Deciduous tooth pulp mesenchymal stem cell population, separation and extraction method and proliferation potential identification method

Abstract

The invention discloses a deciduous tooth pulp mesenchymal stem cell population, a separation and extraction method and a proliferation potential identification method. According to the separation method, SHED is efficiently separated from dental pulp tissues of deciduous teeth through an optimized mixed enzyme digestion method (3 mg / mL of neutral protease and 4 mg / mL of II type collagenase), and the separation success rate reaches 76% or above. According to the identification method, a proliferation-function-aging three-in-one evaluation system is constructed, and the method comprises the following steps: quantitatively evaluating proliferation potential through a clone formation rate (greater than or equal to 10%), an S-phase cell proportion (greater than or equal to 20%) and a Ki-67 positive rate (greater than or equal to 60%); the migration and paracrine capabilities are evaluated through a scratch experiment (the healing rate in 24 hours is greater than or equal to 35%) and a conditioned medium function experiment; the aging trend is evaluated through SA-beta-gal staining (the positive rate is lt; 10%). The quantitative standard of the high-quality SHED is defined for the first time, and reliable technical support is provided for quality control and standardized application of the high-quality SHED in regenerative medicine.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a population of mesenchymal stem cells from deciduous dental pulp, a method for their isolation and extraction, and a method for identifying their proliferation potential. In particular, it relates to a method for high-quality isolation and extraction of mesenchymal stem cells from desquamated deciduous dental pulp and for systematic identification of their proliferation potential. Background Technology

[0002] Mesenchymal stem cells (MSCs), as a type of adult stem cell with multi-lineage differentiation potential, have attracted increasing attention in recent years. Currently, dental pulp stem cells (DPSCs) are becoming a research hotspot, particularly showing great promise in bone, cartilage, and nerve repair, as well as immunomodulatory therapy. At present, MSCs used in research and clinical practice are mainly derived from bone marrow-derived MSCs (BMMSCs) and adipose-derived MSCs (ADSCs). However, these traditional sources have significant limitations: First, the acquisition process is invasive; both bone marrow extraction and fat aspiration are surgical procedures, causing pain, potential infection risks, and recovery periods for the donor. Second, the number of cells, their proliferative activity, and differentiation potential are negatively correlated with the donor's age, and many degenerative diseases (such as osteoarthritis) are prevalent in the elderly, making it difficult to obtain high-quality MSCs from this population. Furthermore, certain ethical controversies arise.

[0003] Since Gronthos et al. first successfully isolated DPSCs in 2000, deciduous tooth DPSCs, periodontal ligament stem cells, and other cells have been discovered. DPSCs can differentiate into various cell types, including dentin cells and vascular endothelial cells, under specific induction, showing broad application prospects. The discovery of Stem Cells from Human Exfoliated Deciduous Teeth (SHED) provides a highly attractive new cell source for regenerative medicine. Compared with BMMSCs and ADSCs, SHEDs have unparalleled advantages: 1. Non-invasive and ethically friendly source: SHED is derived from children's naturally shed baby teeth. This is a physiological process with no additional trauma and does not involve ethical controversies.

[0004] 2. More primitive cells: Studies have shown that stem cells derived from children's tissues typically exhibit stronger proliferative activity and differentiation potential. The proliferation rate and clonogenic capacity of SHEDs have been demonstrated to be higher than those of adult-derived MSCs (such as dental pulp stem cells DPSCs derived from wisdom teeth).

[0005] 3. Easy to obtain and store: The shedding period of deciduous teeth is concentrated, which makes it easy to establish a biobank and realize the long-term preservation and future application of resources.

[0006] Despite the significant advantages of SHED, research on it both domestically and internationally is still in its early stages, requiring extensive experimental and clinical studies. Therefore, finding suitable tooth sources is particularly important, as there is currently no unified standard. Moving from laboratory research to clinical application still faces two major technical bottlenecks: First, the separation methods are not standardized enough, resulting in unstable success rates and cell quality.

[0007] In existing technologies, SHED separation mostly employs enzymatic digestion, but key parameters such as enzyme type, concentration, and digestion time vary greatly. Common methods include using collagenase alone (such as type I or type II) or in combination with dispersants. These methods generally suffer from the following problems: The definition of success rate is vague: Most studies have not given a clear and unified definition and statistics for "successful isolation", but only use "obtaining adherent cells" as the marker. This leads to large fluctuations in the isolation success rate between different laboratories and even between different batches in the same laboratory, and cell yield cannot be guaranteed.

