Methods for improving stemness of mesenchymal stem cells and compounds and compositions therefor

Abstract

The application provides a method for improving stem cell stemness of mesenchymal stem cells and compounds and compositions thereof, the method for improving stem cell stemness of mesenchymal stem cells comprises treating the mesenchymal stem cells with an active substance, activating an integrin-FAK-Src pathway, enhancing the phosphorylation level of FAK and Src proteins, and then activating a downstream PI3K / Akt pathway to improve the cell stemness of the mesenchymal stem cells. The research of the application confirms that COL21 promotes the proliferation of MSCs, and confirms that COL21 promotes the expression of four stemness markers of Oct-4, Klf4, Nanog and Sox2 in MSCs, and improves the stem cell stemness of MSCs.

Description

Technical Field

[0001] This invention relates to the field of cells, and particularly to methods, compounds, and compositions thereof for improving the stem cell stemness of mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of adult pluripotent stem cell originating from the mesoderm. They are one of the most widely researched and maturely applied stem cell types in regenerative medicine and cell therapy. Their core characteristics are adherent growth, multipotent differentiation potential, and immunomodulatory capacity. Furthermore, their wide availability and low ethical controversy make them important seed cells for clinical translation.

[0003] The International Society for Cell Therapy (ISCT) established unified identification criteria for MSCs in 2006. MSCs can be defined as cells meeting the following three criteria: Morphology and growth characteristics: Adherent growth resembling fibroblasts in vitro, forming whorled clonal colonies; Surface marker expression: Positive expression of CD73, CD90, and CD105 (≥95%), and negative expression of CD45, CD34, CD14 / CD11b, CD79α / CD19, and HLA-DR (≤2%); Multi-lineage differentiation potential: Under specific in vitro induction conditions, they can differentiate into osteoblasts, adipocytes, and chondrocytes. MSCs are a type of adult stem cell with self-renewal capacity and multi-lineage differentiation potential, possessing significant research value and clinical application prospects in regenerative medicine, immune regulation, and tissue engineering. In the aforementioned applications of mesenchymal stem cells, maintaining and improving their stem cell stemness is crucial.

[0004] Stem cell stemness is the core biological essence of stem cells, referring to their unique dual capacity for self-renewal and multi-lineage differentiation, and is a key marker distinguishing stem cells from terminally differentiated cells. Its maintenance and regulation are fundamental to embryonic development, tissue regeneration, and homeostasis, and are core targets in regenerative medicine, cell therapy, and tumor research. Stem cell stemness possesses two core capabilities: first, the ability to self-renew, where stem cells continuously produce daughter cells with the same phenotype and function as themselves through division, ensuring that stem cells are not depleted during an individual's lifespan and maintaining the stability of the stem cell pool within tissues; second, the ability to differentiate into multiple lineages, where, under specific induction conditions, stem cells break through the undifferentiated state and differentiate into mature cells of different germ layers or specific tissues, enabling the formation of tissues and organs during embryonic development, as well as the repair and regeneration of damaged tissues in adulthood.

[0005] Type 21 collagen (COL21) is a rare FACIT family collagen first discovered in 2002 with the development of human genomics technology in the early 21st century. It accounts for less than 1% of the adult population and is naturally present in human skin mesenchymal cells, blood vessels, and other tissues, acting as a "signaling collagen" without forming fibers. Current research indicates that it may participate in extracellular matrix assembly during angiogenesis, collagen formation in the heart and aorta, and is associated with vascular remodeling. Its gene expression is regulated by developmental stages and plays a crucial role in tissue development. Furthermore, recent research has found that a low copy number of the COL21A1 gene, which encodes type 21 collagen, can disrupt the integrity of the extracellular matrix during embryonic maxillofacial development, potentially increasing the risk of non-syndromic cleft lip with or without cleft palate (NSCLP).

[0006] The inventors' research group previously focused on the core amino acid sequence of COL21, and through rational design using AI and computational biology, successfully developed 100% human homologous recombinant human COL21. They constructed a eukaryotic expression system and obtained a high-purity, bioactive, and non-toxic protein. CN118949130B discloses the use of homologous recombinant human COL21 as a facial filler for skin damaged by UVA and / or UVB radiation, enhancing the skin's self-repair ability. CN119455070B discloses type 21 recombinant humanized collagen as a raw material for preparing collagen for wound repair. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a method for improving the stem cell stemness of mesenchymal stem cells. The method includes treating the mesenchymal stem cells with an active substance, thereby activating the integrin-FAK-Src pathway, enhancing the phosphorylation of FAK and Src, and subsequently activating the downstream PI3K / Akt pathway, thereby improving the stem cell stemness of the mesenchymal stem cells.

