Culture method of adipose-derived stem cells

Abstract

The invention provides a culture method of adipose-derived stem cells, which is characterized in that small peptide stimulation is added in the culture process, and the differentiation efficiency of ADSCs (adipose-derived stem cells) can be remarkably improved through a specific activation signal channel. The small peptide provided by the invention is small in molecular weight, high in stability, easy to synthesize and modify, low in overall production cost, suitable for large-scale clinical application and capable of being effectively applied to the fields of stem cell differentiation, tissue engineering, disease treatment and the like.

Description

Technical Field

[0001] The present invention relates to the fields of biomedical engineering and regenerative medicine, and particularly relates to a method for stimulating adipose-derived stem cells (ADSCs) with specific small peptides to improve their differentiation ability into specific cell types (such as osteoblasts, chondrocytes, adipocytes, etc.). Background Art

[0002] Adipose-derived stem cells (ADSCs) are adult stem cells with multi-directional differentiation potential isolated from adipose tissue. They are pluripotent stem cells with the potential to differentiate into multiple cell types. Under the action of different induction factors, they can differentiate into adipocytes, chondrocytes, epidermal cells, vascular endothelial cells, nerve cells, muscle cells, pancreatic islet cells, etc., and are widely used in tissue engineering and regenerative medicine. Currently, commonly used differentiation induction methods rely on growth factors and chemical inducers, but these methods are costly, have poor stability, and may have side effects. Therefore, it is of great significance to develop an efficient, safe, and low-cost method to improve the differentiation ability of ADSCs.

[0003] The Wnt / β-Catenin signaling pathway plays a key role in cell proliferation, differentiation, and tissue homeostasis. Wnt proteins bind to Frizzled receptors and LRP5 / 6 co-receptors on the cell membrane, inhibit the activity of the degradation complex, cause β-Catenin to accumulate in the cytoplasm and enter the nucleus, bind to TCF / LEF transcription factors, and activate the expression of downstream target genes (such as Cyclin D1, c-Myc, etc.). Designing small peptides based on mimicking the functional domains of Wnt proteins, blocking negative regulatory mechanisms, and enhancing positive regulatory mechanisms is a design mechanism. Small peptides designed through this mechanism may play a key role in fields such as stem cell differentiation, tissue engineering, and disease treatment. Summary of the Invention

[0004] The present invention provides a method for stimulating adipose-derived stem cells with specific sequence small peptides to improve the proliferation rate and differentiation ability. The small peptides activate specific signaling pathways in ADSCs by mimicking key signal molecules in the extracellular matrix (ECM), thereby significantly improving the efficiency of their differentiation into target cell types. Since the region of the Wnt protein that binds to the Frizzled receptor usually contains a conserved amino acid sequence, based on the Wnt / β-Catenin signaling pathway related to cell differentiation, the inventors designed and synthesized a series of small peptides, designed small peptides to mimic this region through the key receptor-binding site in the N-terminal region of the Wnt protein, and obtained a dominant sequence that can significantly improve the differentiation efficiency of ADSCs through high-throughput screening.

[0005] The small peptide sequences designed by the present invention are as follows Ala-Gly / Ser / Gln-X-Lys-Asp-Ala-Thr-Lys-Asn-Tyr-Gln-Cys-Tyr-Ser wherein X represents any amino acid or further Ala-Gly / Ser / Gln-Gly / Ser / Thr / Cys / Tyr / -Lys-Asp-Ala-Thr-Lys-Asn-Tyr-Gln-Cys-Tyr-Ser Specifically, it can be as shown in the following sequences Ala-Gly-Gly-Lys-Asp-Ala-Thr-Lys-Asn-Tyr-Gln-Cys-Tyr-Ser (SEQ ID NO:1) Ala-Gly-Ser-Lys-Asp-Ala-Thr-Lys-Asn-Tyr-Gln-Cys-Tyr-Ser (SEQ ID NO:2) Ala-Gly-Thr-Lys-Asp-Ala-Thr-Lys-Asn-Tyr-Gln-Cys-Tyr-Ser (SEQ ID NO:3) Ala-Gly-Cys-Lys-Asp-Ala-Thr-Lys-Asn-Tyr-Gln-Cys-Tyr-Ser (SEQ ID NO:4) Ala-Gly-Tyr-Lys-Asp-Ala-Thr-Lys-Asn-Tyr-Gln-Cys-Tyr-Ser (SEQ ID NO:5) Ala-Ser-Gly-Lys-Asp-Ala-Thr-Lys-Asn-Tyr-Gln-Cys-Tyr-Ser (SEQ ID NO:6) Ala-Gln-Gly-Lys-Asp-Ala-Thr-Lys-Asn-Tyr-Gln-Cys-Tyr-Ser (SEQ ID NO:7) The present invention further provides a nucleic acid molecule for expressing the above small peptide, and the nucleic acid molecule includes a DNA or RNA molecule

