Method for isolating and differentiating nucleus pulposus progenitor cells and uses thereof

Abstract

To provide a separation and discrimination method of a nucleus pulposus precursor cell, and application thereof.SOLUTION: Provided is a separation and discrimination method of a nucleus pulposus precursor cell, where a large number of tissue blocks are divided from a nucleus pulposus tissue, and the tissue blocks are left to stand and cultured in a culture dish after performing hydrolysis by enzyme. When cells are formed into the partition blocks on a bottom of the culture dish, a nucleus pulposus precursor cell is screened from the partition blocks by using a specific embryonic stem cell gene (e.g., Nanog, Oct-4, SOX2). Further, the method can screen a nucleus pulposus precursor cell having moving performance and / or mesenchymal stem cell properties by using a specific protein and a gene marker, and further can screen a nucleus pulposus precursor cell having anti-inflammatory performance. Finally, disclosed is application used for producing a pharmaceutical composition treating lower back pain by using the nucleus pulposus precursor cell obtained by the method, and a new therapy strategy to lower back pain is provided.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present invention relates to the technical field of cell isolation and differentiation methods, and more particularly to the technical field of methods for isolating and differentiating nucleus pulposus progenitor cells having migratory and anti-inflammatory abilities from human intervertebral disc nucleus pulposus tissue. [Background technology]

[0002] Intervertebral discs are soft tissue structures located between the vertebrae of the human body, primarily composed of an outer layer called the annulus fibrosus and an inner layer called the nucleus pulpous tissue. These discs provide cushioning and support for the spine, helping to reduce friction and shock between the vertebrae. Degenerative changes in the discs often cause symptoms such as lower back pain in adults, interfering with daily life and work efficiency.

[0003] Traditional treatments have focused on medications, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and steroids, to alleviate pain and inflammation when early symptoms of disc degeneration appear. However, long-term use of these medications can cause gastrointestinal side effects and affect cellular function. When disc degeneration symptoms become severe, more invasive treatments, such as disc replacement, are required, but these procedures are fraught with risk and cost.

[0004] When an intervertebral disc is damaged or degenerated, the nucleus pulposus tissue can become deformed or protrude, which can lead to nerve compression and cause problems such as pain, numbness, muscle weakness, etc. Current treatment methods often do not directly address the underlying problem with the intervertebral disc (i.e., degeneration and damage of the nucleus pulposus tissue). Summary of the Invention [Problem to be solved by the invention]

[0005] In light of the limitations of current intervertebral disc treatment methods, the present invention proposes an innovative method for isolating and differentiating nucleus pulposus progenitor cells with migratory and anti-inflammatory capabilities from a patient's intervertebral disc nucleus pulposus tissue. The method of the present invention involves isolating cells from a patient's nucleus pulposus at an early stage of intervertebral disc degeneration, selecting and differentiating nucleus pulposus progenitor cells with stem cell characteristics and migratory and anti-inflammatory capabilities from the isolated cells, and then maintaining and expanding the cells in vitro. These cells can then be easily transplanted back into the patient, if necessary, to promote the regeneration and repair of intervertebral disc tissue. This innovative method not only allows for direct treatment of intervertebral disc degeneration, but also significantly improves the patient's quality of life. [Means for solving the problem]

[0006] The main object of the present invention is to provide a method for isolating and differentiating nucleus pulposus progenitor cells, the method comprising steps a) to d), in which in step a) nucleus pulposus tissue is provided and divided into a plurality of tissue blocks, in step b) the tissue blocks are hydrolyzed using an enzyme, in step c) the enzyme is removed and the tissue blocks are cultured in a culture dish, and in step d) a plurality of cell populations are allowed to migrate out of the tissue block and form a plurality of compartment blocks at the bottom of the culture dish, and nucleus pulposus progenitor cells are selected from each of the cell populations in each of the compartment blocks using an embryonic stem cell gene, wherein the embryonic stem cell gene is at least one selected from the group consisting of Nanog, Oct-4 and SOX2, and wherein the enzyme used to hydrolyze the tissue blocks is collagenase, trypsin or a combination thereof.

