Method for enhancing regeneration capacity of stem cells and culture preparation
The enhanced culture medium coated with CHIR-99021, RGD tripeptide and KLT peptide was solved by laminin, which greatly improved the regeneration ability of hair follicle mesenchymal stem cells and slowed down aging, and achieved efficient regulation of stem cell function.
Patent Information
- Application Number
- CN202510772156.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-11
- Publication Date
- 2025-08-22
AI Technical Summary
The traditional HFSC culture system relies on fetal bovine serum and macromolecular growth factors, and has large batch differences, high risk of inducing differentiation, strong immune origin, and difficult to meet the needs of precise regulation and clinical transformation. Aging HFSCs show problems such as dry loss and decreased proliferation ability.
Laminin coated with enhanced culture medium containing CHIR-99021, RGD tripeptide and KLT peptide was used to enhance the regeneration ability of hair follicle mesenchymal stem cells and slow aging through the synergistic effect of physical simulation and chemical activation.
It significantly improves the proliferation ability and cell stemness of hair follicle mesenchymal stem cells, inhibits cell aging, and achieves efficient regulation of stem cell functions, which is better than traditional culture systems.
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Figure CN120519383A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of stem cell culture, and in particular relates to a method for enhancing the regeneration ability of stem cells and a culture medium preparation. Background Art
[0002] Hair follicle stem cells (HFSCs), a minority of adult stem cells in skin tissue with high self-renewal and multidirectional differentiation potential, are widely involved in hair follicle regeneration, skin repair, and maintenance of dermal structure. The activity of HFSCs plays a central role in regulating the hair life cycle, wound healing, and even anti-aging skin engineering.
[0003] However, with aging or the accumulation of external stressors (such as oxidative damage and inflammatory stimulation), HFSCs exhibit phenotypes such as loss of stemness, decreased proliferation, and abnormal differentiation tendencies, ultimately leading to hair follicle atrophy and impaired hair regeneration, seriously affecting hair function and appearance, and limiting their potential applications in tissue engineering and regenerative medicine. Therefore, developing a culture system that can effectively activate the regenerative potential of aged HFSCs, maintain their stemness, and mimic their physiological microenvironment has become a current research focus.
[0004] Traditional HFSC culture systems rely heavily on exogenous proteins such as fetal bovine serum (FBS) and epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). However, these components suffer from large batch-to-batch variability, a high risk of inducing differentiation, and strong immunogenicity, making them difficult to meet the demands of precise regulation and clinical translation. Therefore, establishing an efficient, safe, and controllable culture system that is independent of traditional serum and macromolecular growth factors and can activate the regenerative potential of aging HFSCs is a pressing issue. Summary of the Invention
[0005] The purpose of the present invention is to provide a method and culture preparation for enhancing the regenerative ability of stem cells, thereby improving the proliferation ability and cell stemness of aged hair follicle stem cells and inhibiting cell aging.
[0006] To achieve the above object, the present invention provides the following technical solutions: First, the present invention provides a method for enhancing the regenerative capacity of stem cells and slowing down the aging of stem cells, the method comprising the following steps: (a) Preparation of culture vessels coated with laminin The culture vessel is coated with 100-200 μg / mL laminin and incubated to obtain a laminin-coated culture vessel; (b) Preparation of enhanced culture medium The enhanced medium consists of basal medium, KnockOut Serum Replacement, 1.5-2.5 μM CHIR-99021, 80-120 μg / mL RGD tripeptide, and 15-25 μg / mL KLT peptide; (c) Cell culture Inoculating stem cells into the culture vessel coated with laminin, adding the enhanced culture medium, and placing the culture vessel in a cell culture incubator for conventional culture; The CAS number of CHIR-99021 is 252917-06-9, the amino acid sequence of the RGD tripeptide is Arg-Gly-Asp, and the amino acid sequence of the KLT peptide is Lys-Leu-Thr-Trp-Gln-Glu-Leu-Tyr-Gln-Leu-Lys-Tyr-Lys-Gly-Ile.
[0007] Preferably, the stem cells are hair follicle mesenchymal stem cells.
