Example 9: Characterization of the immunophenotype of isolated cells after PAI withdrawal Tissue slices of fat, bone marrow, cardiac muscle, nerve, skeletal muscle, and synovial membrane were invaginated into fibrin or fibrin / collagen hydrogels, and then 3D organ culture was performed. Organ culture medium was added in an amount twice the volume of the hydrogel, and the culture vessel was placed on an orbital shaker and cultured for 14 days while stirring at 30 rpm. To suppress hydrogel degradation, tranexamic acid, a PAI, was added to the culture medium at a concentration of 100 mg / mL.

[0113] After 14 days of 3D organ culture, the culture medium was removed. After adding DMEM, the cells were washed for 30 minutes while stirring at 30 rpm, and this washing process was repeated three times. After washing, the cells that had migrated and grown in the hydrogel were separated and then collected using the PAI withdrawal method. After 14 days of organ culture, the culture medium was removed, and the PAI remaining in the culture medium and hydrogel was removed by washing three times with DMEM. After adding fresh culture medium, the cells were cultured for 1 hour while stirring at 30 rpm, and no PAI was added to the culture medium. The cells that were separated and released from the hydrogel were collected using a transfer pipette, transferred to tubes, and centrifuged at 3,000 rpm for 10 minutes, the supernatant was removed, and the collected cells and tissue slice pellets were suspended in culture medium.

[0114] After PAI withdrawal method or urokinase treatment, the isolated and collected cells were suspended in PBS and analyzed for immunophenotypic characteristics using flow cytometry. The expression rates of uPAR and nestin were evaluated along with the mesenchymal stem cell (MSC) markers CD29, CD44, CD105, and CD140b. The expression rates of CD34 and CD45 as hematopoietic cell markers and CD31 as vascular endothelial cell marker were evaluated.

[0115] As a result of urokinase treatment or PAI withdrawal, cells that migrated and grew out of the hydrogel could be isolated and then recovered. Regardless of the cell recovery method, cells that migrated and grew into the hydrogel from all cultured tissues showed the same immunophenotypic characteristics as mesenchymal stem cells, with CD29, CD73, CD105, and CD140b expression rates of 90% or more (Figure 16A). There was no difference in the expression rates of mesenchymal stem cell markers in cells isolated and recovered from the hydrogel, depending on the isolation method.

[0116] However, in this example, after organ culture of all tissue slices, the expression rates of uPAR and nestin in cells isolated and recovered by PAI withdrawal treatment were over 90%. Meanwhile, in the case of cells recovered after urokinase treatment, the expression rates were in the range of 30-67%. It was confirmed that the high plasmin activity of uPAR+ and nestin+ cells in the hydrogel allowed them to be selectively isolated and recovered through PAI withdrawal. Meanwhile, it was confirmed that urokinase treatment allowed the recovery of a mixture of cells with various immunophenotypic characteristics in the hydrogel (Figure 16B).

[0117] Hematopoietic cells and vascular endothelial cells present in tissues are mixed during the process of isolating cells from tissues, and the degree of mixing of these cells can be confirmed by checking CD31+, CD34+, and CD45+ cells. In the case of cells separated and recovered from hydrogels using the PAI withdrawal method, the expression rates of CD31, CD34, and CD45 were less than 1%, but the cells separated and recovered after Urokinase treatment were 2.8-7.0%, confirming that there was a high degree of mixing of hematopoietic cells and vascular endothelial cells (Figure 17).

[0118] Through this example, it was confirmed that this is a method for selectively isolating and recovering uPAR+ and nestin+ cells that migrated from the tissue into the hydrogel through PAI withdrawal, and at the same time, mixing of hematopoietic cells and vascular endothelial cells was minimized during the isolation process.

[0119] <Example 10> Self-renewal ability of uPAR+ / nestin+ cells separated and collected after PAI withdrawal Tissue slices of adipose tissue, bone marrow tissue, cardiac muscle tissue, nerve tissue, skeletal muscle tissue, and synovial membrane were invaginated into fibrin or fibrin / collagen hydrogels, and then 3D organ culture was performed for 2 weeks. After that, the cells that had migrated and grown within the hydrogels were isolated and collected using the PAI withdrawal and urokinase methods described in Example 6.