[0008] In one embodiment, the method upregulates four stem cell stemness genes—Oct-4, Klf4, Nanog, and SOX2—in mesenchymal stem cells, thereby improving the stemness of the mesenchymal stem cells.

[0009] In one embodiment, the active substance is type 21 recombinant humanized collagen, the collagen comprising the amino acid sequence SEQ ID No. 1.

[0010] In one embodiment, the amino acid sequence of the collagen is SEQ ID No. 1.

[0011] In one embodiment, the concentration of the collagen is 1-50 μg / mL.

[0012] In one embodiment, the present invention provides a method for promoting the upregulation of four stem cell genes—Oct-4, Klf4, Nanog, and SOX2—in mesenchymal stem cells. The method includes treating the mesenchymal stem cells with an active substance, wherein the active substance is recombinant humanized collagen type 21, and the collagen includes the amino acid sequence SEQ ID No. 1.

[0013] In one embodiment, the present invention provides a compound that enhances the stem cell stemness of mesenchymal stem cells, said compound being type 21 recombinant humanized collagen, said collagen comprising the amino acid sequence SEQ ID No. 1.

[0014] In one embodiment, the present invention provides a composition for improving the stem cell stemness of mesenchymal stem cells, the composition comprising type 21 recombinant humanized collagen, the collagen comprising the amino acid sequence SEQ ID No. 1.

[0015] In one embodiment, the present invention provides the use of a compound in the preparation of a stem cell therapy agent that enhances the stemness of mesenchymal stem cells, said compound being recombinant humanized collagen type 21, said collagen comprising the amino acid sequence SEQ ID No. 1. A therapy agent refers to a chemical substance, biological product, or other preparation used for the prevention, diagnosis, treatment of diseases, or regulation of physiological functions. Its primary purpose is to improve the patient's health status, alleviate symptoms, control disease progression, or eradicate the cause of the disease.

[0016] In this invention, it was confirmed that in mesenchymal stem cells, COL21 binds to integrin ITGA2, activating the downstream integrin-FAK-Src pathway. Western blotting experiments confirmed that after COL21 treatment, FAK is recruited and phosphorylated, thereby binding to and activating Src kinase to form a functional complex. This complex further activates the downstream PI3K / Akt pathway, increasing AKT phosphorylation levels and constructing a complete "receptor-pathway-downstream signaling" signaling chain.

[0017] This invention confirms that COL21 promotes MSC proliferation, with the most significant effect observed at a concentration of 10 μg / mL. It also confirms that COL21 promotes the expression of four stem cell markers in MSCs: Oct-4, Klf4, Nanog, and Sox2, thereby enhancing MSC stem cell stemness. Stem cell spheroidization experiments showed that COL21 treatment significantly enhanced the microsphere-forming ability of MSCs, suggesting that COL21 can effectively improve MSC stemness. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In all the drawings, the significance symbols have the following meanings: * indicates P<0.05, indicating that the difference between the two groups of results is statistically significant; ** indicates P<0.01, indicating that the difference is more significant; *** indicates P<0.001, indicating that the difference is extremely significant.

[0019] Figure 1 This is a graph showing the effects of COL21 on four dry biomarkers in MSCs: Oct-4, Klf4, Nanog, and Sox2.

[0020] Figure 2 The graph shows the effects of different collagens on four stem cell markers in MSCs: Oct-4, Klf4, Nanog, and Sox2.

[0021] Figure 3 These are images of stem cell microspheres from a stem cell spheroidization experiment using COL21.

[0022] Figure 4 Statistical results of stem cell microspheres in the stem cell spheroidization experiment of COL21;

[0023] Figure 5 This is a graph showing the changes in the expression levels of candidate receptors after COL21 treatment;

[0024] Figure 6 This is a graph showing the changes in integrin ITGA2 protein expression in a Western blot (WB) experiment.