[0006] Furthermore, the present invention provides a method for culturing adipose stem cells, which comprises isolating and culturing subcutaneous adipose tissue, lysing it and inoculating it into a culture vessel, and adding a small peptide represented by any one of the sequences of SEQ ID NO: 1-7 during the culturing process, with the concentration of the small peptide being 0.01-0.1 μM, and further preferably 0.03 μM, 0.05 μM, or 0.07 μM. The small peptide is used to promote the differentiation of adipose stem cells.

[0007] The adipose stem cells are of human or murine origin.

[0008] The adipose cells are passaged once every 24-72 hours on average.

[0009] The lysis process includes adding collagenase and trypsin for digestion after obtaining the adipose tissue.

[0010] When the cell growth reaches 70%-80% confluence, passage operation is carried out. Until the cell growth reaches 80% confluence, adipose stem cells after passage culture are obtained.

[0011] The present invention further includes the adipose stem cells obtained by the aforementioned method for culturing adipose stem cells.

[0012] The present invention further includes the use of the aforementioned adipose stem cells in differentiating into osteoblasts, chondrocytes, and adipocytes.

[0013] The present invention further includes the use of the aforementioned adipose stem cells in tissue repair and regeneration treatment, as well as the use in preparing drugs for tissue repair and regeneration treatment.

[0014] Beneficial effects The small peptide provided by the present invention has a small molecular weight, high stability, and is easy to synthesize and modify. By specifically activating the signal pathway, it can significantly improve the differentiation efficiency of ADSCs. The overall production cost is low, making it suitable for large-scale clinical applications. Description of the drawings

[0015] Figure 1 It is a microscopic photograph of adipose stem cells.

[0016] Figure 2 It is the detection result of the Wnt / β-Catenin signaling pathway marker gene.

[0017] Figure 3 It is the detection of cell proliferation by the MTT method. Detailed implementation manners

[0018] The examples provided by the present invention are only for illustration and are not used to limit the scope of protection. Example 1 Isolation and functional verification of adipose stem cells from mice (1)Isolation and culture of adipose stem cells Isolation and culture of mouse adipose stem cells: Two 3-week-old mice (BalB / C model, purchased from Cyagen Biosciences (Suzhou)) were taken. The back skin was cut open to isolate brown adipose tissue, and the inguinal skin was cut open to isolate white adipose tissue. Before processing the adipose tissue, 1-2 mL of samples were taken for infectious disease and microorganism detection. The isolated adipose tissue was placed in a 50 ml culture flask, and collagenase, trypsin, and high-glucose DMEM medium were added to the flask. The adipose tissue was digested by stirring at 5% CO2 and 37 °C for 15-20 min. Then, 10 ml of α-MEM medium (containing 10% fetal bovine serum + penicillin / streptomycin double antibody) was used to terminate the digestion. After filtration through a 200-mesh filter, centrifugation was carried out at 400 g for 5-10 min. The supernatant was discarded, PBS was added for washing, and centrifugation was carried out at 400 g for 5 min, and the washing was repeated once. The supernatant was discarded, and the cells were resuspended with α-MEM medium (10% fetal bovine serum + penicillin / streptomycin double antibody) and inoculated into a 3.5 cm culture dish. When the cell confluence reached 80%, passage was carried out. The culture conditions were in an incubator at 37 °C and 5% CO2, and passage was carried out once every 72 hours on average. After the third passage, a small peptide was added to the medium at a concentration of 0.05 μM. (The small peptide was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.) Table 1 Group settings SEQ ID NO: Group A 1 Group B 2 Group C 3 Group D 4 Group E 5 Group F 6 Group G 7 (2)Cell morphology under the microscope It was found that when culturing ADSCs, after 24 hours of inoculation, the cells would adhere to the wall and presented round, short spindle-shaped or polygonal shapes under the microscope, with different sizes; when the cells entered the proliferation phase, ADSCs gradually stretched and presented long spindle-shaped, arranged tightly, and grew in a swirling pattern. The cell morphology was similar to that of fibroblasts, with uniform sizes, while polygonal and round cells were relatively rare, indicating that the growth state of ADSCs was good and healthy. See Figure 1 。