[0120] The cells isolated and recovered from the hydrogel were transferred to tubes and centrifuged at 3,000 rpm for 10 minutes, after which the supernatant was removed. The cell pellet was suspended in culture medium and adjusted to a cell density of 1.0E+06 per mL. The self-replication ability of the cells isolated and recovered from the hydrogel was evaluated through a colony formation assay (CFU). 6,000 cells were seeded in a 100 mm culture vessel and cultured for 2 weeks. After culture, the cells were stained with 1% crystal violet. After washing, the number of colonies with a diameter of 2 mm or more was counted.

[0121] Although the CFU formation frequency varied depending on the tissue type, in all tissue sections, the cells isolated and recovered from hydrogels using PAI withdrawal were significantly higher than the cells recovered after urokinase treatment (Figure 18). In particular, cells derived from cardiac, nervous, and skeletal muscles showed higher colony formation ability than cells derived from other tissues. In the case of cells isolated after urokinase treatment, the CFU frequency was 3.1-8.4%, while the cells isolated using the PAI withdrawal method had a higher CFU frequency of 8.8-37.9%.

[0122] Self-renewal ability is one of the main characteristics of stem cells. 3D organ culture can activate stem cells in tissues and induce migration and growth into the hydrogel. Stem cells with high self-renewal ability can be selectively isolated and recovered from the hydrogel using the PAI withdrawal method. This result proves that uPAR+ / nestin+ cells with high plasmin activity are high-potency stem cells (Figure 18).

[0123] Example 11: In vitro growth ability of isolated cells after PAI withdrawal Tissue slices of adipose tissue, bone marrow tissue, cardiac muscle tissue, nerve tissue, skeletal muscle tissue, and synovial membrane were invaginated into fibrin or fibrin / collagen hydrogels, and then 3D organ culture was performed for 2 weeks. After that, the cells that had migrated and grown within the hydrogels were isolated and collected using the PAI withdrawal and urokinase methods described in Example 6.

[0124] The cells isolated and recovered from the hydrogel were transferred to tubes and centrifuged at 3,000 rpm for 10 minutes, after which the supernatant was removed. The cell pellet was suspended in culture medium and adjusted to a cell density of 1.0E+06 per mL. To verify the in vitro growth potential of the cells isolated and recovered from the hydrogel, the population doubling time (PDT) and population doubling level (PDL) were analyzed and evaluated. 2 After seeding 3,000 cells per well, they were cultured for 7 days, treated with trypsin / EDTA, and the cells were harvested and the total cell count was calculated using a hemocytometer. The seeded cell count, total cell count recovered, and PDT and PDL were calculated and compared throughout the culture period. PDT was calculated using the following formula: PDT=[(culture time) / ((logN2-logN1) / log2)], where N1 is the number of cells at seeding and N2 is the number of cells recovered after culture. PDL was calculated using the following formula: PDL=[PDL0+3.322(logN2-logN1)], where N1 is the number of cells at seeding and N2 is the number of cells recovered after cell culture.

[0125] The higher the in vitro growth potential, the higher the expression rate of Ki-67, a cell cycle marker, in the cultured cells. In vitro cell growth potential was analyzed by analyzing anti-Ki67 expression rate using flow cytometry.

[0126] After urokinase or PAI withdrawal treatment, the cells that migrated and grew from the hydrogel were isolated, and the in vitro growth ability of the recovered cells was compared. The PDT was significantly lower and the PDL was significantly higher for the cells isolated and recovered through PAI withdrawal compared to the cells secured after urokinase treatment. Following the same trend as the self-renewal ability, it was confirmed that the cells isolated and recovered through PAI withdrawal had excellent in vitro growth ability with low PDT and high PDL (Figure 19).

[0127] To verify the high growth potential of the cells isolated and recovered from the hydrogel after PAI withdrawal treatment, the Ki-67 expression rate was analyzed by flow cytometry. The Ki-67 marker indicates cells in the cell cycle, so a higher Ki-67 expression rate is an indicator of higher cell growth potential. The cells isolated through PAI withdrawal showed a high Ki-67 expression rate of 58.5-75.4%, which was significantly higher than the 33.6-48.7% in the cells isolated after urokinase treatment (Figure 20).

[0128] From the above results, it was confirmed that PAI withdrawal is a method for selectively isolating cells with high in vitro growth potential, and this result supports the idea that uPAR+ / nestin+ cells with high plasmin activity are cells with high growth potential.

