Separation and identification method of nucleus pulposus precursor cells and application of nucleus pulposus precursor cells

Abstract

The invention mainly discloses a separation and identification method of nucleus pulposus precursor cells, which comprises the following steps of: cutting a plurality of tissue blocks from nucleus pulposus tissues, hydrolyzing by enzyme, and culturing the tissue blocks in a culture dish; when cells form blocks at the bottom of a culture dish, specific embryonic stem cell genes (such as Nanog, Oct-4 and SOX2) are used for screening out nucleus pulposus precursor cells from the blocks. Furthermore, the method can be used for screening nucleus pulposus precursor cells with mobility and / or mesenchymal stem cell characteristics by using specific protein and gene markers, and further screening nucleus pulposus precursor cells with anti-inflammatory ability. Finally, the invention discloses an application of the nucleus pulposus precursor cell obtained by the method for preparing a pharmaceutical composition for treating lower back pain, and a new strategy is provided for lower back pain treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of cell separation and identification methods, and in particular to the technical field of a method for separating and identifying nucleus pulposus precursor cells with migration and anti-inflammatory capabilities from human intervertebral disc nucleus pulposus tissue. Background Art

[0002] Intervertebral discs are soft tissue structures located between the human vertebrae. They are primarily composed of an outer layer (annulus fibrosus) and an inner layer (nucleus pulposus). These discs provide cushioning and support within the spine, helping to reduce friction and impact between bones. Degenerative disc disease often causes lower back pain in adults, impacting their daily lives and work performance.

[0003] Traditional treatments for early-stage disc degeneration often rely on medications, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and steroids, to relieve pain and inflammation. However, long-term use of these medications can cause gastrointestinal problems and affect cell function. For severe disc degeneration, more invasive treatments, such as disc replacement surgery, may be necessary, but these approaches carry risks and are expensive.

[0004] When a disc is damaged or degenerates, the nucleus pulposus may become deformed or herniated, which can cause nerve compression and lead to pain, numbness, or muscle weakness. Current treatments often fail to directly address the underlying disc problem, which is the degeneration and damage of the nucleus pulposus.

[0005] Given the limitations of existing intervertebral disc treatment methods, the present invention proposes an innovative method to isolate and identify mobile and anti-inflammatory nucleus pulposus precursor cells from a patient's nucleus pulposus tissue. This method involves isolating cells from the patient's nucleus pulposus during the early stages of disc degeneration, screening and identifying nucleus pulposus precursor cells with stem cell characteristics, mobility, and anti-inflammatory abilities from the isolated cells, and culturing and amplifying these cells in vitro so that they can be re-implanted into the patient when needed, thereby promoting regeneration and repair of the intervertebral disc tissue. The innovation of this method lies in its ability to directly treat disc degeneration and significantly improve the patient's quality of life. Summary of the Invention

[0006] The main purpose of the present invention is to provide a method for isolating and identifying nucleus pulposus precursor cells, which comprises:

[0007] a) providing a nucleus pulposus tissue, and cutting the nucleus pulposus tissue into a plurality of tissue blocks;

[0008] b) hydrolyzing the tissue mass with an enzyme;

[0009] c) removing the enzyme and culturing the tissue piece in a culture dish; and

[0010] d) when the multiple cell populations crawl out from the tissue block and form multiple blocks at the bottom of the culture dish, screening the nucleus pulposus precursor cells from each of the cell populations in each of the blocks using an embryonic stem cell gene;

[0011] wherein the embryonic stem cell gene is at least one selected from the group consisting of Nanog, Oct-4, and SOX2;

[0012] Wherein, the enzyme used to hydrolyze the tissue block is collagenase, trypsin or a combination thereof.

[0013] The above-mentioned method for isolating and identifying nucleus pulposus progenitor cells may further utilize at least one protein selected from the group consisting of N-Cadherin, Vimentin, β-Catenin and Snail protein to screen out the nucleus pulposus progenitor cells with migration ability.

[0014] The above-mentioned method for isolating and identifying 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 screen out the nucleus pulposus progenitor cells with mesenchymal stem cell characteristics.

[0015] The above-mentioned method for isolating and identifying nucleus pulposus progenitor cells can further utilize at least one selected from the group consisting of CD34, CD44, CD73, CD90, CD105 and CD133 stem cell surface antigens to screen out the nucleus pulposus progenitor cells with mesenchymal stem cell characteristics.