[0008] Impaired cell viability: Digestion conditions (such as excessively high enzyme concentration or excessively long time) may cause irreversible damage to cell membrane surface proteins and activity, affecting initial cell adhesion and subsequent cell expansion.

[0009] Interference from impurity cells: Incomplete digestion or insufficient washing may lead to the overgrowth of impurity cells such as fibroblasts, affecting the purity and functional studies of SHED.

[0010] Second, there is a lack of a systematic and objective system and quality standards for assessing proliferation potential.

[0011] Proliferative potential is a key indicator determining whether MSCs can be effectively expanded in vitro to meet therapeutic dosage requirements. Currently, there are serious deficiencies in the quality assessment of isolated SHEDs, especially in the identification of their proliferative potential: The methods are simplistic and subjective: conventional identification methods are mostly limited to observing cell morphology (spindle-shaped, fibroblast-shaped) or performing simple cell counting and doubling time calculations. These methods are easily influenced by subjective judgment and cannot deeply reflect the heterogeneity of cell populations and the true proliferative state.

[0012] Lack of prospective screening indicators: Morphological observation and growth curves are post-hoc validation methods, unable to accurately predict and screen the expansion potential of cell populations at an early stage (e.g., before P3 generation), leading to potential expansion failures due to poor "seed cell" quality. Lack of unified quantitative standards: What level of proliferation capacity can be considered "high-quality" SHED? Currently, there is a lack of a universally accepted quantitative standard encompassing multiple core indicators. This makes it difficult to compare results from different studies and fails to provide crucial evidence for quality control of cell therapy products.

[0013] In summary, current technologies lack both an optimized separation scheme for reliably obtaining highly active SHED and a system for early, objective, and quantitative identification of its proliferation potential and quality grading. This gap severely restricts in-depth basic research and clinical translational applications of SHED. Therefore, developing a systematic method integrating efficient separation and scientific identification has become an urgent technical need in this field. Summary of the Invention

[0014] The primary objective of this invention is to overcome the shortcomings of existing technologies, such as unstable success rate and inconsistent cell quality in the isolation of deciduous tooth pulp mesenchymal stem cells (SHED), and to provide a standardized and highly successful method for the isolation and extraction of SHED.

[0015] Another objective of this invention is to address the subjective and one-sided nature of existing SHED quality assessment methods, provide a systematic, quantitative, and reproducible system for identifying proliferation potential, and for the first time clearly define the quantitative criteria for high-quality SHEDs, providing key technical support for stem cell quality control and clinical application screening.

[0016] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for high-quality isolation and extraction of deciduous tooth pulp mesenchymal stem cells (SHED), characterized by comprising the following steps: 1. Sample pretreatment: Take the child's naturally shed intact deciduous teeth and, within 24 hours of removal from the body, thoroughly clean and disinfect the surface using PBS containing antibiotics and 75% ethanol.

[0017] 2. Tissue Acquisition and Processing: Under aseptic conditions, the tooth is drilled open to expose the pulp chamber. The pulp tissue is completely removed using a pulp plucking needle and then cut into pieces approximately 1 mm using sterile ophthalmic scissors. 3 A tissue block of a certain size.

[0018] 3. Optimize enzyme digestion: Transfer the shredded tissue pieces to a mixed enzyme solution consisting of 3 mg / mL neutral protease and 4 mg / mL type II collagenase, and digest in a shaker at 37°C for 45 minutes.

[0019] 4. Cell collection and primary culture: Add serum-containing complete culture medium to stop digestion, filter through a 70 μm cell sieve, centrifuge to collect cells, resuspend and seed in culture flasks, and incubate in a 37℃, 5% CO2 incubator.

[0020] 5. Criteria for successful isolation: Successful isolation is defined as the formation of typical cell colonies within 21 days of primary culture and successful passage to the third generation (P3). Using this method, the isolation success rate of SHED is no less than 76%.

[0021] Further, in step 1, the antibiotic is preferably a penicillin-streptomycin bispecific antibiotic solution.