[0025] Figure 7 This is a graph showing the recruitment and phosphorylation activation results of FAK;

[0026] Figure 8 This is a graph showing the combination of Src and the complete activation of FAK;

[0027] Figure 9 This is a diagram showing the activation results of AKT phosphorylation in the downstream pathway. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application.

[0029] Example 1: Effect of Type 21 recombinant humanized collagen on mesenchymal stem cell proliferation

[0030] I. Experimental Materials

[0031] The inventors' research group previously focused on the core amino acid sequence of COL21 and, through rational design using AI and computational biology, successfully developed a 100% human homologous recombinant human COL21. They constructed a eukaryotic expression system and obtained a high-purity, biologically active protein that is non-toxic to cells. Its amino acid sequence is SEQ ID No. 1: GKPGLQGPKGDPGLPGNPGYPGQPGQDGKPGYQGIAGTPGVPGSPGIQGARGLPGYKGEPGRDGDKGDRGLPGFPGLHGMPGSKGEMGAKGDKGSPGFYGKKGAKGEKGNAGFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGAPGQDGTRGEPGIPGFPGNRGLMGQKGEIGPPGQQGKKGAPGMPGLMGSNGSPGQPGTPGSKGSKGEPGIQGMPGASGLKGEPGATG.

[0032] Unless otherwise specified, all experimental materials, including human mesenchymal stem cells (MSCs), protein (COL21), DMEM / F12 basal medium, fetal bovine serum, penicillin-streptomycin antibiotics, trypsin, recombinant human epidermal growth factor (EGF), and plant-derived recombinant human basic fibroblast growth factor (bFGF), were commercially available reagents.

[0033] Culture medium preparation: 89% basal medium, 1% penicillin-drug antibiotics, 10% fetal bovine serum, add EGF and bFGF to a final concentration of 10 ng / mL. Mix thoroughly to prepare complete culture medium, store at 4°C, and use within one month.

[0034] COL21 preparation: Weigh 0.1g of protein powder, dissolve it fully in ultrapure water, and bring the volume to 10 mL to prepare a 10mg / mL stock solution. When using, dilute to the required concentration using a serial dilution method.

[0035] MSC resuscitation and culture: Remove the cryovials from the liquid nitrogen tank and immediately place them in a 37°C constant temperature water bath for 1-1.5 min for rapid thawing. Then, slowly add the cell suspension to a 15 mL centrifuge tube containing preheated complete culture medium for dilution. Centrifuge at 1000 rpm (200-300 g) for 5 min, discard the supernatant, and resuspend the cells in complete culture medium. Seed the cells into T25 culture flasks and culture them in a carbon dioxide incubator with 5% CO2, 37°C, and saturated humidity. Change the complete culture medium every 2-3 days during culture. When the cell confluence reaches 80-90%, passage the cells. After discarding the old culture medium, wash once with PBS buffer, add an appropriate amount of 0.25% trypsin, and incubate at 37°C for 1-2 min until the cells become rounded and detach. Add complete culture medium to stop digestion, gently pipette to form a single-cell suspension, collect the suspension by centrifugation, resuspend the cells in complete culture medium, and seed them into new culture flasks at a 1:5 ratio. Continue passage culture to maintain cell viability and stemness.

[0036] The effect of COL21 on MSC cell proliferation: COL21 was serially diluted to concentrations of 0, 1, 2.5, 5, 10, 25, 50, and 100 μg / mL in complete culture medium. Well-grown MSCs were digested, counted using a cell counter, and seeded into 96-well plates at 2000-3000 cells per well. The next day, after cell attachment, the culture medium was removed and replaced with complete culture medium containing different concentrations of COL21. At 24, 48, and 72 hours, MSC cell proliferation was assessed using the MTS colorimetric assay with a microplate reader. The specific experimental results are shown in Table 1. Table 1 shows the effect of type 21 collagen on MSC proliferation; the four data sets for each concentration in Table 1 represent four replicates of the same treatment at the same time point.

[0037] Table 1

[0038] .

[0039] The results above show that COL21 promotes MSC proliferation within the concentration range of 1-50 μg / mL, with the most significant proliferation effect observed at a concentration of 10 μg / mL.

[0040] Example 2: Effects of COL21 on four dry biomarkers in MSCs: Oct-4, Klf4, Nanog, and Sox2.

[0041] The effects of COL21 treatment on stemness gene expression in MSCs were detected using qPCR. GAPDH was used as an internal reference gene to measure the mRNA expression levels of stemness-related markers such as Oct-4, Klf4, Nanog, and Sox2.