[0007] The above-mentioned method for isolating and differentiating nucleus pulposus progenitor cells can further utilize at least one selected from the group consisting of N-Cadherin, Vimentin, β-Catenin, and Snail protein to select the nucleus pulposus progenitor cells with migration ability.

[0008] The above-mentioned method for isolating and differentiating nucleus pulposus progenitor cells can further utilize at least one gene selected from the group consisting of STRO-1, C-KIT, β-catenin, Jagged, and Delta4 genes to select the nucleus pulposus progenitor cells having mesenchymal stem cell characteristics.

[0009] The above-mentioned method for isolating and differentiating nucleus pulposus progenitor cells can further utilize at least one selected from the group consisting of stem cell surface antigens CD34, CD44, CD73, CD90, CD105, and CD133 to select the nucleus pulposus progenitor cells having mesenchymal stem cell characteristics.

[0010] Furthermore, the above-mentioned method for isolating and differentiating nucleus pulposus progenitor cells can utilize differentiation ability to select nucleus pulposus progenitor cells having the differentiation ability, and the differentiation ability is at least one selected from the group consisting of chondrogenesis, osteogenesis, and adipogenesis.

[0011] In addition, the above-mentioned method for isolating and differentiating nucleus pulposus progenitor cells can further utilize at least one selected from the group consisting of IL-1β, COX-2, and MMP3 cellular inflammatory genes to select the nucleus pulposus progenitor cells with anti-inflammatory ability.

[0012] At the same time, a secondary object of the present invention is to provide a use of the nucleus pulposus progenitor cells obtained by the above method for producing a pharmaceutical composition for treating lower back pain, wherein the pharmaceutical composition includes a pharmaceutically acceptable carrier.

[0013] The use of the nucleus pulposus progenitor cells described above for producing a pharmaceutical composition for treating lower back pain can further utilize at least one selected from the group consisting of N-Cadherin, Vimentin, β-Catenin, and Snail protein to select the nucleus pulposus progenitor cells with migration ability, and / or can utilize at least one selected from the group consisting of stem cell surface antigens CD34, CD44, CD73, CD90, CD105, and CD133 to select the nucleus pulposus progenitor cells with mesenchymal stem cell characteristics.

[0014] The use of the nucleus pulposus progenitor cells described above for producing a pharmaceutical composition for treating lower back pain can further utilize their differentiation ability to select the nucleus pulposus progenitor cells having the differentiation ability, and the differentiation ability can be at least one selected from the group consisting of chondrogenesis, osteogenesis, and adipogenesis, and / or at least one selected from the group consisting of IL-1β, COX-2, and MMP3 inflammatory genes can be used to select the nucleus pulposus progenitor cells having anti-inflammatory ability. [Effects of the Invention]

[0015] According to the pharmaceutical composition, the nucleus pulposus progenitor cells obtained from the patient's nucleus pulposus tissue can be expanded and cultured, and then re-transplanted into the affected area of ​​the patient (i.e., the site of degenerative intervertebral disc degeneration), where they can grow and grow in the affected area, thereby achieving the therapeutic goal through the regeneration of nucleus pulposus tissue and avoiding the possibility of an immune rejection reaction in the patient receiving the transplant. [Brief explanation of the drawings]