[0129] <Example 12> Differentiation potential of isolated cells after PAI withdrawal Tissue slices of adipose tissue, bone marrow tissue, cardiac muscle tissue, nerve tissue, skeletal muscle tissue, and synovial membrane were invaginated into fibrin or fibrin / collagen hydrogels, and then 3D organ culture was performed for 2 weeks. After that, the cells that had migrated and grown within the hydrogels were isolated and collected using the PAI withdrawal and urokinase methods described in Example 6.

[0130] The cells separated and collected from the hydrogel were transferred to tubes, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was removed. The cell precipitate was suspended in culture medium and adjusted to a cell density of 1.0E+06 per mL. The differentiation ability of the cells in the hydrogel into adipocytes and osteoblasts was evaluated. 2.0E+05 cells were seeded in a 24-well culture vessel, and then a differentiation medium containing 90% DMEM, 10% CS, 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), 80 μM indomethacin (Sigma), 1 μM DEX, and 5 μg / mL insulin was added, and the cells were cultured for 14 days. The degree of differentiation of the stem cells into adipocytes was evaluated by staining with 0.5% Oil Red O (Sigma) solution, which is an indicator of fat accumulation in the cytoplasm, at room temperature for 1 hour, and the triglyceride content in the cytoplasm was evaluated using the Triglyceride-Glo kit (Promega) after 2 weeks of differentiation induction. To evaluate the differentiation potential into osteoblasts, 2.0E+05 cells were seeded in a 24-well culture vessel, and α-MEM containing 1μM DEX, 50μM Ascorbic Acid, 10mM β-glycerol phosphate, and 10% calf serum was added and cultured for 2 weeks to induce differentiation. The presence or absence of differentiation into osteoblasts was determined after 2 weeks of differentiation induction by staining with Alizarin Red (Sigma), and the degree of mineral deposition was measured by measuring the absorbance at 520nm and comparative analysis of optical density.

[0131] In Figure 21, after inducing differentiation of cells isolated and recovered from bone marrow into osteoblasts (left) and adipocytes (right), calcium phosphate crytal accumulation and deposition can be confirmed through alizarin red staining, and in cells induced to differentiate into adipocytes, fat accumulation in the cytoplasm can be confirmed through oil red O staining. It was confirmed that all cells derived from any tissue section had the ability to differentiate into osteoblasts and adipocytes.

[0132] After dissolving the deposited or accumulated mineral cryostals and fat vacuoles, the differentiation ability into osteoblasts and adipocytes was compared and evaluated through the measured OD value. It was confirmed that the differentiation ability into osteoblasts and adipocytes of the cells isolated and recovered from hydrogels using PAI withdrawal in all tissue sections was significantly higher than that of the cells isolated and recovered after urokinase treatment (Figure 22). The differentiation into osteoblasts was high in cells derived from bone marrow, cardiac muscle, and skeletal muscle, while the differentiation into adipocytes was high in fat, skeletal muscle, and synovial membrane. However, as it was confirmed that the differentiation ability into osteoblasts and adipocytes was significantly higher in the cells isolated and recovered from hydrogels using the PAI withdrawal method regardless of the tissue section origin, it was confirmed that uPAR+ / nestin+ cells with high plasmin activity were stem cells with high multi-differentiation ability.

[0133] Example 13: Induction of tissue regeneration by isolated cells after PAI withdrawal Tissue slices of adipose tissue, bone marrow tissue, cardiac muscle tissue, nerve tissue, skeletal muscle tissue, and synovial membrane were invaginated into fibrin or fibrin / collagen hydrogels, and then 3D organ culture was performed for 2 weeks. After that, the cells that had migrated and grown within the hydrogels were isolated and collected using the PAI withdrawal and urokinase methods described in Example 6.