[0016] Furthermore, the above-mentioned method for isolating and identifying nucleus pulposus progenitor cells can utilize a differentiation ability to screen out the nucleus pulposus progenitor cells with the differentiation ability, and the differentiation ability is at least one selected from the group consisting of chondrogenesis, osteogenesis, and adipogenesis.

[0017] In addition, the above-mentioned method for isolating and identifying nucleus pulposus progenitor cells can further utilize at least one selected from the group consisting of IL-1β, COX-2 and MMP3 cell inflammation genes to screen out the nucleus pulposus progenitor cells with anti-inflammatory ability.

[0018] Meanwhile, another object of the present invention is to provide a use of the nucleus pulposus precursor cells obtained by the above method for preparing a pharmaceutical composition for treating low back pain, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0019] The use of the nucleus pulposus progenitor cells for manufacturing a pharmaceutical composition for treating low back pain can further utilize at least one selected from the group consisting of N-Cadherin, Vimentin, β-Catenin, and Snail proteins to screen for nucleus pulposus progenitor cells with mobility, and / or utilize at least one selected from the group consisting of CD34, CD44, CD73, CD90, CD105, and CD133 stem cell surface antigens to screen for nucleus pulposus progenitor cells with mesenchymal stem cell characteristics.

[0020] The use of the nucleus pulposus progenitor cells for producing a pharmaceutical composition for treating low back pain can further utilize differentiation ability to screen for nucleus pulposus progenitor cells having the differentiation ability, wherein the differentiation ability is at least one selected from the group consisting of chondrogenesis, osseogenesis, and adipogenesis; and / or utilize at least one selected from the group consisting of IL-1β, COX-2, and MMP3 cell inflammation genes to screen for nucleus pulposus progenitor cells having anti-inflammatory ability.

[0021] Through this pharmaceutical composition, the nucleus pulposus precursor cells obtained from a patient's nucleus pulposus tissue can be expanded and cultured and then re-transplanted into the patient's affected area, i.e., the site of intervertebral disc degeneration, to allow them to grow in the affected area, thereby achieving the purpose of treatment through the regeneration of the nucleus pulposus tissue and avoiding the possibility of immune rejection in the patient receiving the transplant. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shown are the states of the tissue block at day 1 and day 4 of culture, in which the cell populations climb out of the tissue block and grow at the bottom of the culture dish;

[0023] Figures 2A-2D The results of using embryonic stem cell genes Nanog, Oct-4, and SOX2 to identify nucleus pulposus progenitor cells;

[0024] Figures 3A-3B Shows the results of a migration ability test on screened nucleus pulposus precursor cells;

[0025] Figures 4A-4D Demonstrate the identification of selected cell populations based on the characteristics of mesenchymal stem cells (MSCs);

[0026] Figures 5A-5D showing the results of the anti-inflammatory capacity test to identify the selected cell populations; and

[0027] Figure 6 Shown is a flow chart of the method for isolating and identifying nucleus pulposus precursor cells of the present invention.

[0028] Wherein, the reference numerals:

[0029] S101~S107: Steps DETAILED DESCRIPTION

[0030] Unless otherwise defined, all technical and scientific terms used in this specification have the same meanings as commonly understood by those skilled in the art. The present invention is illustrated and explained with reference to the following examples, which are intended to be illustrative and non-limiting. The present invention is not limited by these examples. Unless otherwise noted, the materials used in this invention are commercially available and readily available. The following examples are merely examples of available sources.

[0031] Example 1 Processing of Nucleus Pulposus Tissue Specimens

[0032] 1. First, in the operating room of a hospital, a sample of nucleus pulposus tissue from a patient with intervertebral disc degeneration or herniated disc is surgically removed under sterile conditions. The sample is placed in a sterile, sealed container containing an antibiotic composition and normal saline solution. The sample is then processed under a sterile operating table in a Good Tissue Practice (GTP) laboratory.