[0016] [Figure 1] The photographs show the cell population creeping out of the tissue block and its growth on the bottom of the culture dish after the tissue block has been cultured for one day and four days, respectively. [Figure 2A]FIG. 2A shows the results of differentiation of nucleus pulposus progenitor cells using embryonic stem cell genes Nanog, Oct-4, and SOX2. [Figure 2B] FIG. 2B shows the results of differentiation of nucleus pulposus progenitor cells using embryonic stem cell genes Nanog, Oct-4, and SOX2. [Figure 2C] FIG. 2C shows the results of differentiation of nucleus pulposus progenitor cells using embryonic stem cell genes Nanog, Oct-4, and SOX2. [Figure 2D] FIG. 2D shows the results of differentiation of nucleus pulposus progenitor cells using embryonic stem cell genes Nanog, Oct-4, and SOX2. [Figure 3A] FIG. 3A shows the results of a migration ability test performed on the selected nucleus pulposus progenitor cells. [Figure 3B] FIG. 3B shows the results of a migration ability test performed on the selected nucleus pulposus progenitor cells. [Figure 4A] FIG. 4A shows the results of differentiation of cell populations selected using mesenchymal stem cell (MSC) characteristics. [Figure 4B] FIG. 4B shows the results of differentiation of cell populations selected using mesenchymal stem cell (MSC) characteristics. [Figure 4C] FIG. 4C shows the results of differentiation of cell populations selected using mesenchymal stem cell (MSC) characteristics. [Figure 4D] FIG. 4D shows the results of differentiation of cell populations selected using mesenchymal stem cell (MSC) characteristics. [Figure 5A] FIG. 5A shows the results of identifying selected cell populations using an anti-inflammatory ability test. [Figure 5B] FIG. 5B shows the results of identifying selected cell populations using an anti-inflammatory ability test. [Figure 5C] FIG. 5C shows the results of identifying selected cell populations using an anti-inflammatory ability test. [Figure 5D]FIG. 5D shows the results of identifying selected cell populations using an anti-inflammatory ability test. [Figure 6] 1 is a flowchart showing the method for isolating and differentiating nucleus pulposus precursor cells of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Hereinafter, the details of the present invention will be explicitly explained using examples, but these examples are merely illustrative and not limiting, and the present invention is not limited to these examples. Unless otherwise specified, all materials used in the present invention are preferably commercially available and readily available, and the available routes shown below are merely examples. [Example]

[0018] <Processing of Nucleus Pulposus Tissue Samples> 1. First, in a hospital operating room, a sample of nucleus pulposus tissue from a patient with degenerative disc disease or herniated disc is surgically removed in a sterile manner, placed in a sterile, sealed carrier containing saline containing an antibiotic composition, and then transferred to a sterile workbench in a laboratory that complies with Good Tissue Practice (GTP) standards for handling human cells and tissues for processing. 2. Record the weight of the nucleus pulposus tissue specimen and basic information about the patient of said specimen. 3. Place the specimen in a 10 cm petri dish containing 2 mL of medium, and then immerse the specimen in a 1 mm 3The tissue is cut into small tissue blocks smaller than 1000 μg, and each 10 cm dish contains approximately 5 g of tissue blocks. If there are a relatively large number of tissue blocks, the tissue blocks are divided into several culture dishes in a certain proportion, and the medium contains DMEM (Dulbecco's Modified Eagle's Medium), human platelet lysate, and an antibiotic. 4. Preparation of collagenase solution: 7 mL of medium + 1 mL of type I collagenase, that is, the ratio of medium to type I collagenase in the collagenase solution is 7:1 (volume ratio). 5. Add the prepared collagenase solution to the culture dishes containing the tissue blocks, add 8 ml of collagenase solution to each culture dish, and culture in an incubator at 37°C with 5% CO2 for 4 to 24 hours. 6. After 4 to 24 hours of culture, the tissue blocks in each culture dish are washed with 10 mL of DPBS (Dulbecco's phosphate-buffered saline) to remove the collagenase solution. 7. The tissue block is transferred to a 10 cm petri dish containing 10 mL of medium and cultured for 1 to 7 days. 8. When multiple cell populations are allowed to crawl out of the tissue block and multiple compartment blocks are formed on the bottom of the culture dish, the cell populations in each compartment block are collected and transferred to different culture dishes for individual culture. 9. Using embryonic stem cell genes, the nucleus pulposus progenitor cells are selected from each of the cell populations collected and cultured in each of the compartment blocks.

[0019] Figure 1 shows the tissue block cultured for one day and four days. As can be clearly seen, after four days of culture, the number of cell populations that emerged from the tissue block and grew to the bottom of the culture dish increased significantly, while the increase in the number of cell populations after one day of culture was relatively small. Next, each of these cell populations that emerged and grew to different compartments at the bottom of the culture dish was collected and transferred to different culture dishes for further expansion. Finally, these cells were differentiated (qualified), and nucleus pulposus progenitor cells were selected using, for example, embryonic stem cell genes Nanog, Oct-4, and SOX2. [Example]

[0020] <Identification of nucleus pulposus progenitor cells by embryonic stem cell genes> In this example, each cell population grown in a separate compartment at the bottom of the culture dish was collected and expanded. Nucleus pulposus progenitor cells were then selected from these cell populations using the embryonic stem cell (prototype) genes Nanog, Oct-4, and SOX2. Mesenchymal stem cells (MSCs) and primary nucleus pulposus cell parental lines were used as controls to compare the expression ratios (quantities) of these cell populations with those of embryonic stem cells. A gene expression ratio greater than 1.5 was set as the selection threshold; that is, when the expression ratio of an embryonic stem cell gene in each cell population was greater than 1.5, it was considered to have reached the selection threshold. Furthermore, only those genes selected from cell populations that reached the selection threshold (Nanog, Oct-4, and SOX2) were subjected to further analysis.