[0134] The cells separated and collected from the hydrogel were transferred to tubes, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was removed. The cell pellet was suspended in culture medium and adjusted to a cell density of 1.0E+06 per mL. 5.0E+06 cells were seeded in a T175 flask and cultured for 3 days, after which 1 mL of TRIzol was added, and the cell lysate was collected and frozen at -20°C until total RNA was isolated. Total RNA was extracted and purified using the PureLink RNA kit (Thermo Fisher), and cDNA was synthesized using reverse transcriptase. The mRNA expression of basic FGF (bFGF), HGF, IGF, and SDF-1, which play a major role in tissue protection and regeneration, was calculated and compared with that of cord blood-derived mesenchymal stem cells (CB-MSCs) by expression rate calculation. The target mRNA gene was amplified through real-time gene amplification using a target mRNA specific primer and a SYBR Green Real-Time PCR kit, and the mRNA expression rate was evaluated by the 2-ΔΔCt method compared to cord blood-derived mesenchymal stem cells (CB-MSCs).

[0135] After urokinase or PAI withdrawal treatment, the cells that migrated and grew from the hydrogel were isolated, and the tissue regeneration induction ability of the collected cells was compared through the expression of related mRNA. Gene expression inducing tissue regeneration differed depending on the tissue of origin, and these gene expressions were high in cells derived from cardiac muscle, nerve, and skeletal muscle, and HGF and SDF-1 mRNA were high in cardiac muscle-derived cells, while IGF mRNA expression was high in nerve-derived cells (Figure 23). Expression of all tissue regeneration genes was significantly higher in cells isolated and collected using PAI withdrawal compared to cells isolated and collected after urokinase treatment. These results confirm that uPAR expression is proportionally related to high tissue regeneration regulating gene expression.

[0136] Example 14: Plasmin activity and nestin expression rate of uPAR+ cells After invaginating tissue slices of fat, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial membrane into fibrin or fibrin / collagen hydrogel, 3D organ culture was performed for 2 weeks, and then cells that migrated and grew in the hydrogel were isolated and collected by the urokinase method described in Example 6. Then, uPAR+ (UK+uPAR+) and uPAR- (UK+uPAR-) cells were isolated using FACSAria (BD Bioscience) and cultured in a monolayer environment for 7 days, and plasmin activity and nestin expression rate were evaluated. Since the cells isolated and collected from the hydrogel using the PAI withdrawal method had a uPAR expression rate of 90% or more, secondary uPAR+ cells were not isolated. Plasmin activity was performed according to the method described in Example 6, and nestin expression rate was evaluated using flow cytometry.

[0137] Significantly higher plasmin activity was confirmed in uPAR+ cells (Figure 24, left). UK+uPAR+ cells isolated and cultured after urokinase treatment and cells isolated and cultured after PAI withdrawal showed significantly higher plasmin activity than UK+uPAR- cells. Plasmin activity of cells varied depending on the tissue of origin, being high in cardiac, nervous and skeletal muscles, indicating that plasmin activity of tissue-resident cells is determined by the tissue of origin.

[0138] These results confirmed a close correlation between plasmin activity and nestin expression in uPAR+ cells.

[0139] Example 15: Self-renewal and in vitro growth potential of uPAR+ cells After invaginating tissue slices of fat, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial membrane into fibrin or fibrin / collagen hydrogel, 3D organ culture was performed for 2 weeks, and then cells that migrated and grew in the hydrogel were isolated and collected by the urokinase method described in Example 6. Then, uPAR+ (UK+uPAR+) and uPAR- (UK+uPAR-) cells were isolated using FACSAria (BD Bioscience) and cultured in a monolayer environment for 7 days, and their self-renewal and growth ability were evaluated. Since the cells isolated and collected from the hydrogel using the PAI withdrawal method had a uPAR expression rate of 90% or more, secondary uPAR+ cells were not isolated. Self-renewal ability was performed according to the method described in Example 10, and growth ability was evaluated by flow cytometry using Ki-67 expression rate.

[0140] A significantly higher self-renewal ability was confirmed in uPAR+ cells. The CFU frequency was 12.5-37.5% in UK+uPAR+ cells and PAI withdrawal cells, but was 3.1-8.8% in UK+uPAR- cells, confirming that uPAR+ cells had a significantly higher self-renewal ability (Figure 25, left).

[0141] Self-renewal ability and in vitro growth ability are closely related, and it was confirmed that uPAR+ cells had a Ki-67 expression rate of 40.8-82.4%, which was significantly higher than the Ki-67 expression rate of 28.1-45.7% in uPAR- cells (Figure 25, right). uPAR+ cells derived from cardiac, nervous, and skeletal muscles had higher CFU formation ability and Ki-67 expression rates compared to tissues of other origins.