[0022] Preferably, in step 3, the digestion is carried out gently by oscillation at 100 rpm in a constant-temperature shaker to improve digestion efficiency. More preferably, the volume ratio of the mixed enzyme solution to the tissue block is 3:1 to 5:1.

[0023] Secondly, the present invention provides a method for identifying the proliferation potential of deciduous tooth pulp mesenchymal stem cells (SHEDs), characterized in that the P3 generation SHEDs isolated by the above method are subjected to the following three tests: 1. Colony formation rate determination: Cells were seeded at a density of 1000 cells / well, cultured for 14 days, and then stained and counted. The colony formation rate was not less than 10%.

[0024] 2. Cell cycle distribution analysis: Cell cycle was detected by flow cytometry, and the proportion of cells in S phase was not less than 20%.

[0025] 3. Detection of Ki-67 positivity rate: Ki-67 expression was detected by immunofluorescence, and the proportion of Ki-67 positive cells was not less than 60%.

[0026] Furthermore, in the colony formation rate determination, the dye used for staining is a 0.1% crystal violet solution.

[0027] Preferably, the staining solution used in the cell cycle distribution analysis is a propidium iodide (PI) / RNase A mixed staining solution.

[0028] Furthermore, the anti-Ki-67 antibody used in the Ki-67 positivity rate detection is a mouse anti-human monoclonal antibody.

[0029] Thirdly, this invention further provides a method for identifying SHED migration ability and paracrine function: 1. Cell migration ability assay: The lateral migration ability of cells is assessed by scratch assay, or the directional migration ability of cells is assessed by Transwell assay. The healing rate of high-quality SHEDs should be no less than 35% after 24 hours of scratch assay, and the number of migrating cells in Transwell assay should be significantly higher than that of control (such as adult-derived DPSCs).

[0030] 2. Verification of paracrine function: SHED conditioned medium was collected and used to culture human umbilical vein endothelial cells. Its promoting effect on endothelial cell proliferation was determined by the MTT assay, its migration-promoting ability was determined by the scratch assay, or its angiogenesis-promoting ability was assessed by the tube formation assay. High-quality SHED conditioned medium should possess significant paracrine proliferative and / or migration-promoting activities.

[0031] Fourthly, this invention provides an early assessment method for SHED aging trends, used to predict the long-term expansion stability of cell populations: 1. Cell morphology analysis: The morphology of P3 generation SHED cells was observed under a microscope. High-quality cells should maintain a uniform, slender spindle shape. If a large number of flattened, enlarged cells with increased cytoplasmic granulation are present, it indicates a significant aging trend.

[0032] 2. SA-β-Gal Activity Assay: P3 generation SHED cells were stained using an SA-β-Gal staining kit (Shanghai Beyotime Biotechnology Co., Ltd.) (Product No.: C0602). In a high-quality SHED population, the proportion of SA-β-Gal-positive cells should be less than 10%.

[0033] Analysis of aging-related protein expression: The expression levels of aging-related markers such as P16, P21, or P53 in P3 generation SHEDs were detected by immunofluorescence or Western blotting. High-quality SHEDs should show low levels of aging-related protein expression.

[0034] Fifthly, the present invention provides a criterion for determining high-quality deciduous tooth pulp mesenchymal stem cells (SHED), characterized in that the following three conditions are met simultaneously: The clone formation rate is no less than 10%; The proportion of cells in S phase should not be less than 20%; The proportion of Ki-67 positive cells should not be less than 60%.

[0035] Preferably, to obtain cells with better function and more stable expansion potential, at least one of the following enhancement conditions is also met: (4) The healing rate of the scratch test after 24 hours is not less than 35%; (5) The proportion of SA-β-Gal positive cells is less than 10%.