[0021] Figure 2A shows the results of an analysis of different cell populations (NPP2, 3, 4, 7, 9, 10, 14, 15, 17, 19, and 20) using the Nanog gene, showing that among these, cell populations NPP2, 3, 4, 7, 9, 10, 19, and 20 reached the selection threshold with an expression ratio greater than 1.5.

[0022] Figure 2B shows the results of analysis of different cell populations (NPP2, 3, 4, 7, 9, 10, 14, 15, 17, 19, and 20) using the Oct-4 gene, and shows that among them, cell populations NPP3, 4, 7, 10, 15, and 19 reached the selection threshold with an expression ratio greater than 1.5.

[0023] Figure 2C shows the results of an analysis of different cell populations (NPP2, 3, 4, 7, 9, 10, 14, 15, 17, 19, and 20) using the SOX2 gene, showing that the cell populations NPP3, 7, 9, 19, and 20 reached the selection threshold with an expression ratio greater than 1.5.

[0024] The diagram in Figure 2D shows the results of initial selection of nucleus pulposus progenitor cells from these cell populations using the embryonic stem cell genes Nanog, Oct-4, and SOX2, and subsequent analysis is performed only on cell populations that reach the selection threshold for all three genes (Nanog, Oct-4, and SOX2). The selected cell population is expected to have properties most similar to stem cells, The most promising genes were Nanog, Oct-4, and SOX2, which are generally associated with stem cell maintenance and pluripotency, and thus could be used in nucleus pulposus tissue regeneration to achieve therapeutic goals. In Figure 2D, open circles represent genes whose expression ratios reached the selection threshold of greater than 1.5, while filled circles represent genes whose expression ratios were all above the selection threshold. The final cell population selected for further analysis included NPP3, NPP7, and NPP19. [Example]

[0025] <Mobility Test> In this example, the migration ability of the cell populations NPP3, NPP7, and NPP19 selected based on embryonic stem cell genes was further tested to measure their migration ability. In this experiment, the migration ability of cells was evaluated using Transwell culture dishes. Transwell experiments are a common method for measuring cell migration ability. Cells are permeated downward through a Transwell membrane, adhere to the membrane, and migrate from one chamber to another, simulating the migration of cells in tissues.

[0026] The bar graph in Figure 3A shows the percentage of membrane penetration for the primary nucleus pulposus cell parental line and the NPP3, NPP7, and NPP19 cell populations. NPP19 showed the highest percentage of penetration, indicating that NPP19 had the strongest migratory ability in this test, followed by NPP7 and then NPP3. The migratory ability of the primary nucleus pulposus cell parental line was significantly reduced. Images of hematoxylin-stained cells are displayed below the bar graph; this staining was used to clearly visualize the cell nuclei and therefore calculate the number of cells that penetrated the membrane.

[0027] In this migration assay, Western blotting was also used to detect the expression of proteins related to cell migration. The proteins measured included N-cadherin, Vimentin, β-catenin, and Snail, all of which are important proteins related to cell adhesion, cytoskeletal structure, and cell migration. As shown in Figure 3B, NPP3, NPP7, and NPP19 cell populations all express these proteins. The loading amount of sample protein was ensured to be consistent with that of β-actin, which was used as an internal control protein. [Example]