[0142] <Example 16> Anti-inflammatory activity of uPAR+ cells After invaginating tissue slices of fat, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial membrane into fibrin or fibrin / collagen hydrogel, 3D organ culture was performed for 2 weeks, and then cells that migrated and grew in the hydrogel were isolated and collected using the urokinase method described in Example 6. Then, uPAR+ (UK+uPAR+) and uPAR- (UK+uPAR-) cells were isolated using FACSAria (BD Bioscience) and cultured in a monolayer environment for 7 days, and anti-inflammatory activity was evaluated. Since the cells isolated and collected from the hydrogel using the PAI withdrawal method had a uPAR expression rate of 90% or more, secondary uPAR+ cells were not isolated.

[0143] The anti-inflammatory activity was evaluated using conditioned media containing factors secreted from the cells. To prepare the conditioned media, UK+uPAR+, UK+uPAR- and PAI withdrawal cells were cultured in T175 flasks. 2 After adding serum-free DMEM / F12 medium to which 40,000 cells were seeded per well, the cells were cultured for one week.

[0144] RAW 264.7 cells were suspended in 90% RPMi 1640 / 10% calf serum and then cultured in a 30-well plate. 2 100,000 cells were seeded per 12-multiwell plate and cultured for 1 day. 100μg / mL LPS was added to the culture medium to deplete RAW 264.7 cells. At the time of LPS depletion, conditioned medium was added to the culture medium at a ratio of 1:10 to evaluate anti-inflammatory activity. Anti-inflammatory evaluation index was evaluated by measuring the levels of TNFα and IL-1β secreted by RAW 264.7 cells using ELISA.

[0145] After stimulation with LPS, RAW 264.7 cells showed a 5-fold increase in TNFα and IL-1β secretion levels. Substances secreted from tissue-resident stem cells isolated and recovered from hydrogels were confirmed to have the ability to suppress inflammatory cytokine secretion by RAW 264.7 cells by 30-70% (Figure 26). Conditioned media prepared from UK+uPAR+ cells and PAI withdrawal cells showed significantly higher TNFα and IL-1β secretion suppression abilities than UK+uPAR- cells. Regardless of the tissue of origin, uPAR+ cells were confirmed to have higher anti-inflammatory efficacy than uPAR- cells.

[0146] Example 17: Efficacy of uPAR+ cells in inducing growth of vascular endothelial cells and fibroblasts After invaginating tissue slices of fat, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial membrane into fibrin or fibrin / collagen hydrogel, 3D organ culture was performed for 2 weeks, and then cells that migrated and grew in the hydrogel were isolated and collected using the urokinase method described in Example 6. Then, uPAR+ (UK+uPAR+) and uPAR- (UK+uPAR-) cells were isolated using FACSAria (BD Bioscience) and cultured in a monolayer environment for 7 days, and anti-inflammatory activity was evaluated. Since the cells isolated and collected from the hydrogel using the PAI withdrawal method had a uPAR expression rate of 90% or more, secondary uPAR+ cells were not isolated.

[0147] Human umbilical vein endothelial cells (HUVEC) and human dermal fibroblasts (DF) were used as vascular endothelial cells and human dermis-derived fibroblasts, respectively. The efficacy of the conditioned medium in inducing the growth of the cells was examined. The conditioned medium was prepared by the method described in Example 16, and its efficacy was examined. HUVEC cells and DF were suspended in a 99% DMEM / 1% calf serum culture medium, and then cultured in a CM 24,000 cells were seeded per well in a 24-multiwell plate. Conditioned medium was added to the culture medium at a ratio of 1:9 and cultured for 3 days. After culturing, cell numbers were measured using a PicoGreen dsDNA quantitation kit (Invitrogen). Cell growth efficacy was analyzed by calculating the fold increase compared to the initial cell number seeded.

[0148] The conditioned medium prepared from UK+uPAR+ and PAI withdrawal cells was confirmed to have the ability to promote HUVEC and DF growth, and was significantly more effective than the conditioned medium prepared from UK+uPAR- cells by 30% or more, but there was no difference in the effectiveness between UK+uPAR+ and PAI withdrawal cells (Figure 27). In particular, the conditioned medium obtained from myocytes derived from cardiac, neural, and skeletal muscles was confirmed to have a higher growth induction efficacy than stem cells derived from other tissues.