[0008] Preferably, in step (a), the incubation condition is 37° C. for 2 h; In the step (b), the basal culture medium is DMEM / F12 culture medium, and the concentration of the KnockOut Serum Replacement is 10%; In the step (c), the conventional culture is cultured at 37° C. and 5% CO 2 for 3 days.
[0009] Preferably, the concentration of laminin is 100 μg / mL; The concentration of CHIR-99021 was 2 μM, the concentration of RGD tripeptide was 100 μg / mL, and the concentration of KLT peptide was 20 μg / mL.
[0010] Secondly, the present invention provides an enhanced culture preparation for enhancing the regenerative ability of stem cells and slowing down the aging of stem cells, wherein the enhanced culture preparation comprises the following components: Basal medium, KnockOut Serum Replacement, 1.5-2.5 μM CHIR-99021, 80-120 μg / mL RGD tripeptide, and 15-25 μg / mL KLT peptide; The CAS number of CHIR-99021 is 252917-06-9, the amino acid sequence of the RGD tripeptide is Arg-Gly-Asp, and the amino acid sequence of the KLT peptide is Lys-Leu-Thr-Trp-Gln-Glu-Leu-Tyr-Gln-Leu-Lys-Tyr-Lys-Gly-Ile.
[0011] Preferably, the basal culture medium is DMEM / F12 culture medium, and the concentration of the KnockOut Serum Replacement is 10%; The stem cells are hair follicle mesenchymal stem cells.
[0012] Preferably, the concentration of CHIR-99021 is 2 μM, the concentration of RGD tripeptide is 100 μg / mL, and the concentration of KLT peptide is 20 μg / mL.
[0013] In addition, the present invention provides a culture system for synergistically enhancing the regenerative ability of hair follicle mesenchymal stem cells and slowing down the aging of hair follicle mesenchymal stem cells, wherein the culture system is composed of a laminin-coated culture vessel and an enhanced culture preparation; The enhanced culture preparation comprises the following components: DMEM / F12 medium, 10% KnockOut Serum Replacement, 1.5-2.5 μM CHIR-99021, 80-120 μg / mL RGD tripeptide, and 15-25 μg / mL KLT peptide; The CAS number of CHIR-99021 is 252917-06-9, the amino acid sequence of the RGD tripeptide is Arg-Gly-Asp, and the amino acid sequence of the KLT peptide is Lys-Leu-Thr-Trp-Gln-Glu-Leu-Tyr-Gln-Leu-Lys-Tyr-Lys-Gly-Ile.
[0014] Preferably, the laminin-coated culture vessel is obtained by coating with 100-200 μg / mL laminin and incubating at 37° C. for 2 hours.
[0015] Preferably, the concentration of laminin is 100 μg / mL; The concentration of CHIR-99021 was 2 μM, the concentration of RGD tripeptide was 100 μg / mL, and the concentration of KLT peptide was 20 μg / mL.
[0016] The beneficial effects of the present invention are: This invention significantly enhances the regenerative capacity of hair follicle mesenchymal stem cells and slows their aging through the synergistic effect of laminin 521 coating and a culture medium containing CHIR-99021, RGD, and KLT peptides. Compared to single-ingredient treatments, this invention achieves highly efficient regulation of stem cell function through the organic combination of physical simulation and chemical activation, demonstrating significant benefits over traditional culture systems. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 The differences in proliferation ability of hair follicle mesenchymal stem cells after being cultured using different methods; Figure 2 The relative expression levels of Sox2 gene in hair follicle mesenchymal stem cells after different culture methods Figure 3 This is a comparison of the SA-β-gal staining positive rates of hair follicle mesenchymal stem cells after treatment with different culture methods. DETAILED DESCRIPTION
[0018] We will now provide a detailed description of specific embodiments of the present invention. It should be noted that the examples provided are intended to illustrate the concepts and applications of the present invention and are not intended to limit the potential applications or improvements of the present invention. These examples are intended to serve as a reference for further exploration and improvement by those skilled in the art and should not be construed as limiting the present invention. Example 1 A method for enhancing the regenerative ability of hair follicle mesenchymal stem cells 1. Preparation of culture dishes coated with laminin 521 The culture dish was coated with 100 μg / mL laminin 521 (Thermo Fisher Scientific, catalog number: A29249) solution (evenly covering the culture dish) and incubated at 37°C for 2 hours to obtain a culture dish coated with laminin 521; 2. Preparation of Enhanced Culture Medium The culture medium composition is as follows: DMEM / F12 medium, 10% KnockOut Serum Replacement (ThermoFisher Scientific, Catalog No. 10828028), 2 μM CHIR-99021 (MCE, Catalog No.: HY-10182, CAS No.: 252917-06-9), 100 μg / mL RGD tripeptide (MCE, Catalog No.: HY-P0278, amino acid sequence: Arg-Gly-Asp), and 20 μg / mL KLT peptide (MCE, Catalog No.: HY-P5558, amino acid sequence: Lys-Leu-Thr-Trp-Gln-Glu-Leu-Tyr-Gln-Leu-Lys-Tyr-Lys-Gly-Ile).