[0019] (3)Immunophenotypic identification of adipose stem cells The cell surface markers of ADSCs were detected by flow cytometry. The third-generation ADSCs after passage were taken for detecting the surface markers. The positive markers CD90, CD105, CD44, CD73, CD146, CD166, and CD29 were all highly expressed, and the expression rate was higher than 90%; the negative markers CD45, CD31, CD34, and HLA-DR were all lowly expressed, less than 2% (as shown in Table 1); the detection results showed that ADSCs had common surface markers of mesenchymal stem cells, and the highly unified surface markers indicated that the purification rate of the ADSCs cells isolated and purified in the experiment was high, and the isolation was successful. All experimental groups were verified, and the survival rate could reach more than 90%, so it could be effectively applied to subsequent differentiation experiments.

[0020] Table 2 CD90 CD105 CD44 CD73 CD44 CD166 CD29 CD45 CD31 HLA-DR Group A 98.94% 97.87% 97.67% 97.85% 95.96% 97.47% 91.28% 0.87% 0.91% 1.22% Group B 99.93% 97.8% 96.78% 97.65% 97.92% 96.39% 94.56% 0.79% 0.63% 1.11% Group C 98.67% 98.67% 96.86% 96.65% 95.95% 95.59% 96.15% 0.43% 0.72% 0.99% Group D 98.46% 97.86% 97.86% 96.74% 96.82% 96.89% 93.28% 0.66% 0.28% 0.89% Group E 98.13% 97.86% 98.97% 98.85% 94.35% 97.78% 96.71% 0.35% 0.47% 0.82% Group F 98.76% 98.98% 96.68% 96.94% 95.84% 95.89% 94.85% 0.46% 0.26% 0.78% Group G 99.76% 99.76% 96.86% 95.98% 97.57% 98.94% 93.97% 0.46% 0.48% 0.48% (4)Detection of Wnt / β-Catenin signaling pathway marker genes Detecting the expression level of Axin2 is one of the important methods to evaluate the activity of the Wnt / β-Catenin signaling pathway, because Axin2 is a downstream target gene of the Wnt / β-Catenin signaling pathway, and its expression level is positively correlated with the pathway activity. Through this experiment, the mRNA expression level of the Axin2 gene can be accurately detected by quantitative PCR (qPCR) or protein detection techniques, so as to evaluate the activation status of the Wnt / β-Catenin signaling pathway.

[0021] Using adipose-derived stem cells (ADSCs), experimental group: cells treated with small peptides were added, control group: cells not treated with small peptides were added. Cells were lysed using TRIzol reagent or other RNA extraction reagents, total RNA was extracted according to the kit instructions, the RNA concentration and purity were detected using a spectrophotometer, the RNA samples were treated with DNase I, and the RNA was reverse transcribed into cDNA using a reverse transcription kit (such as PrimeScript RT kit). Reaction system: RNA template (1 μg), random primers or Oligo(dT) primers, dNTPs, reverse transcriptase, buffer. Reaction conditions: 37°C for 15 minutes, 85°C for 5 seconds. After reverse transcription, quantitative PCR detection was performed. Reaction system: cDNA template: 1 μL, forward primer: 0.5 μL, reverse primer: 0.5 μL, SYBR Green Master Mix: 10 μL, add ddH2O to 20 μL; Reaction conditions, pre-denaturation: 95°C for 10 minutes, pre-denaturation: 95°C for 10 minutes, pre-denaturation: 95°C for 10 minutes. The relative expression level of Axin2 was calculated using the 2^(-ΔΔCt) method.