[0149] Example 18: Protective effect of uPAR+ cells on vascular endothelial cells and fibroblasts After invaginating tissue slices of fat, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial membrane into fibrin or fibrin / collagen hydrogel, 3D organ culture was performed for 2 weeks, and then cells that migrated and grew in the hydrogel were isolated and collected using the urokinase method described in Example 6. Then, uPAR+ (UK+uPAR+) and uPAR- (UK+uPAR-) cells were isolated using FACSAria (BD Bioscience) and cultured in a monolayer environment for 7 days, and anti-inflammatory activity was evaluated. Since the cells isolated and collected from the hydrogel using the PAI withdrawal method had a uPAR expression rate of 90% or more, secondary uPAR+ cells were not isolated.

[0150] After withdrawal of UK+uPAR- cells, UK+uPAR+ cells, and PAI, the efficacy of cell protection was tested using the conditioned media prepared from the cells separated and collected. HUVEC and DF were suspended in 99% DMEM / 1% calf serum culture medium, and then 25,000 cells were seeded per well in a 24-multiwell plate. One hour before induction of H2O2-mediated cell damage, conditioned medium was added at a ratio of 1:9 to culture medium. Cell damage was induced by adding 0.01% H2O2. Six hours after induction, the culture medium was removed and the cells were washed twice with PBS. The degree of cell damage was evaluated by annexin V expression rate. After reacting the cells with FITC-conjugated annexin V (BD Biosciences), the dead cells were analyzed by flow cytometry.

[0151] The results confirmed that the conditioned medium prepared from UK+uPAR+ cells and cells recovered after PAI withdrawal treatment could significantly protect H2O2-mediated HUVEC and DF cell death compared to the conditioned medium derived from UK+uPAR- cells (Figure 28). Differences in the efficacy of cytoprotection were observed depending on the tissue of origin, and the efficacy of HUVEC and DF cell protection was high in cells derived from cardiac, neuronal and skeletal muscles. The annexin V expression rate in HUVEC and DF treated with UK+uPAR+ cell conditioned medium was 28.7-45.1%, which was significantly higher than the 42.1-71.3% in the case of cells treated with UK+uPAR- cell conditioned medium, but no significant difference was observed with the conditioned medium derived from PAI withdrawal cells. These results confirmed that uPAR expression is related to high cytoprotective efficacy, and confirmed the basis for isolating and culturing cells with high cytoprotective efficacy using only PAI withdrawal.

[0152] The present invention is carried out through an engineered provisional matrix-supported organ culture. A tissue slice is invaginated into the artificial provisional matrix, and the integrin-FAK cell signaling pathway is activated through the provisional matrix to induce cell division and growth of tissue-resident stem cells. Through the organ culture process, the migration and growth of tissue-resident stem cells into the provisional matrix increases in proportion to the activity of uPAR-uPA-plasmin. In order to control the result that the provisional matrix is ​​decomposed and lost due to excessive uPAR-uPA-plasmin activity, and stem cell migration is inhibited and stopped, excessive degradation of the provisional matrix can be regulated by adding PAI. After the migration and growth of stem cells in the provisional matrix reaches a target level, a technology is provided that can isolate uPAR+ stem cells by stopping the addition of PAI. The result is that uPAR+ stem cells are dissociated from the provisional matrix and released into the culture medium due to the activation of uPAR-uPA-plasmin and MMP by stopping the addition of PAI. The present invention provides a technology that utilizes the migration and degradation properties of the provisional matrix due to uPAR expression to isolate and culture uPAR+ stem cells without the use of specific tissue decomposition proteins or stem cell purification processes using markers.

[0153] The present invention can be applied to the isolation and culture of uPAR+ stem cells present in solid tissues such as bone marrow, adipose tissue, skeletal muscle, heart, peripheral nerve, spinal cord, brain, lung, liver, synovial membrane, umbilical cord, placenta, periodontium, etc. The present invention provides a method for isolating stem cells without specific tissue decomposition enzyme treatment, thereby completely preserving the structure of tissue slices used for organ culture and for isolating and culturing stem cells through repeated organ culture.