[0033] 2. Record the weight of the nucleus pulposus tissue specimen and the basic information of the patient;

[0034] 3. Place the specimen in a 10 cm petri dish containing 2 mL of medium and cut the specimen into smaller pieces with an average size of less than 1 mm. 3 Small pieces of tissue; one 10 cm dish contains about 5 g of tissue. If there are more tissue pieces, divide the tissue pieces into several culture dishes according to this ratio. The medium includes the following components: DMEM (Dubeka's modified Eagle's medium), Human Platelet Lysate (human platelet lysate), and antibiotics;

[0035] 4. Prepare collagenase solution: 7 mL medium + 1 mL type I collagenase (i.e., the ratio of medium to type I collagenase in the collagenase solution is 7:1 (volume ratio);

[0036] 5. Add the prepared collagenase solution to the culture dishes containing tissue pieces, adding 8 ml of collagenase solution to each dish, and incubate in a 37°C, 5% CO2 incubator for 4 to 24 hours;

[0037] 6. After 4 to 24 hours of culture, wash the tissue pieces in each dish with 10 mL of Dulbecco's phosphate-buffered saline (DPBS) to remove the collagenase solution.

[0038] 7. Transfer the tissue block to a 10 cm petri dish containing 10 mL of medium and culture for 1 to 7 days;

[0039] 8. When multiple cell populations climb out of the tissue block and form multiple blocks at the bottom of the culture dish, each cell population in each block is collected and transferred to a different culture dish for separate culture; and

[0040] 9. Using an embryonic stem cell gene, the nucleus pulposus precursor cells are screened out from each of the cell populations collected and cultured from each of the blocks.

[0041] Figure 1 The tissue blocks are shown in Figure 1 and 4 after culturing. We can clearly see that after 4 days of culture, the number of cell populations that crawled out of the tissue blocks and grew at the bottom of the culture dish increased significantly. In contrast, the number of cell populations that were cultured for 1 day was relatively small. Then, the cell populations that crawled out and grew in different blocks at the bottom of the culture dish were collected and moved to different culture dishes for further amplification and culture. Finally, these cells were identified (qualitatively), for example, using the embryonic stem cell genes Nanog, Oct-4, and SOX2 to screen out nucleus pulposus precursor cells.

[0042] Example 2: Genetic Identification of Nucleus Pulposus Progenitor Cells from Embryonic Stem Cells

[0043] In this example, the cell populations growing in different areas at the bottom of the culture dish were collected and amplified for culture. The embryonic stem cell (ESC) genes Nanog, Oct-4, and SOX2 were then used to screen for nucleus pulposus progenitor cells from these cell populations. Mesenchymal stem cells (MSCs) and the original parent population of nucleus pulposus cells were used as controls to compare the embryonic stem cell gene expression ratios (amounts) of these cell populations. A gene expression ratio greater than 1.5 was set as the selection threshold. That is, when the embryonic stem cell gene expression ratio in each cell population was greater than 1.5, the selection threshold was reached. Furthermore, only cell populations that reached the selection threshold for all three genes (Nanog, Oct-4, and SOX2) were selected for subsequent analysis.

[0044] Figure 2A The results of Nanog gene analysis for different cell populations (NPP2, 3, 4, 7, 9, 10, 14, 15, 17, 19, and 20) are shown. Among them, the cell populations NPP2, 3, 4, 7, 9, 10, 19, and 20 reached the selection threshold of an expression ratio greater than 1.5.

[0045] Figure 2B The results of Oct-4 gene analysis of different cell populations (NPP2, 3, 4, 7, 9, 10, 14, 15, 17, 19, and 20) are shown. Among them, cell populations NPP3, 4, 7, 10, 15, and 19 reached the selection threshold of an expression ratio greater than 1.5.

[0046] Figure 2C The results of SOX2 gene analysis for different cell populations (NPP2, 3, 4, 7, 9, 10, 14, 15, 17, 19, and 20) are shown. Among them, cell populations NPP3, 7, 9, 19, and 20 reached the selection threshold of an expression ratio greater than 1.5.

[0047] Figure 2D The chart shows the results of preliminary screening of nucleus pulposus progenitor cells from these cell populations using the embryonic stem cell genes Nanog, Oct-4, and SOX2. Only cell populations that meet the selection threshold for all three genes (Nanog, Oct-4, and SOX2) are selected for subsequent analysis. These selected cell populations are most likely to possess stem cell-like properties and have the greatest potential for therapeutic applications in nucleus pulposus tissue regeneration, as Nanog, Oct-4, and SOX2 are genes commonly associated with stem cell maintenance and pluripotency. Figure 2DThe hollow circles indicate that the expression ratio of the gene reached the selection threshold of greater than 1.5, while the solid circles indicate that the expression ratio of all three genes exceeded the selection threshold and the cell populations that were finally screened out and subsequently subjected to subsequent analysis included NPP3, NPP7 and NPP19.