[0019] 3. Cell Culture P9 hair follicle mesenchymal stem cells were seeded into culture dishes coated with laminin 521 and enhanced culture medium (2×10 4 Each cell corresponds to 2 mL of enhanced culture medium) and was placed in a cell culture incubator at 37°C and 5% CO2 for 3 days.
[0020] Example 2 A method for enhancing the regenerative ability of hair follicle mesenchymal stem cells 1. Preparation of culture dishes coated with laminin 521 The culture dish was coated with 200 μg / mL laminin 521 solution and incubated at 37°C for 2 hours to obtain a culture dish coated with laminin 521; 2. Preparation of Enhanced Culture Medium The culture medium composition is as follows: DMEM / F12 medium, 10% KnockOut Serum Replacement, 1.5 μM CHIR-99021, 80 μg / mL RGD tripeptide, and 25 μg / mL KLT peptide.
[0021] 3. Cell Culture P9 hair follicle mesenchymal stem cells were seeded into a culture dish coated with laminin 521, and enhanced culture medium was added. The dish was placed in a cell culture incubator at 37°C and 5% CO2 for 3 days.
[0022] Example 3 A method for enhancing the regenerative ability of hair follicle mesenchymal stem cells 1. Preparation of culture dishes coated with laminin 521 The culture dish was coated with 100 μg / mL laminin 521 solution and incubated at 37°C for 2 hours to obtain a culture dish coated with laminin 521; 2. Preparation of Enhanced Culture Medium The culture medium composition is as follows: DMEM / F12 medium, 10% KnockOut Serum Replacement, 2.5 μM CHIR-99021, 120 μg / mL RGD tripeptide, and 15 μg / mL KLT peptide.
[0023] 3. Cell Culture P9 hair follicle mesenchymal stem cells were seeded into a culture dish coated with laminin 521, and enhanced culture medium was added. The dish was placed in a cell culture incubator at 37°C and 5% CO2 for 3 days.
[0024] Comparative Example 1 P9 hair follicle mesenchymal stem cells were seeded into ordinary cell culture dishes, and DMEM / F12 culture medium containing 10% KnockOut Serum Replacement was added. The dishes were placed in a cell culture incubator and cultured at 37°C and 5% CO2 for 3 days.
[0025] Comparative Example 2 1. Preparation of culture dishes coated with laminin 521 After coating the culture dish with 200 μg / mL laminin 521 solution, the dish was incubated at 37°C for 2 hours to obtain a culture dish coated with laminin 521; 2. Cell Culture P9 hair follicle mesenchymal stem cells were seeded into a culture dish coated with laminin 521, and DMEM / F12 medium containing 10% KnockOut Serum Replacement was added. The dish was placed in a cell culture incubator at 37°C and 5% CO2 for 3 days.
[0026] Comparative Example 3 1. Prepare Enhanced Culture Medium The culture medium composition is as follows: DMEM / F12 medium, 10% KnockOut Serum Replacement, 2 μM CHIR-99021, 100 μg / mL RGD tripeptide, and 20 μg / mL KLT peptide.
[0027] 2. Cell Culture P9 hair follicle mesenchymal stem cells were seeded into a culture dish coated with laminin 521, and DMEM / F12 medium containing 10% KnockOut Serum Replacement was added. The dish was placed in a cell culture incubator at 37°C and 5% CO2 for 3 days.