[0036] The novelty and advantages of this invention are as follows: High separation efficiency and good stability: By optimizing the enzyme digestion system and clarifying the success criteria, efficient and stable separation of SHED is achieved, with a success rate significantly increased to over 76%, solving the problem of unstable sources; Scientific and objective identification system: For the first time, colony-forming ability, cell cycle activity, and specific proliferation markers are combined to construct a multi-dimensional and quantitative proliferation potential identification system, with accurate and reliable results, overcoming the subjectivity and one-sidedness of traditional methods; The newly added assessment of migration ability, paracrine function, and aging trend upgrades the single proliferation potential identification into a comprehensive quality assessment system integrating "proliferation-function-aging," which can more accurately predict cell behavior and long-term expansion stability in the therapeutic environment, providing more core technical support for the quality control of cell therapy products. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the overall process of SHED separation, extraction and identification in this invention; Figure 2 The images show typical cell morphology photographs (left, inverted microscope, scale bar: 100μm) and a statistical chart of isolation success rate (right) from the primary SHED culture in Example 1. Figure 3 The images show the clonal formation of SHED in Example 2 (left, stained with crystal violet) and a statistical chart of clonal formation rate (right). Figure 4 The image shows the cell cycle flow cytometry (left) and the statistical distribution of each phase of the cell cycle in Example 2 (right). Figure 5 This is the Ki-67 immunofluorescence staining image of SHED in Example 2 (green: Ki-67, blue: DAPI, scale bar: 50μm) and the positive rate statistics (right). Figure 6 These are typical comparison photos (left, scale bar: 200μm) of SHED scratch test at 0 hours and 24 hours in Example 4, and a statistical chart of scratch healing rate (right). Figure 7 These are typical photographs (scale bar: 100 μm) showing the effect of SHED conditioned medium (SHED-CM) on HUVEC tube formation in Example 4. Figure 8 This is a typical photograph of SA-β-Gal staining of P3 generation SHED cells in Example 4 (left, blue indicates positive cells, scale bar: 50μm). Detailed Implementation

[0038] The present invention will be further described below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0039] Example 1: High-quality separation and extraction of SHED and success rate statistics First, naturally shed healthy deciduous teeth from children are collected and processed within 24 hours in a laminar flow hood. After rinsing with PBS containing 1% antibiotics and surface disinfection with 75% ethanol, the pulp chamber is opened along the cementoenamel junction using a sterile high-speed dental handpiece. The intact pulp tissue is then removed non-invasively with a pulp plucking needle and immediately minced into approximately 1 mm pieces using ophthalmic scissors. 3 Tissue blocks were collected. The tissue blocks were transferred to centrifuge tubes containing 5 times their volume of a mixed enzyme solution (3 mg / mL neutral protease + 4 mg / mL type II collagenase, dissolved in a general serum-free α-MEM solution). The cells were digested at 37°C and 100 rpm for 45 minutes with gentle pipetting every 15 minutes. After digestion, an equal volume of α-MEM complete medium containing 15% fetal bovine serum was added to terminate the reaction. The cell suspension was filtered through a 70 μm filter, centrifuged to collect cells, resuspended, and seeded into T25 culture flasks. The flasks were then incubated at 37°C in a 5% CO2 incubator. The medium was completely changed for the first time after 48 hours, and thereafter every 2-3 days. Daily observation under an inverted microscope revealed that approximately 76% (19 / 25 cases) of the samples showed typical spindle cell adhesion and colony formation within 7-14 days (see [link to relevant documentation]). Figure 2 (Left), and all can be successfully digested with trypsin and stably passaged to the 3rd generation (P3) within 21 days. This is considered a successful isolation. See the statistics on isolation success rate for details. Figure 2 right.

[0040] Example 2: Systematic identification of SHED proliferation potential The three successfully isolated P3 generation SHEDs (numbered #05, #12, and #19) were subjected to multidimensional identification of their propagation potential.

[0041] (1) Colony formation rate determination: Cells were seeded at a density of 1000 cells / well in 6-well plates and cultured for 14 days. After fixation with 4% paraformaldehyde and staining with 0.1% crystal violet, clones with more than 50 cells were counted (see Figure 3 (Left). The average colony formation rate of the three cell lines was calculated to be 17.3% (17.2%, 16.5%, and 18.1%, respectively), which is significantly higher than the standard of 10%.

[0042] (2) Cell cycle distribution analysis: After cells were collected and fixed with 70% ice-cold ethanol, they were stained with PI / RNase A. Flow cytometry analysis showed that the average proportion of S phase in the three cell lines was 24.5% (24.8%, 23.1%, and 25.5%, respectively), which exceeded the threshold of 20% (see flow cytometry). Figure 4 See left for statistical results. Figure 4 right).