[0048] Example 3 Mobility Test

[0049] In this example, cell populations NPP3, NPP7, and NPP19, selected based on embryonic stem cell genes, were further tested for their mobility. This experiment used Transwell culture dishes to assess cell mobility. The Transwell assay is a common method for measuring cell migration. Cells penetrate the permeable membrane beneath the Transwell and attach to it, migrating from one chamber to another, a process that simulates cell movement within a tissue.

[0050] Figure 3A The bar graph shows the percentage of cells that penetrated the membrane for the parental population of nucleus pulposus cells, as well as the NPP3, NPP7, and NPP19 cell populations. NPP19 showed the highest percentage of penetration, indicating that it had the greatest mobility in this assay, followed by NPP7, while NPP3 had lower mobility. The mobility of the parental population of nucleus pulposus cells was significantly lower. The images below the bar graph show cells stained with hematoxylin, a stain used to clearly visualize the cell nucleus, allowing for the counting of cells that penetrated the membrane.

[0051] In this example's motility assay, Western blot analysis was performed to examine the expression of proteins associated with cell motility. The proteins measured included N-cadherin, vimentin, β-catenin, and Snail, all of which are important proteins involved in cell adhesion, cytoskeletal structure, and cell motility. Figure 3B These proteins are expressed in all NPP3, NPP7, and NPP19 cell lines. β-actin was used as an internal control protein to ensure consistent protein loading across samples.

[0052] Example 4 Identification of Mesenchymal Stem Cells (MSCs) Characteristics

[0053] See also Figures 4A-4D In this example, the cell populations NPP3, NPP7, and NPP19 screened above were again tested for identification based on their mesenchymal stem cell characteristics. Figure 4AThe proliferation of different cell populations (Parental and cell populations NPP3, NPP7, and NPP19) on days 1, 3, 5, and 7 of culture is shown. All cell populations proliferated over time, as indicated by increasing ratios. On day 1, the proliferation ratios of all cell populations were close to 1, indicating that they were roughly at the same starting point at the beginning of culture. Comparing the different time points, NPP7 exhibited higher proliferation ratios on days 5 and 7 compared to the other cell populations. In particular, on day 7, NPP7's proliferation ratio reached approximately 10, significantly higher than that of the other cell populations, indicating a stronger proliferative capacity at these time points. On days 5 and 7, the proliferation ratio of NPP19 was intermediate between that of NPP7 and NPP3. On day 5, the proliferation ratio of NPP3 was no different from that of the original parent population of nucleus pulposus cells; however, on day 7, the proliferation ratio of NPP3 was slightly higher than that of the original parent population of nucleus pulposus cells. The higher proliferation ratios of NPP7 and NPP19 indicate that they have stronger proliferation capabilities and can provide the required number of cells in a shorter period of time. Therefore, they can more effectively repair damaged nucleus pulposus tissue and achieve the therapeutic goal of nucleus pulposus tissue regeneration.

[0054] Figure 4B The cell populations NPP3, NPP7, and NPP19 were identified based on their expression of the highly mesenchymal stem cell-associated genes STRO-1, C-KIT, β-catenin, Jagged, and Delta4, with the GAPDH gene serving as an internal reference gene. All three cell populations, NPP3, NPP7, and NPP19, expressed these five mesenchymal stem cell genes, indicating that they retain mesenchymal stem cell characteristics. Compared with the original parent population of nucleus pulposus cells, NPP3, NPP7, and NPP19 had higher expression of mesenchymal stem cell genes. The expression levels of all five genes in NPP3 and NPP7 were generally higher than those in NPP19, with particularly large differences in the expression of STRO-1, C-KIT, and β-catenin. This suggests that NPP3 and NPP7 have more mesenchymal stem cell characteristics and may be more effectively involved in the repair and regeneration of nucleus pulposus tissue.

[0055] Figure 4CFlow cytometric analysis of the expression of six different stem cell surface antigens (CD markers): CD34, CD133, CD44, CD73, CD90, and CD105, was performed on the selected cell populations NPP3, NPP7, and NPP19. NPP3, NPP7, and NPP19 express the antigens CD44, 73, 90, and 105 on their cell surfaces, which are typical surface markers of mesenchymal stem cells. This indicates that the cell populations NPP3, NPP7, and NPP19 share characteristics similar to mesenchymal stem cells and may possess similar functions and differentiation potential, potentially enabling applications in the repair and regeneration of nucleus pulposus tissue. Furthermore, NPP3, NPP7, and NPP19 do not express surface antigens such as CD34 and 133, which are characteristic of hematopoietic stem cells. This further confirms their similarity to mesenchymal stem cells, rather than their lack of hematopoietic stem cell characteristics.