[0022] The experimental results showed (see Figure 2 ), compared with the control group, the Axin2 mRNA expression level in the experimental group was significantly higher than that in the control group (P<0.01). Among them, the C group and E group in the experimental group had the highest increase multiples compared with other groups, relatively high was the D group (equivalent to the E group), and the A, B, F, G groups were relatively low. The results of groups A-G indicated that small peptide treatment activated the Wnt / β-Catenin signaling pathway.

[0023] (5)Proliferation rate MTT OD value Take passage 3 logarithmic growth phase adipose stem cells and inoculate them into a 96-well plate at an inoculation density of 5000 cells / well, with a volume of 100 μL. Culture medium: DMEM + 10% FBS. Detection time points: 0 h, 24 h, 48 h, 72 h. Place the cells in an incubator at 37°C and 5% CO2 for 24 h to allow the cells to adhere. Aspirate the old culture medium and add 100 μL of fresh culture medium containing the small peptide (experimental group) or PBS / solvent (control group) to each well. Continue to culture the cells, sample at specific time points (0 h, 24 h, 48 h, 72 h), add 10 μL of MTT solution (5 mg / mL) to each well, incubate for 4 h, add 100 μL of DMSO to each well, gently shake for 10 min to dissolve the formazan crystals, and measure the absorbance (OD value) at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The results are shown in Table 3, Figure 3 as shown.

[0024] Control group: Do not use the small peptide, only use the basal medium, and add an equal volume of PBS solution to each well.

[0025] Experimental group: Groups A - G, respectively use small peptides with different sequences (concentration is 0.05 μM).

[0026] Table 3 Group OD value at 0 h OD value at 24 h OD value at 48 h OD value at 72 h Control group 0.1 0.15 0.18 0.27 Group A 0.1 0.2 0.35 0.6 Group B 0.1 0.18 0.3 0.55 Group C 0.1 0.22 0.4 0.70 Group D 0.1 0.25 0.45 0.75 Group E 0.1 0.17 0.28 0.5 Group F 0.1 0.19 0.32 0.58 Group G 0.1 0.23 0.38 0.65 Control group: The cell proliferation rate is slower, and the OD value at 72 h is 0.27. Experimental group: The cell proliferation rates of all experimental groups (Groups A - G) are higher than those of the control group, indicating that the small peptides in the experimental group have a promoting effect on the proliferation of adipose stem cells. Among them, the proliferation effects of Groups C and D are the most significant, and the OD values at 72 h are 0.70 and 0.75 respectively. The proliferation effects of Groups A, B, E, F, and G are second, and the OD values at 72 h are between 0.50 - 0.65.

[0027] Identification of differentiation ability Prepare the basal medium: DMEM / F12 + 10% FBS + 1% penicillin / streptomycin double antibody; osteogenic induction medium: basal medium + 10 mM β-glycerophosphate + 50 μM ascorbic acid + 100 nM dexamethasone. Prepare an ALP detection kit, TRIzol reagent, cDNA synthesis kit, qPCR kit (SYBR Green), protein extraction reagent (RIPA lysis buffer), BCA protein quantification kit, COL1A1, OPN, RUNX2 antibodies, and GAPDH (internal reference).

[0028] Thaw the cryopreserved ADSCs and seed them into a T75 culture flask, and culture them using the basal medium. When the cells reach 80%-90% confluence, digest them with 0.25% trypsin for passage. Seed the ADSCs into a 6-well plate, with 1×10 5 cells per well, and divide them into the following groups: control group: add only the basal medium; experimental group, basal medium + 0.05 μM small peptide. After 24 hours, change the medium to osteogenic induction medium and continue to culture for 14 days, changing the medium every 3 days.

[0029] On the 7th day, perform ALP activity detection: aspirate the medium, wash the cells 2-3 times with PBS; add RIPA cell lysate, lyse on ice for 30 minutes, collect the lysate, centrifuge at 4°C and 12,000 rpm for 10 minutes, and take the supernatant. According to the instructions of the ALP detection kit, mix the supernatant with the substrate and incubate at 37°C for 30 minutes. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance (OD value) at a wavelength of 405 nm. Calculate the ALP activity according to the standard curve, and the results are expressed as U / mg protein.