[0028] Comparative Example 4 1. Preparation of culture dishes coated with laminin 521 The culture dish was coated with 200 μg / mL laminin 521 solution and incubated at 37°C for 2 hours to obtain a culture dish coated with laminin 521; 2. Preparation of Enhanced Medium Lacking RGD and KLT The culture medium composition is as follows: DMEM / F12 medium, 10% KnockOut Serum Replacement, 2 μM CHIR-99021.
[0029] 3. Cell Culture P9 hair follicle mesenchymal stem cells were seeded into culture dishes coated with laminin 521, and enhanced culture medium lacking RGD and KLT was added. The culture dishes were placed in a cell culture incubator at 37°C and 5% CO2 for 3 days.
[0030] Comparative Example 5 1. Preparation of culture dishes coated with laminin 521 The culture dish was coated with 200 μg / mL laminin 521 solution and incubated at 37°C for 2 hours to obtain a culture dish coated with laminin 521; 2. Preparation of Enhanced Medium Lacking CHIR-99021 The culture medium composition is as follows: DMEM / F12 medium, 10% KnockOut Serum Replacement, 120 μg / mL RGD tripeptide, and 15 μg / mL KLT peptide.
[0031] 3. Cell Culture P9 hair follicle mesenchymal stem cells were seeded into culture dishes coated with laminin 521, and enhanced culture medium lacking RGD and KLT was added. The culture dishes were placed in a cell culture incubator at 37°C and 5% CO2 for 3 days.
[0032] Example 4 Examining the differences in cell proliferation ability after treatment with different methods 1. The culture medium of the hair follicle mesenchymal stem cells obtained by different culture methods in Examples 1-3 and Comparative Examples 1-5 was removed, the cells were washed twice with PBS, and the cells were digested with trypsin. The cells were then resuspended in DMEM / F12 medium containing 10% KnockOut Serum Replacement to prepare a cell suspension. 2. Inoculate hair follicle stem cells cultured using different culture methods into 96-well plates at a density of 10,000 cells per well, with five replicate wells for each culture method. 3. Place the 96-well plate in a cell culture incubator. After 72 hours of culture, use the Cell Counting Kit-8 reagent to detect the differences in the proliferation capacity of hair follicle mesenchymal stem cells cultured by different culture methods. The results are shown in Table 1 and Figure 1 shown.
[0033] Table 1 Differences in proliferation ability of hair follicle mesenchymal stem cells cultured using different culture methods as detected by CCK-8
[0034] From Table 1 and Figure 1 The results show that compared with comparative example 1, the proliferation improvement rate of comparative example 2 is 11.82%, indicating that the use of laminin alone can improve the proliferation ability of aging hair follicle mesenchymal stem cells, but the improvement effect is relatively limited.
[0035] The proliferation enhancement rate of Comparative Example 3 (without laminin coating and containing enhanced culture medium) (27.73%) was significantly higher than that of Comparative Example 2, but significantly lower than that of Examples 1-3 (72.58%-83.79%), indicating that the enhanced culture medium needs to rely on the laminin coating to achieve maximum effectiveness.
[0036] The proliferation enhancement rates of Comparative Examples 4 and 5 were 41.97% and 39.24%, respectively. Although relatively good results were achieved, they were still significantly lower than those of Examples 1-3. This indicates that when the enhanced culture medium lacks RGD and KLT or CHIR-99021, the combination with laminin can only exert a partial synergistic effect.
[0037] The proliferation enhancement rate of Example 1 is improved to a certain extent compared with Examples 2 and 3, and is significantly higher than the effect of the sum of Comparative Examples 2 and 3, indicating that under the conditions of Example 1, the best synergistic effect is achieved.