[0043] (3) Ki-67 positivity rate detection: Immunofluorescence assay (using mouse anti-human Ki-67 monoclonal primary antibody (commonly used in the industry) and FITC-labeled secondary antibody (standard reagent) combined with DAPI counterstaining of nuclei, counting showed that the average Ki-67 positivity rate of the three cell lines reached 71.5% (71.5%, 69.8%, and 73.2%, respectively), which is higher than the standard requirement of 60% (see typical fluorescence images). Figure 5 See left for statistical results. Figure 5 right).

[0044] The above three indicators together confirm that the SHED obtained by the method of this invention has excellent proliferation potential.

[0045] Example 3: Comprehensive assessment of SHED migration ability, paracrine function, and aging trend Supplementary identification was performed on the same batch of SHEDs (numbers #12, #23, and #25) separated in Example 1.

[0046] (1) Cell migration ability assay: Cell migration ability was assessed using a scratch assay. Cells were cultured in 6-well plates until complete confluence. A 200 μL sterile pipette tip was used to make a straight scratch on the cell monolayer. After washing with PBS, the culture medium was replaced with fresh medium. Images were taken under a microscope at 0 and 24 hours (see typical comparison photos). Figure 6 (Left) The scratch area was measured and the healing rate was calculated using ImageJ software. The results showed that the scratch healing rates of the three SHED strains after 24 hours were 51.6%, 44.9%, and 49.2%, respectively, with an average of 48.6% (see statistical results). Figure 6 (Right), far exceeding the 35% of the present invention standard.

[0047] (2) Paracrine function verification: Serum-free conditioned medium (SHED-CM) was collected from P3 generation SHED cells cultured for 48 hours. Human umbilical vein endothelial cells (HUVECs) were cultured in this medium, with conventional endothelial cell culture medium as a control. MTT assay showed that the proliferation activity of HUVECs in the SHED-CM treatment group was significantly higher than that in the control group (p<0.01) (see typical photographs). Figure 7 Further evidence from HUVEC scratch experiments confirmed that SHED-CM can significantly accelerate the migration and healing of HUVECs.

[0048] (3) Assessment of aging trend: P3 generation SHED cells were stained with aging-related β-galactosidase (SA-β-Gal). The procedure was strictly followed according to the kit instructions. After staining, blue positive cells were counted under an optical microscope (see typical staining photographs). Figure 8 The results showed that the proportions of SA-β-Gal positive cells in the three SHED strains were 6.1%, 5.2%, and 4.9%, respectively, with an average of 5.4%, far below the warning threshold of 10%. Meanwhile, the cells exhibited uniform morphology, maintaining a typical spindle shape, without obvious flattening, enlargement, or other senescent morphologies.

[0049] The comprehensive evaluation results show that the high-quality SHED isolated by the method of this invention not only has strong proliferation potential, but also excellent migration and paracrine functions, and the proportion of senescent cells is extremely low, demonstrating its great potential as a high-quality "off-the-shelf" cell therapy product.

[0050] Example 4: Comparison of proliferative potential with adult DPSCs To highlight the proliferative advantage of SHEDs obtained in this invention, dental pulp stem cells (DPSCs) were isolated from five adult orthodontic wisdom teeth using the same isolation method as in Example 1, and their P3 generation cells were identified using the same standards. The results showed that the average colony formation rate of DPSCs was only 8.1%, the average proportion of S-phase cells was 15.3%, and the average Ki-67 positivity rate was 45.7%, all three core indicators being significantly lower than those of SHEDs isolated in this invention. (Relevant comparative data can be found in...) Figure 3-5 (As shown in the statistical chart) This comparison powerfully demonstrates the unique advantages of the method of the present invention in obtaining cells with high proliferative potential, and also verifies the effectiveness and necessity of the quality judgment criteria established by the present invention in distinguishing between superior and inferior cells.

Claims

1. A method for isolating and extracting mesenchymal stem cells from the pulp of deciduous teeth, characterized in that, Includes the following steps: 1) Sample pretreatment: Take deciduous teeth and disinfect them by rinsing with disinfectant solution; 2) Tissue Acquisition: The pulp tissue is removed from the pulp cavity and minced; 3) Enzymatic digestion: The shredded dental pulp tissue is placed in a mixed enzyme solution containing neutral protease and collagenase for digestion; 4) Termination of digestion and culture: Add complete culture medium to terminate digestion, filter, and then inoculate for culture; 5) Determining successful isolation: If cell colonies are formed within 21 days and successfully passaged to the 3rd generation, the isolation is considered successful.