[0028] <Identification of Mesenchymal Stem Cell (MSC) Characteristics> Referring to Figures 4A-4D, in this example, we again performed a differentiation test on the above-mentioned cell populations NPP3, NPP7, and NPP19, which were selected for their mesenchymal stem cell characteristics. Figure 4A shows the proliferation status of different cell populations (Parental and cell populations NPP3, NPP7, and NPP19) cultured on days 1, 3, 5, and 7. All cell populations proliferated with increasing time, as evidenced by increasing ratios. On day 1, the proliferation ratios of all cell populations were close to 1, indicating that they originated from approximately the same time point at the start of culture. Comparing different time points, NPP7 showed a higher proliferation ratio than the other cell populations on days 5 and 7. In particular, the proliferation ratio of NPP7 on day 7 was approximately 10, significantly higher than the other cell populations, indicating that NPP7 had a stronger proliferation ability at these time points. The proliferation rate of NPP19 on days 5 and 7 is intermediate between that of NPP7 and NPP3. At day 5, the proliferation rate of NPP3 is no different from that of the primary nucleus pulposus cell parent line, and at day 7, the proliferation rate of NPP3 is slightly higher than that of the primary nucleus pulposus cell parent line. The higher the proliferation rates of NPP7 and NPP19, the stronger their proliferation ability, making it possible to provide the required number of cells in a relatively short period of time, thereby more effectively repairing damaged nucleus pulposus tissue and achieving the therapeutic goal of nucleus pulposus tissue regeneration.

[0029] Figure 4B shows the differentiation of cell populations NPP3, NPP7, and NPP19, selected based on the expression of genes highly associated with mesenchymal stem cells (STRO-1, C-KIT, β-catenin, Jagged, and Delta4), with the GAPDH gene used as an internal control. All three cell populations, NPP3, NPP7, and NPP19, expressed these five mesenchymal stem cell genes, demonstrating that all retained mesenchymal stem cell characteristics. Compared with the primary nucleus pulposus cell parent line, NPP3, NPP7, and NPP19 exhibited relatively high expression of mesenchymal stem cell genes. The expression levels of all five genes in NPP3 and NPP7 were generally higher than those of NPP19, with particularly significant differences in the expression of STRO-1, C-KIT, and β-catenin, demonstrating that NPP3 and NPP7 possess more mesenchymal stem cell characteristics and may be more effectively involved in the repair and regeneration of nucleus pulposus tissue.

[0030] Figure 4C shows the results of flow cytometric analysis of the expression of six different stem cell surface antigen markers (CD markers: CD34, CD133, CD44, CD73, CD90, and CD105) in the cell populations NPP3, NPP7, and NPP19 selected for selection. NPP3, NPP7, and NPP19 express CD34, CD73, CD90, and CD105 on their cell surface, which are typical surface markers of mesenchymal stem cells. Therefore, the characteristics of the cell populations NPP3, NPP7, and NPP19 are similar to those of mesenchymal stem cells and may have similar functions and differentiation potential, making them suitable for use in the repair and regeneration of nucleus pulposus tissue. Furthermore, NPP3, NPP7, and NPP19 do not express surface antigens such as CD34 and CD133, which are found on hematopoietic stem cells, further confirming their similarity to mesenchymal stem cells but lacking the characteristics of hematopoietic stem cells.

[0031] Figure 4D shows the gene expression ratios of the selected cell populations NPP3, NPP7, and NPP19 under three different differentiation conditions. The black bars represent the expression levels in the control group, and the gray bars represent the expression levels in the induction group. The cell populations NPP3, NPP7, and NPP19 were able to increase the expression ratios of genes associated with chondrogenesis, osteogenic differentiation, and adipogenic differentiation under induction conditions for chondrogenesis, osteogenic differentiation, and adipogenic differentiation, respectively. This multidirectional differentiation potential is important for tissue engineering and regenerative medicine, and these cells can be used to repair or replace damaged nucleus pulposus tissue. [Example]

[0032] <Anti-inflammatory ability test> 5A to 5D, in this Example, the above-mentioned selected cell populations NPP3, NPP7, and NPP19 were further differentiated using an anti-inflammatory ability test. The anti-inflammatory ability test included measuring the survival percentage of the primary nucleus pulposus cell parent line and the NPP3, NPP7, and NPP19 cell populations under an inflammatory environment (treatment with IL-1β (interleukin-1β) + TNF-α (tumor necrosis factor-α) and LPS (lipopolysaccharide)), and measuring the expression levels of inflammatory genes (IL-1β, COX-2, and MMP3) of the primary nucleus pulposus cell parent line and the NPP3, NPP7, and NPP19 cell populations under an inflammatory environment.