[0042] 1. COL21 treatment of MSCs: Human mesenchymal stem cells (MSCs) in good growth condition were digested with trypsin, resuspended, and counted using a cell counter at a density of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates. After cell attachment, the original culture medium was discarded. The experimental group was replaced with fresh complete culture medium containing 10 μg / mL COL21, while the control group was only replaced with fresh complete culture medium. After 24 hours, the medium was replaced again with the corresponding concentration of COL21. After 48 hours of treatment, the culture medium was discarded, and the cells were gently washed once with PBS. A suitable amount of trypsin was added to digest the cells, and digestion was terminated with complete culture medium. The cell suspension was collected, centrifuged, and the supernatant was discarded. The resulting cell pellet was resuspended in PBS and washed again by centrifugation. The final cell pellet was stored at -80°C for subsequent total RNA extraction.

[0043] 2. RNA Extraction: RNA was extracted using a commercially available animal tissue / cell total RNA extraction kit. The experimental steps are as follows:

[0044] ① Gently tap the cells at the bottom of the centrifuge tube to loosen them, add 350 μL of lysis buffer RLA and 10 μL of proteinase K, and vortex to mix them thoroughly.

[0045] ② The above lysate was centrifuged at 12,000 rpm (~13,400×g) for 2-5 min, and the supernatant was collected.

[0046] ③ Transfer the obtained supernatant to a genomic DNA removal column, centrifuge at 12,000 rpm for 30 seconds, and collect the filtrate.

[0047] ④ Slowly add 70% ethanol (1 volume of supernatant) to the filtrate, mix well, and transfer the resulting solution and precipitate into an RNase-Free adsorption column CR4. Centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube.

[0048] ⑤ Add 700 μL of protein removal solution RW3 to the RNase-Free adsorption column CR4, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and put the adsorption column back into the collection tube.

[0049] ⑥ Add 500 μL of wash buffer RW to the RNase-Free adsorption column CR4 (check if ethanol has been added before use), let stand at room temperature for 2 min, centrifuge at 12,000 rpm for 30-60 s, discard the waste liquid, and put the adsorption column back into the collection tube.

[0050] ⑦ Repeat the previous step.

[0051] ⑧ Centrifuge at 12,000 rpm for 2 minutes and discard the waste liquid. Place the RNase-Free adsorption column CR4 at room temperature for 2 minutes to thoroughly dry any residual washing liquid in the adsorption material.

[0052] 9. Transfer the RNase-Free adsorption column CR4 into a new RNase-Free centrifuge tube, add 60 μL of RNase-Free ddH2O dropwise to the middle of the adsorption membrane, incubate at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min to obtain the RNA solution.

[0053] 3. RT-PCR: A commercially available one-step reverse transcription kit for genomic DNA removal was used, integrating the reverse transcription and genomic DNA removal steps into the same reaction system. The RT-PCR system is shown in Table 2 below.

[0054] Table 2

[0055] Composition Usage 5xFastKing-RT SuperMix 4μL Total RNA 1μg <![CDATA[RNase-Free ddH2O]]> To bring the volume up to 20 μL .

[0056] Perform the reverse transcription reaction according to Table 3 below. Table 3 is the RT-PCR procedure.

[0057] Table 3

[0058] reaction temperature reaction time illustrate 42℃ 15 min Genome removal and reverse transcription 95℃ 3 min Enzyme inactivation process .

[0059] After the reaction was complete, 80 μL of RNase-Free ddH2O was added to dilute the cDNA product 5-fold for subsequent qPCR detection.

[0060] 4. qPCR analysis: A commercially available qPCR SYBR Green Master Mix (LOW Rox Plus) kit was used. The reaction mixture was prepared on ice, as shown in Table 4 below.

[0061] Table 4

[0062] Components Volume (μL) qPCR SYBR Green Master Mix 10 Forward primer (10 μM) 0.4 Reverse primer (10 μM) 0.4 Template DNA 2 <![CDATA[RNase Free H2O]]> Add to 20 .

[0063] Perform amplification according to the procedure shown in Table 5.

[0064] Table 5

[0065] .

[0066] The experiment was conducted with at least three biological replicates, and each biological replicate had three technical replicates. Quantitative cycle values ​​were used for data analysis, employing the ΔΔCt method (2⁻ΔΔCt method). Stem cell gene markers and internal reference gene primers are shown in Table 6 below.