2. The method for isolating and extracting mesenchymal stem cells from deciduous tooth pulp as described in claim 1, characterized in that, In step 3), the concentration of the neutral protease is 3 mg / mL, and the collagenase is type II collagenase with a concentration of 4 mg / mL.

3. The method for isolating and extracting mesenchymal stem cells from deciduous tooth pulp as described in claim 1, characterized in that, In step 4), the complete culture medium is α-MEM complete culture medium.

4. The method for isolating and extracting mesenchymal stem cells from deciduous tooth pulp as described in claim 1, characterized in that, In step 5), the success rate of separation is no less than 76%.

5. A method for identifying the proliferation potential of mesenchymal stem cells in deciduous dental pulp, characterized in that, The third-generation deciduous tooth pulp mesenchymal stem cells isolated by the method for isolating and extracting deciduous tooth pulp mesenchymal stem cells as described in any one of claims 1 to 4 were identified as follows: 1) Clonal formation rate determination; 2) Cell cycle distribution analysis; 3) Detection of the positive rate of the proliferation marker Ki-67; This allows us to assess the cell's proliferation potential.

6. The method for identifying the proliferation potential of mesenchymal stem cells in deciduous tooth pulp as described in claim 5, characterized in that, The colony formation rate determination specifically includes: seeding deciduous tooth pulp mesenchymal stem cells at a density of 1000 cells per well in a 6-well plate, culturing for 14 days, staining and counting the cells.

7. The method for identifying the proliferation potential of mesenchymal stem cells in deciduous tooth pulp as described in claim 5, characterized in that, The cell cycle distribution analysis was performed using flow cytometry.

8. The method for identifying the proliferation potential of mesenchymal stem cells in deciduous tooth pulp as described in claim 5, characterized in that, The positive rate of the proliferation marker Ki-67 was detected using immunofluorescence assay.

9. The method for identifying the proliferation potential of mesenchymal stem cells in deciduous dental pulp as described in claim 5, characterized in that, When all three of the following conditions are met: 1) The clonal formation rate is not less than 10%; 2) The proportion of cells in the S phase of the cell cycle is not less than 20%; 3) The proportion of Ki-67 positive cells is not less than 60%; It was determined to be high-quality mesenchymal stem cells from the pulp of deciduous teeth.

10. The method for identifying the proliferation potential of mesenchymal stem cells in deciduous dental pulp as described in claim 5, characterized in that, The aforementioned identification also includes any one of the following: 4) Cell migration ability assay: The lateral migration ability of cells is assessed by scratch assay, or the directional migration ability of cells is assessed by Transwell assay. 5) Paracrine function verification: The ability of its conditioned medium to promote the proliferation and / or migration of human umbilical vein endothelial cells was assessed. 6) Assessment of aging trends, using cell morphology observation and SA-β-Gal activity assay.

11. The method for identifying the proliferation potential of mesenchymal stem cells in deciduous dental pulp as described in claim 10, characterized in that, When all three of the following conditions are met: 1) The clone formation rate is not less than 10%; 2) The proportion of cells in the S phase of the cell cycle is not less than 20%; 3) The proportion of Ki-67 positive cells is not less than 60%; And it meets at least one of the following conditions: 4) The healing rate after 24 hours of scratch testing should not be less than 35%; 5) The positive rate of SA-β-Gal is less than 10%; These were identified as high-quality, functionally superior, and more stable deciduous tooth pulp mesenchymal stem cells.

12. A kit for identifying the proliferation potential of mesenchymal stem cells in deciduous dental pulp, characterized in that, It includes crystal violet staining solution for colony formation rate determination, PI / RNase A staining solution for cell cycle analysis, and anti-Ki-67 antibody for Ki-67 positivity rate detection.

13. A population of mesenchymal stem cells from the pulp of deciduous teeth, characterized in that, Its P3 generation cells simultaneously satisfy: 1) The positive rate of SA-β-Gal is less than 10%; 2) The healing rate after 24 hours of scratch testing should not be less than 35%; 3) The clone formation rate is not less than 10%.

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