[0033] Figure 5A shows the survival percentages of the primary nucleus pulposus cell parental line (Parental) and the NPP3, NPP7, and NPP19 cell populations under normal conditions (CTRL, control group) and under inflammatory conditions (treated with IL-1β, TNF-α, and LPS). As can be seen from Figure 5A, under inflammatory conditions, the primary nucleus pulposus cell parental line exhibited suppression of cell growth, resulting in a decrease in its survival rate. However, the survival rates of the cell populations (NPP3, NPP7, and NPP19) were not significantly reduced. This indicates that the cell populations (NPP3, NPP7, and NPP19) exhibited relatively good resistance to the inflammatory environment and were capable of sustained growth and proliferation in that environment.

[0034] Figure 5B shows the expression ratio of the inflammatory gene IL-1β in the primary nucleus pulposus cell parent line (Parental) and NPP3, NPP7, and NPP19 cell populations under normal conditions (CTRL, control group) and under an inflammatory environment (IL-1β + TNF-α and LPS treatment). It was suggested that the primary nucleus pulposus cell parent line significantly increased the expression ratio of the IL-1β gene under an inflammatory environment, and this increase was less apparent in the cell populations (NPP3, NPP7, and NPP19), indicating that the cell populations (NPP3, NPP7, and NPP19) exhibit a certain degree of inhibitory effect on the inflammatory response and are less susceptible to interference.

[0035] Figure 5C shows the expression ratio of the inflammatory gene COX-2 in the parental primary nucleus pulposus cell line and NPP3, NPP7, and NPP19 cell populations under normal conditions (CTRL, control group) and inflammatory conditions (IL-1β + TNF-α and LPS treatment). Similar to the IL-1β gene, the parental primary nucleus pulposus cell line increased COX-2 expression under inflammatory conditions, and the increase in COX-2 expression in the NPP3, NPP7, and NPP19 cell populations was obscured, further supporting the anti-inflammatory properties of the NPP3, NPP7, and NPP19 cell populations.

[0036] Figure 5D shows the expression ratio of the inflammatory gene MMP3 in the primary nucleus pulposus cell parental line (Parental) and NPP3, NPP7, and NPP19 cell populations under normal conditions (CTRL, control group) and inflammatory conditions (IL-1β + TNF-α and LPS treatment). Consistent with the expression trends of the previous two inflammatory genes (IL-1β and COX-2), the primary nucleus pulposus cell parental line significantly increased the expression ratio of MMP3 under inflammatory conditions, while having a relatively small effect on the expression of the cell populations (NPP3, NPP7, and NPP19).

[0037] As can be seen from the above anti-inflammatory ability test, the nucleus pulposus progenitor cells selected according to the present invention (cell populations NPP3, NPP7, and NPP19) exhibit relatively strong survival ability and relatively low expression of inflammatory genes under inflammatory conditions compared to the primary nucleus pulposus cell parent line. This indicates that the cell populations NPP3, NPP7, and NPP19 have relatively high anti-inflammatory potential, which is extremely valuable in clinical applications, especially when it is necessary to control inflammatory responses (e.g., in the repair and treatment of nucleus pulposus tissue).

[0038] FIG. 6 is a flow chart showing the method for isolating and differentiating nucleus pulposus progenitor cells of the present invention, which includes the following steps:

[0039] In step S101 (providing nucleus pulposus tissue), a sample of nucleus pulposus tissue from a patient with degenerative disc disease or herniated disc is surgically removed in a sterile manner.

[0040] In step S102 (division into a plurality of tissue blocks), the nucleus pulposus tissue specimen is then divided into a plurality of tissue blocks.

[0041] In step S103 (enzyme treatment), the tissue block is hydrolyzed using an enzyme such as collagenase or trypsin.

[0042] In step S104 (culture of undigested tissue blocks), the enzyme is removed by washing with DPBS, and then the undigested tissue blocks are cultured in a culture dish for 1 to 7 days.

[0043] In step S105 (extraction of cell populations), the undigested tissue block is cultured in a culture dish for 1 to 7 days, after which multiple cell populations are extruded from the tissue block and multiple compartment blocks are formed at the bottom of the culture dish.