[0067] Table 6

[0068] .

[0069] Test results as follows Figure 1 As shown, by Figure 1 It was found that after MSCs were treated with 10 μg / mL COL21, the stemness genes were significantly upregulated, with Klf4 upregulated by 7.94-fold, Oct-4 by 1.9-fold, Nanog by 2-fold, and Sox2 by 1.6-fold.

[0070] Example 3: Effects of different collagens on four stem cell markers (Oct-4, Klf4, Nanog, and Sox2) in MSCs

[0071] Effects of different collagen treatments on MSC stemness: Following the COL21 treatment method, MSCs were treated with type I collagen (COL1), type III collagen (COL3), type IV collagen (COL4), type VII collagen (COL7), type XVII collagen (COL17), and type XXI collagen (COL21) at a concentration of 10 μg / mL. Changes in the expression of KLf4, Oct-4, Nanog, and Sox2 were detected. The results are as follows: Figure 2 As shown.

[0072] The results above show that among the treatments of MSCs with COL1, COL3, COL4, COL7, COL17, and COL21, only COL21 can improve cell stemness.

[0073] Example 4: COL21 stem cell spheroidization experiment

[0074] Stem cell spheroidization is the spontaneous aggregation of stem cells into three-dimensional (3D) spherical cell clusters under specific non-adherent culture conditions (such as on low-adhesion surfaces) in vitro, regulated by intercellular adhesion and autocrine / paracrine signaling. This structure better mimics the physiological microenvironment of stem cell nests in vivo and serves as an important in vitro model for maintaining stem cell stemness, regulating differentiation, and simulating function. Compared to traditional two-dimensional (2D) adherent culture, spheroidization culture exhibits significant advantages in maintaining stem cell stemness and regulating differentiation direction, and has become one of the core methods for basic stem cell research, drug screening, and clinical translation in regenerative medicine. The size, cellular composition, and function of the spheroids vary depending on the type of stem cells and culture conditions, and their diameter typically needs to be controlled within tens to hundreds of micrometers (e.g., 50-500 μm) to ensure the survival and function of the internal cells.

[0075] This study evaluated the influence of COL21 on stem cell stemness through a stem cell spheroidization assay. The spheroidization assay simulates the in vivo stem cell microenvironment by seeding stem cells in a serum-free or low-serum, low-adhesion three-dimensional culture system. Stem cell stemness is the core basis for their formation of microspheres. Only stem cells with strong self-renewal capacity and in an undifferentiated state can aggregate to form structurally stable three-dimensional microspheres through autocrine signals and intercellular adhesion. Conversely, cells with weakened stemness or those that have differentiated are unlikely to form effective microspheres. Simultaneously, the formed microsphere structure can conversely maintain stem cell stemness by reducing differentiation signal contact, enriching stemness-related factors, inhibiting stem cell differentiation into mature cells, and further preserving their multi-directional differentiation potential and self-renewal characteristics. Therefore, this experiment not only directly reflects the strength of stem cell stemness through spheroidization rate, spheroid morphology, and stability, but also provides an in vitro functional verification system for maintaining stemness.

[0076] Experimental Procedure: First, prepare spheroid culture medium by adding 2% FBS, 1% penicillin-streptomycin 10 ng / mL bFGF, 10 ng / mL EGF, 4 μg / mL heparin, and 0.48 μg / mL hydrocortisone to DMEM / F12 basal medium. Then, seed MSCs at a density of 5000 cells per well in ultra-low adsorption six-well plates, with three replicates each for the collagen group and the control group. Before seeding, thoroughly mix the cells and filter through a 70 μm cell sieve to ensure single-cell suspension before accurate counting and plating. The final concentration of COL21 in the collagen group's medium was 10 μg / mL, while no COL21 was added to the control group. The culture period was 7–10 days, during which the medium was replaced every 2–3 days using a half-medium replacement method: first, slowly aspirate 1 mL of the old medium, then slowly add 1 mL of fresh spheroidizing medium containing the appropriate concentration of COL21. When the cell spheroids reached a diameter of approximately 150 μm, image acquisition was performed. Finally, ImageJ software was used to quantitatively analyze the diameter of the microspheres, and GraphPad Prism software was used for statistical analysis and graph plotting.