[0056] Figure 4D The gene expression ratios of the selected cell populations NPP3, NPP7, and NPP19 under three different differentiation conditions are shown. Black bars represent expression levels in the control group, and gray bars represent expression levels in the induced group. Cell populations NPP3, NPP7, and NPP19 were able to increase the expression ratios of genes associated with chondrogenesis, osseous differentiation, and adipogenesis, respectively, under the induction conditions. This multidirectional differentiation ability is of great significance for tissue engineering and regenerative medicine, as these cells can be used to repair or replace damaged nucleus pulposus tissue.

[0057] Example 5 Anti-inflammatory ability test

[0058] See also Figures 5A-5D In this example, the above-screened cell populations NPP3, NPP7, and NPP19 were further identified using an anti-inflammatory assay. The anti-inflammatory assay included testing the survival percentage of the parental nucleus pulposus cell population and the NPP3, NPP7, and NPP19 cell populations under an inflammatory environment (treated with IL-1β (interleukin-1β) + TNF-α (tumor necrosis factor-α) and LPS (lipopolysaccharide)); and testing the expression levels of inflammatory genes (IL-1β, COX-2, and MMP3) in the parental nucleus pulposus cell population and the NPP3, NPP7, and NPP19 cell populations under an inflammatory environment.

[0059] Figure 5AShown are the survival percentages of the original parent population of nucleus pulposus cells (Parental) and the NPP3, NPP7, and NPP19 cell populations under normal conditions (CTRL, control group) and in an inflammatory environment (IL-1β+TNF-α and LPS treatment). Figure 5A It can be seen that in an inflammatory environment, the cell growth of the original mother population of nucleus pulposus cells will be inhibited, and thus its survival rate will decrease, while the survival rate of the cell population (NPP3, NPP7, NPP19) has not decreased significantly. This indicates that the cell population (NPP3, NPP7, NPP19) has a good resistance to the inflammatory environment and can continue to grow and proliferate in it.

[0060] Figure 5B The figure shows the expression ratio of the inflammatory gene IL-1β in the parental nucleus pulposus cell population and the NPP3, NPP7, and NPP19 cell populations under normal conditions (CTRL, control group) and under inflammatory conditions (IL-1β + TNF-α and LPS treatment). The parental nucleus pulposus cell population showed a significant increase in the expression ratio of the IL-1β gene under inflammatory conditions, while this increase was not significant in the cell populations (NPP3, NPP7, and NPP19). This may indicate that the cell populations (NPP3, NPP7, and NPP19) have a certain inhibitory effect on the inflammatory response and are not easily disturbed.

[0061] Figure 5C Shown are the expression ratios of the inflammatory gene COX-2 in the parental nucleus pulposus cell population and the 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, COX-2 expression increased in the parental nucleus pulposus cell population under inflammatory conditions, while the increases in the NPP3, NPP7, and NPP19 cell populations were less pronounced, further supporting the anti-inflammatory properties of the NPP3, NPP7, and NPP19 cell populations.

[0062] Figure 5D The figure shows the expression ratios of the inflammatory gene MMP3 in the parental nucleus pulposus cell population and the NPP3, NPP7, and NPP19 cell populations under normal conditions (CTRL, control group) and under inflammatory conditions (IL-1β + TNF-α and LPS treatment). Consistent with the expression trends of the first two inflammatory genes (IL-1β and COX-2), the expression ratio of MMP3 in the parental nucleus pulposus cell population increased significantly under inflammatory conditions, while the expression of the cell populations (NPP3, NPP7, and NPP19) was less affected.

[0063] The anti-inflammatory activity tests demonstrated that the nucleus pulposus precursor cells (populations NPP3, NPP7, and NPP19) screened in this study exhibited enhanced survival and lower expression of inflammatory genes under inflammatory conditions compared to the original nucleus pulposus cell population. This indicates that populations NPP3, NPP7, and NPP19 possess significant anti-inflammatory potential, which is highly valuable in clinical applications, particularly in conditions requiring controlled inflammatory responses, such as nucleus pulposus tissue repair and treatment.