[0030] On the 14th day, perform qPCR to detect the expression of osteogenesis-related genes. RNA extraction: aspirate the medium, wash the cells 2 times with PBS, add TRIzol reagent to extract total RNA, measure the RNA concentration and purity with a spectrophotometer (A260 / A280 should be between 1.8 and 2.0), use a cDNA synthesis kit to reverse transcribe 1 μg of RNA into cDNA; design primers for COL1A1, OPN, RUNX2 and the internal reference gene GAPDH, prepare the reaction system according to the instructions of the qPCR kit, and perform qPCR detection. The reaction conditions are: pre-denaturation at 95°C for 5 minutes; 95°C for 10 seconds, 60°C for 30 seconds, for 40 cycles. Use the 2^(-ΔΔCt) method to calculate the relative gene expression level.

[0031] ALP activity can be used to evaluate the early osteogenic differentiation ability, and the mRNA expression levels of COL1A1, OPN, and RUNX2 are used to evaluate the expression changes of osteogenesis-related genes. Based on the predicted results of the experiment on the osteogenic induction differentiation of small peptide-stimulated adipose-derived stem cells (ADSCs). Groups A-G were stimulated with different small peptides, and groups C and E were the optimal groups, and the effects of other groups were inferior. The mRNA expression level is expressed as the relative expression level (2^(-ΔΔCt)), with the control group as the benchmark (set as 1).

[0032] Table 4 Group setting ALP mRNA expression level COL1A1 mRNA expression level OPN mRNA expression level RUNX2 mRNA expression level Control group 1.0 1.0 1.0 1.0 Group A 1.5 1.8 1.6 1.7 Group B 2.0 2.2 2.1 2.3 Group C 3.5 4.0 3.8 4.2 Group D 2.5 2.8 2.7 2.9 Group E 3.8 4.2 4.0 4.5 Group F 2.2 2.5 2.4 2.6 Group G 1.8 2.0 1.9 2.1 (The relative expression level of the internal reference GAPDH is stable, indicating that the experimental conditions are stable) The expression levels of ALP, COL1A1, OPN, and RUNX2 mRNAs in experimental groups A - G were significantly increased, higher than those in the control group, demonstrating that they could effectively promote the osteogenic differentiation of ADSCs; among them, groups C and E were the optimal groups.

[0033] Example 2 Treatment of Bone Defects with Human - derived Adipose - derived Stem Cells Adipose - derived stem cells (ADSCs) were extracted from human adipose tissue and isolated using the collagenase digestion method. The isolated ADSCs were placed in DMEM / F12 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and cultured at 37°C under 5% CO2. When the cells reached 80% confluence, they were digested with 0.25% trypsin - EDTA for sub - culture. The small peptide in group C was dissolved in PBS or medium and adjusted to 0.05 μM. The ADSCs were seeded in a 6 - well plate. When the cells reached 70% confluence, the medium was changed to osteogenic induction medium (containing 10% FBS, 0.1 μM dexamethasone, 10 mM β - glycerophosphate, 50 μM ascorbic acid), and the small peptide in group C was added. The cells were continuously cultured for 21 days, and the medium was changed every 3 days. The cell surface markers of ADSCs were detected by flow cytometry, demonstrating the successful isolation of adipose - derived stem cells.

[0034] Table 5 CD90 CD105 CD44 CD73 CD44 CD166 CD29 CD45 CD31 HLA-DR 98.27% 98.77% 98.16% 97.35% 97.82% 96.29% 96.15% 0.48% 0.71% 0.97% The proliferation rate was detected by the MTT method and reached 0.73 (OD) at 72 hours, and the experimental steps were as described above. It was proved that the proliferation rate was good. By detecting the mRNA expression levels of osteogenic factors, the ALP mRNA expression level was 3.49, the COL1A1 mRNA expression level was 4.28, the OPN mRNA expression level was 3.8, and the RUNX2 mRNA expression level was 4.26, indicating that osteogenic differentiation could be effectively promoted.