[0154] The present invention shows that uPAR-positive tissue-resident stem cells have the characteristic that normal stem cell markers are positively expressed together with uPAR. In the case of bone marrow, fat, muscle, heart, synovium, umbilical cord, and placenta, mesenchymal stem cell markers CD29, CD44, CD73, CD90, and CD105 are positively expressed, but hematopoietic stem cell or vascular endothelial cell markers are negative. The present invention shows that uPAR-positive stem cells derived from peripheral nerve, spinal cord, and brain tissues have the characteristic that markers such as nestin, p75, Sox10, and Sox2 are simultaneously expressed.

[0155] The uPAR-positive stem cells of the present invention exhibit biological properties such as high secretion capacity of bioactive factors acting on tissue regeneration, such as migration and growth factors, anti-inflammatory factors, and stem cell recruitment factors.

[0156] The uPAR-positive stem cells of the present invention have pluripotency and are highly capable of differentiating into tissue-constituting cells.

Claims

1. (1) preparing a wound repair matrix mimicking hydrogel; (2) invaginating an isolated tissue section into the wound repair matrix-mimicking hydrogel; (3) three-dimensionally culturing the wound repair matrix-mimicking hydrogel into which the tissue slice has been invaginated in a culture medium containing PAI; (4) removing the 3D culture medium and washing to remove PAI; (5) re-culturing the culture from which the PAI has been removed in a culture medium not containing PAI to degrade the wound repair matrix-mimicking hydrogel; (6) isolating the stem cells released into the reculture medium; and A method for isolating and culturing tissue-resident uPAR+ and nestin+ stem cells, comprising:

2. 2. The method for isolating and culturing tissue-resident uPAR+ and nestin+ stem cells according to claim 1, wherein the wound repair matrix mimicking hydrogel is a fibrin hydrogel obtained by mixing a fibrinogen solution having a concentration of 0.25-2.5% and a thrombin solution having a concentration of 0.5-5 I.U. / mL, a fibrin / collagen mixed hydrogel obtained by mixing the fibrin hydrogel with a collagen solution having a concentration of 0.1-0.5%, or a fibrin / gelatin mixed hydrogel obtained by mixing the fibrin hydrogel with a gelatin solution having a concentration of 0.1-0.5%.

3. The method for isolating and culturing tissue-resident uPAR+ and nestin+ stem cells according to claim 1, wherein the tissue is adipose tissue, bone marrow tissue, cardiac muscle tissue, peripheral nerve tissue, skeletal muscle tissue, or synovial tissue.

4. The method for isolating and culturing tissue-resident uPAR+ and nestin+ stem cells according to claim 1, wherein the PAI is tranexamic acid or aminomethylbenzoic acid.

5. The method for isolating and culturing tissue-resident uPAR+ and nestin+ stem cells according to claim 1, characterized in that step (5) induces increased uPAR expression in the tissue and degrades the wound repair matrix-mimicking hydrogel through increased plasmin activity.

6. The method for isolating and culturing tissue-resident uPAR+ and nestin+ stem cells according to claim 1, characterized in that the tissue-resident uPAR+ and nestin+ stem cells have enhanced self-renewal ability, in vitro growth ability, differentiation ability or tissue regeneration induction ability.

7. The method for isolating and culturing tissue-resident uPAR+ and nestin+ stem cells according to claim 1, further comprising repeating steps (2) to (5) 1 to 10 times for the tissue slices recovered from the reculture medium in step (5).

8. A tissue-resident uPAR+ and nestin+ stem cell isolated and cultured by the method according to any one of claims 1 to 7, or a culture medium thereof.

9. A pharmaceutical composition for preventing or treating an inflammatory disease, comprising the tissue-resident uPAR+ and nestin+ stem cell or a culture medium thereof according to claim 8 as an active ingredient.

10. A pharmaceutical composition for preventing or treating an autoimmune disease, comprising the tissue-resident uPAR+ and nestin+ stem cell or a culture medium thereof according to claim 8 as an active ingredient.

11. A pharmaceutical composition for wound treatment comprising the tissue-resident uPAR+ and nestin+ stem cells or a culture medium thereof according to claim 8 as an active ingredient.

12. A pharmaceutical composition for promoting vascular regeneration, comprising the tissue-resident uPAR+ and nestin+ stem cells or a culture medium thereof according to claim 8 as an active ingredient.

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