[0064] Figure 6 Shown is a flow chart of the method for isolating and identifying nucleus pulposus precursor cells of the present invention, which comprises the following steps:

[0065] S101 (Providing a nucleus pulposus tissue): A nucleus pulposus tissue specimen is surgically removed aseptically from a patient with intervertebral disc degeneration or intervertebral disc herniation;

[0066] S102 (cutting into multiple tissue blocks): then cutting the nucleus pulposus tissue specimen into multiple tissue blocks;

[0067] S103 (enzyme treatment): hydrolyzing the tissue block with an enzyme, such as collagenase or trypsin;

[0068] S104 (Undigested tissue blocks culture): Wash with DPBS to remove the enzyme, and then culture the undigested tissue blocks in a culture dish for 1 to 7 days.

[0069] S105 (cell colonies climbing out): After the unconsumed tissue mass is cultured in the culture dish for 1 to 7 days, multiple cell colonies will climb out of the tissue mass and form multiple blocks at the bottom of the culture dish;

[0070] S106 (collecting each cell population in each block): each cell population in each block is collected and moved to different culture dishes for amplification and culture; and

[0071] S107 (Cell Characterization): The embryonic stem cell genes Nanog, Oct-4, and SOX2 are then used to screen nucleus pulposus progenitor cells from each cell population using the method described in Example 2. The selected nucleus pulposus progenitor cells are then further identified for their mobility, mesenchymal stem cell (MSC) properties, and anti-inflammatory ability using the methods described in Examples 3 to 5 to obtain the nucleus pulposus progenitor cells most suitable for nucleus pulposus tissue regeneration.

[0072] The above description fully and clearly illustrates the present invention's method for isolating and identifying nucleus pulposus precursor cells and its uses. It must be emphasized that the above detailed description is a specific description of a feasible embodiment of the present invention, but the embodiment is not intended to limit the scope of the present invention. Any equivalent implementation or modification that does not depart from the spirit of the present invention should be included in the scope of the present application.

Claims

1. A method for isolating and identifying nucleus pulposus precursor cells, comprising: a) providing a nucleus pulposus tissue, and cutting the nucleus pulposus tissue into a plurality of tissue blocks; b) hydrolyzing the tissue mass with an enzyme; c) removing the enzyme and culturing the tissue piece in a culture dish; as well as d) when the multiple cell populations crawl out from the tissue block and form multiple blocks at the bottom of the culture dish, screening the nucleus pulposus precursor cells from each of the cell populations in each of the blocks using an embryonic stem cell gene; The embryonic stem cell gene is at least one selected from the group consisting of Nanog, Oct-4 and SOX2.

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

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

4. The method according to claim 1, wherein The nucleus pulposus precursor cells having mesenchymal stem cell characteristics can be further screened using at least one selected from the group consisting of CD34, CD44, CD73, CD90, CD105, and CD133 stem cell surface antigens.

5. The method according to claim 1, wherein The nucleus pulposus progenitor cells with differentiation ability can be further screened using a differentiation ability, and the differentiation ability is at least one selected from the group consisting of chondrogenesis, osseogenesis, and adipocytogenesis.

6. The method according to claim 1, wherein The nucleus pulposus progenitor cells with anti-inflammatory ability can be further screened using at least one selected from the group consisting of IL-1β, COX-2 and MMP3 cell inflammation genes.

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

8. Use of the nucleus pulposus precursor cells obtained by the method according to claim 1 in preparing a pharmaceutical composition for treating low back pain.

9. The use according to claim 8, wherein The nucleus pulposus progenitor cells with mobility can be further screened using at least one selected from the group consisting of N-Cadherin, Vimentin, β-Catenin, and Snail proteins, and / or the nucleus pulposus progenitor cells with mesenchymal stem cell characteristics can be screened using at least one selected from the group consisting of CD34, CD44, CD73, CD90, CD105, and CD133 stem cell surface antigens.

10. The use according to claim 9, wherein The nucleus pulposus progenitor cells having differentiation ability can be further screened using a differentiation ability, wherein the differentiation ability is at least one selected from the group consisting of chondrogenesis, osseogenesis, and adipogenesis; and / or the nucleus pulposus progenitor cells having anti-inflammatory ability can be screened using at least one selected from the group consisting of IL-1β, COX-2, and MMP3 cell inflammation genes.

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