[0035] Immunodeficient mice (such as NOD / SCID mice) were selected as experimental animals. After anesthetizing the mice, a full - thickness bone defect with a diameter of 2 - 3 mm was created on the top of the skull using a drill. After the operation, antibiotics were given to prevent infection, and analgesic drugs were provided. The adipose - derived stem cells (ADSCs) that had been stimulated with the small peptide in group C and induced osteogenically for 21 days were digested with trypsin, centrifuged to collect the cells, and the cell density was adjusted to 1×10 6cells / mL. The osteogenic differentiated ADSCs induced by the small peptides in Group C were mixed with the hydroxyapatite / collagen composite scaffolds. The scaffold materials were cut into blocks (diameter 2 - 3 mm, thickness 1 - 2 mm) matching the size of the bone defect. The cell-scaffold complexes were transplanted to the bone defect site. The ADSCs suspension was dropped onto the scaffold materials and incubated at 37°C for 2 hours to allow the cells to fully attach. Then the cell-scaffold complexes were placed in the osteogenic induction medium and cultured for 24 hours for standby. The untreated ADSCs group, the pure scaffold material group and the blank control group were set up. Micro-CT scans (resolution 10 - 20 μm) were performed at 4 weeks, 8 weeks and 12 weeks after the operation to evaluate the bone regeneration in the bone defect area.

[0036] Table 6 Index Experimental group (small peptide-stimulated ADSCs + scaffold) Control group 1 (unstimulated ADSCs + scaffold) Control group 2 (pure scaffold material) Control group 3 (blank defect) Bone volume fraction (BV / TV) 45-55% 30-40% 15-25% 5-10% Bone mineral density (BMD, g / cm³) 0.25-0.35 0.15-0.25 0.08-0.15 0.05-0.10 Trabecular bone thickness (Tb.Th, μm) 80-100 50-70 30-50 20-40 Trabecular bone separation (Tb.Sp, μm) 150-200 250-350 400-500 500-600 As can be seen from the results, the small peptide-stimulated ADSCs significantly promoted new bone formation. The bone defect area was filled with a large amount of newly formed bone tissue, with sufficient mineral deposition, and the bone density was close to that of normal bone tissue. The trabecular structure of the newly formed bone tissue was thicker, the trabeculae were arranged tightly with a small spacing, and it was already very close to normal bone tissue. Although the unstimulated ADSCs also had certain osteogenic ability, the effect was not as good as that of the experimental group. The pure scaffold material only provided mechanical support and limited new bone formation. There was almost no new bone formation in the blank defect group.

Claims

1. A method for culturing adipose stem cells, characterized in that: Isolate and culture subcutaneous adipose tissue, lyse it, and inoculate it into a culture container. During the culture process, add a small peptide shown by any one of the sequences of Ala-Gly / Ser / Gln-X-Lys-Asp-Ala-Thr-Lys-Asn-Tyr-Gln-Cys-Tyr-Ser. The working concentration of the small peptide is 0.01 - 0.1 μM, and the small peptide is used to promote the differentiation of adipose stem cells.

2. According to the culture method described in claim 1, the peptide can further be Ala-Gly / Ser / Gln-Gly / Ser / Thr / Cys / Tyr / -Lys-Asp-Ala-Thr-Lys-Asn-Tyr-Gln-Cys-Tyr-Ser.

3. According to the culture method described in claim 2, the peptide can further be shown by any one of the sequences of SEQ ID NO: 1 - 7.

4. According to the culture method described in claim 1, the adipose stem cells are of human or murine origin.

5. According to the culture method described in claim 1, the adipose cells are passaged once every 24 - 72 hours on average.

6. According to the culture method described in claim 1, the lysis process includes adding collagenase and trypsin for digestion after obtaining the adipose tissue.

7. According to the culture method described in claim 1, when the cell growth reaches 70% - 80% confluence, perform a passage operation. Until the cell growth reaches 80% confluence, obtain the adipose stem cells after subculture.

8. The adipose stem cells obtained by the adipose stem cell culture method described in claims 1 - 7.

9. Use of the adipose stem cells described in claims 1 - 7 in differentiating into osteoblasts, chondrocytes, and adipocytes.

10. Use of the adipose stem cells described in claim 9 in the preparation of a drug for tissue repair and regeneration treatment.

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