[0044] In step S106 (collection of each cell population in each compartment block), each of the cell populations in each compartment block is collected, and then transferred to different culture dishes and cultured individually.

[0045] In step S107 (cell qualification), nucleus pulposus progenitor cells are then selected from each cell population using the embryonic stem cell genes Nanog, Oct-4, and SOX2 according to the method described in Example 2 above, and then the selected nucleus pulposus progenitor cells are further differentiated for their migration ability, mesenchymal stem cell (MSC) properties, and anti-inflammatory ability according to the methods shown in Examples 3 to 5, thereby obtaining nucleus pulposus progenitor cells optimal for nucleus pulposus tissue regeneration.

[0046] As explained above, the method for isolating and differentiating nucleus pulposus progenitor cells according to the present invention and its uses have been fully and clearly explained. It should be emphasized that the above detailed description specifically describes feasible embodiments of the present invention, but the scope of the present invention is not limited to these embodiments, and any equivalent implementations or modifications thereof that do not deviate from the technical spirit of the present invention are still included in the scope of the claims of this application. [Explanation of symbols]

[0047] S101~S107: Step

Claims

1. a) providing nucleus pulposus tissue divided into a plurality of tissue blocks; b) hydrolyzing the tissue block using an enzyme; Step c) of removing the enzyme and culturing the tissue block in a culture dish; a step d) of extracting a plurality of cell populations from the tissue block and forming a plurality of compartment blocks on the bottom of the culture dish, and selecting nucleus pulposus progenitor cells from each of the cell populations in each of the compartment blocks using embryonic stem cell genes; Including, The embryonic stem cell genes are Nanog, Oct-4, and SOX2 A method for isolating and differentiating nucleus pulposus progenitor cells, comprising:

2. The method according to claim 1, wherein the nucleus pulposus progenitor cells having migration ability are selected by further using at least one selected from the group consisting of N-Cadherin, Vimentin, β-Catenin and Snail protein.

3. The method of claim 1, wherein the nucleus pulposus progenitor cells having mesenchymal stem cell characteristics are selected by further using at least one gene selected from the group consisting of STRO-1, C-KIT, β-catenin, Jagged, and Delta4 genes.

4. The method of claim 1, further comprising selecting the nucleus pulposus progenitor cells having mesenchymal stem cell characteristics by further using at least one selected from the group consisting of surface antigens of stem cells: CD34, CD44, CD73, CD90, CD105, and CD133.

5. The method according to claim 1, characterized in that the nucleus pulposus progenitor cells having the differentiation ability are selected by further utilizing the differentiation ability, and the differentiation ability is at least one selected from the group consisting of cartilage differentiation (chondrogenesis), bone differentiation (osteogenesis), and fat differentiation (adipogenesis).

6. The method of claim 1, further comprising selecting the nucleus pulposus progenitor cells having anti-inflammatory ability by further using at least one selected from the group consisting of IL-1β, COX-2, and MMP3 cellular inflammatory genes.

7. 2. The method of claim 1, wherein the enzyme used to hydrolyze the tissue block is collagenase, trypsin, or a combination thereof.

8. 10. A use of nucleus pulposus progenitor cells, wherein the nucleus pulposus progenitor cells obtained by the method of claim 1 are used to manufacture a pharmaceutical composition for treating lower back pain.

9. The use according to claim 8, wherein the nucleus pulposus progenitor cells having migration ability are selected by further using at least one selected from the group consisting of N-Cadherin, Vimentin, β-Catenin, and Snail protein, and / or the nucleus pulposus progenitor cells having mesenchymal stem cell characteristics are selected by further using at least one selected from the group consisting of stem cell surface antigens CD34, CD44, CD73, CD90, CD105, and CD133.

10. The use according to claim 9, wherein differentiation ability is further utilized to select the nucleus pulposus progenitor cells having said differentiation ability, and said differentiation ability is at least one selected from the group consisting of cartilage differentiation (chondrogenesis), bone differentiation (osteogenesis), and fat differentiation (adipogenesis), and / or at least one selected from the group consisting of IL-1β, COX-2, and MMP3 cellular inflammatory genes is utilized to select the nucleus pulposus progenitor cells having anti-inflammatory ability.

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