[0038] Example 5 Detection of differences in the expression of the stemness marker Sox2 gene in cells treated with different methods 1. The culture medium of the hair follicle mesenchymal stem cells obtained by different culture methods in Examples 1-3 and Comparative Examples 1-5 was removed, the cells were washed twice with PBS, and the cells were digested with trypsin. The cells were then resuspended in DMEM / F12 medium containing 10% KnockOut Serum Replacement to prepare a cell suspension. 2. Inoculate hair follicle stem cells cultured using different culture methods into 6-well plates at a density of 100,000 cells per well, with three replicate wells for each culture method. 3. Place the 6-well plate in a cell culture incubator. After 72 hours of culture, use TRIzol to lyse the cells and extract RNA. 4. After RNA quality is tested, reverse transcribe the RNA into cDNA using a reverse transcription kit; 5. Detect Sox2 gene expression using the following qPCR reaction system: cDNA template: 2 μL (diluted to 100 ng / μL); Forward primer (10 μM): 0.5 μL; Reverse primer (10 μM): 0.5 μL; SYBR Green Master Mix: 10 μL; Add nuclease-free water to make up to 20 μL.
[0039] 6. Calculate the relative expression level by ΔΔCt method. Taking Comparative Example 1 as the benchmark (100%), compare the Sox2 expression levels of each group. The results are shown in Table 2 and Figure 2 shown.
[0040] Table 2 Differences in expression of stemness marker Sox2 gene in cells treated with different methods
[0041] From Table 2 and Figure 2 The experimental results show that the relative expression levels of the stemness marker Sox2 gene of hair follicle mesenchymal stem cells in Examples 1-3 were significantly higher than those in the control groups (P<0.01), indicating that the treatment method provided by the present invention - laminin coating combined with an enhanced serum-free culture medium containing CHIR-99021, RGD tripeptide, and KLT peptide - can effectively enhance the stemness of aged hair follicle stem cells and have significant functional recovery and regeneration potential.
[0042] A further comparison between the examples and the comparative examples reveals that the relative expression of the Sox2 gene in Example 1 reached 194.51%, a 94.51% increase compared to Comparative Example 1 (basal medium). This increase not only significantly surpassed the effects of Comparative Example 2 (laminin coating alone, a 20.53% increase) and Comparative Example 3 (active ingredient addition without laminin coating, a 32.91% increase), but also significantly exceeded the combined effects of both (20.53% + 32.91% = 53.44%). This demonstrates that the laminin coating combined with enhanced culture medium method provided by the present invention achieves stemness enhancement not through a simple combination of physical coating and chemical stimulation, but rather through a synergistic interaction between the two in terms of structural mimicry and signal activation, resulting in a more efficient reversal of aging-related stemness loss.
[0043] Example 6 Detect the senescence ratio of cells treated with different culture methods after passage (1) The culture medium of the hair follicle mesenchymal stem cells obtained by different culture methods in Example 1 and Comparative Example 1 was removed, the cells were washed twice with PBS, trypsin was added to digest the cells, and the cells were resuspended in DMEM / F12 medium containing 10% KnockOut Serum Replacement to prepare a cell suspension; (2) The cells were seeded into a 10 mm small culture dish and continued to be cultured. The cells were passaged every 3 days until P15. The cells in different treatment groups were cultured in DMEM / F12 medium containing 10% KnockOut Serum Replacement. At the same time, P6 hair follicle mesenchymal stem cells were selected as the control group. (3) After culturing the P6 cells of the control group, the P15 cells of the comparative example 1 group, and the P15 cells of the example 1 group for 3 days, the culture medium was removed, and a staining fixative was added and fixed at room temperature for 15 minutes; (4) Discard the staining fixative and stain with β-galactosidase (SA-β-gal) for 20 minutes. Wash the cells with PBS and place them under a microscope. Randomly select 5 fields of view and count the positive cell rate.
[0044] from Figure 3 The experimental results show that at P6, hair follicle mesenchymal stem cells in the control group showed virtually no signs of aging, with the proportion of SA-β-gal-positive cells remaining low, indicating that the cells were still in a relatively youthful physiological state. However, in the control group 1, which did not receive any special treatment, the proportion of SA-β-gal-positive cells increased significantly to 60.75%, indicating that hair follicle mesenchymal stem cells developed a distinct aging phenotype during long-term in vitro expansion under conventional culture conditions.
[0045] Further observation of the results of each experimental group revealed that the SA-β-gal positive cell rate in Example 1 (using laminin 521 coating combined with an enhanced serum-free culture medium containing CHIR-99021, RGD tripeptide, and KLT peptide) at P15 was only 23.57%, which was significantly different from that in Comparative Example 1 (p<0.0001), indicating that the co-culture system provided by the present invention can effectively inhibit the occurrence of cell senescence.
Claims
1. A method for enhancing the regenerative capacity of stem cells and slowing down the aging of stem cells, characterized in that: The method comprises the following steps: (a) Preparation of culture vessels coated with laminin The culture vessel is coated with 100-200 μg / mL laminin and incubated to obtain a laminin-coated culture vessel; (b) Preparation of enhanced culture medium The enhanced medium consists of basal medium, KnockOut Serum Replacement, 1.5-2.5 μM CHIR-99021, 80-120 μg / mL RGD tripeptide, and 15-25 μg / mL KLT peptide; (c) Cell culture Inoculating stem cells into the culture vessel coated with laminin, adding the enhanced culture medium, and placing the culture vessel in a cell culture incubator for conventional culture; The CAS number of CHIR-99021 is 252917-06-9, the amino acid sequence of the RGD tripeptide is Arg-Gly-Asp, and the amino acid sequence of the KLT peptide is Lys-Leu-Thr-Trp-Gln-Glu-Leu-Tyr-Gln-Leu-Lys-Tyr-Lys-Gly-Ile.
2. The method according to claim 1, characterized in that The stem cells are hair follicle mesenchymal stem cells.
3. The method according to claim 2, characterized in that In the step (a), the incubation condition is 37° C. for 2 h; In the step (b), the basal culture medium is DMEM / F12 culture medium, and the concentration of the KnockOut Serum Replacement is 10%; In the step (c), the conventional culture is cultured at 37° C. and 5% CO 2 for 3 days.
4. The method according to claim 3, characterized in that The concentration of the laminin was 100 μg / mL; The concentration of CHIR-99021 was 2 μM, the concentration of RGD tripeptide was 100 μg / mL, and the concentration of KLT peptide was 20 μg / mL.
5. An enhanced culture preparation for enhancing the regenerative capacity of stem cells and slowing down the aging of stem cells, characterized in that: The enhanced culture preparation comprises the following components: Basal medium, KnockOut Serum Replacement, 1.5-2.5 μM CHIR-99021, 80-120 μg / mL RGD tripeptide, and 15-25 μg / mL KLT peptide; The CAS number of CHIR-99021 is 252917-06-9, the amino acid sequence of the RGD tripeptide is Arg-Gly-Asp, and the amino acid sequence of the KLT peptide is Lys-Leu-Thr-Trp-Gln-Glu-Leu-Tyr-Gln-Leu-Lys-Tyr-Lys-Gly-Ile.
6. The enhanced culture preparation according to claim 5, characterized in that The basal culture medium is DMEM / F12 culture medium, and the concentration of the KnockOut Serum Replacement is 10%; The stem cells are hair follicle mesenchymal stem cells.
7. The enhanced culture preparation according to claim 6, characterized in that The concentration of CHIR-99021 was 2 μM, the concentration of RGD tripeptide was 100 μg / mL, and the concentration of KLT peptide was 20 μg / mL.
8. A culture system for synergistically enhancing the regenerative capacity of hair follicle mesenchymal stem cells and slowing down the aging of hair follicle mesenchymal stem cells, the culture system comprising a laminin-coated culture vessel and an enhanced culture preparation; The enhanced culture preparation comprises the following components: DMEM / F12 medium, 10% KnockOut Serum Replacement, 1.5-2.5 μM CHIR-99021, 80-120 μg / mL RGD tripeptide, and 15-25 μg / mL KLT peptide; The CAS number of CHIR-99021 is 252917-06-9, the amino acid sequence of the RGD tripeptide is Arg-Gly-Asp, and the amino acid sequence of the KLT peptide is Lys-Leu-Thr-Trp-Gln-Glu-Leu-Tyr-Gln-Leu-Lys-Tyr-Lys-Gly-Ile.
9. The culture system according to claim 8, characterized in that The laminin-coated culture vessel is obtained by coating with 100-200 μg / mL laminin and incubating at 37° C. for 2 hours.
10. The culture system according to claim 9, characterized in that The concentration of the laminin was 100 μg / mL; The concentration of CHIR-99021 was 2 μM, the concentration of RGD tripeptide was 100 μg / mL, and the concentration of KLT peptide was 20 μg / mL.