[0077] The results are as follows Figure 3 and Figure 4 As shown in the results of the microsphere formation experiment, in the control group, the number of microspheres formed by MSCs was relatively small, and the volume of each microsphere was also relatively small. After treatment with 10 μg / mL COL21, the number of microspheres formed by MSCs increased significantly, and the volume of each microsphere was also fuller. Microsphere formation ability is one of the core characteristics of MSC self-renewal capacity and a classic functional indicator for evaluating its stemness level. The above experimental phenomena indicate that 10 μg / mL COL21 treatment can significantly enhance the microsphere formation ability of MSCs, suggesting that COL21 can effectively improve the stemness of MSCs.

[0078] Statistical results show that the number of MSCs treated with COL21 was 1.52 times that of the control group. MSCs treated with COL21 also had more stem cell microspheres with a diameter greater than 150 μm, indicating that COL21 can better maintain the stemness of MSCs.

[0079] Example 5: Screening of COL21 receptors on cell surfaces

[0080] According to existing literature, collagen receptors are mainly divided into two categories: integrins and discoid domain proteins. To investigate the effect of COL21 on collagen receptor expression, this study selected representative integrin receptors (such as ITGA1, ITGA2, and ITGB1) and discoid domain protein receptors (such as DDR1 and DDR2), and designed specific primers targeting their encoding genes (see Table 7 for details). qPCR experiments were used to detect changes in the mRNA expression levels of these receptors in cells before and after COL21 treatment, in order to analyze the regulatory role of COL21 in collagen receptor expression. The results are shown in Table 7. Figure 5 .

[0081] Table 7

[0082] .

[0083] The qPCR results showed that the expression of integrin-type candidate receptor mRNAs increased to varying degrees, while the expression of discoid domain protein-type candidate receptor mRNAs remained unchanged. This suggests that the receptor for COL21 on the cell surface is an integrin-type receptor. To further verify this hypothesis at the protein level, Western blotting (WB) was used to detect changes in the protein expression of the relevant receptors. The WB experimental procedure is as follows:

[0084] Human mesenchymal stem cells (MSCs) were cultured at a density of 1.5 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in six-well plates. The experimental group received culture medium supplemented with 10 μg / mL COL21, while the control group received no supplementation. Each group had three replicates. Cells were cultured at 37°C and 5% CO2 for 48 hours, with the medium replaced with fresh medium containing the appropriate concentration of COL21 every 24 hours. After culture, cells were digested with trypsin, the cell pellet was resuspended in PBS, and washed by centrifugation. The resulting cell pellet was stored at -80°C for later use.

[0085] Protein sample extraction: The entire process must be performed on ice. First, prepare the cell lysis buffer by adding 10 μL of protease inhibitor, 10 μL of phosphatase inhibitor, and 5 μL of PMSF to 475 μL of lysis buffer and mixing thoroughly. Add 80-120 μL of the mixed lysis buffer to each tube of cell pellet, vortex to resuspend, and lyse on ice for 30 minutes, vortexing every 10 minutes during lysis. Centrifuge the lysis buffer at 4°C and 12,000 rpm for 25 minutes, and collect the supernatant for subsequent experiments. Protein concentration is determined using the BCA method, and a standard curve is constructed using a commercial BSA standard kit for sample quantification. Mix the protein sample with the loading buffer, denature at 98°C for 10 minutes, aliquot, and store at -80°C for later use.

[0086] SDS-PAGE Gel Preparation: Prepare a 12% separating gel according to the formula. Assemble the electrophoresis apparatus and add Tris-glycine electrophoresis buffer. Add an equal mass (20 μg) of denatured protein sample to each well, and reserve 1-2 wells for pre-stained protein molecular weight standards (markers). Connect the power supply and set the initial voltage to 80V for stacking gel electrophoresis. After the bromophenol blue indicator enters the separating gel, adjust the voltage to 120V and continue electrophoresis until the bromophenol blue reaches about 1 cm from the bottom of the separating gel.

[0087] Transfer: Beforehand, soak the PVDF membrane in methanol for 10-30 seconds to activate the active groups on the membrane. At the same time, prepare the transfer buffer (containing 20% ​​methanol) and pre-cool it. Stack the gel, PVDF membrane, and filter paper after electrophoresis in the following order: "negative electrode → filter paper → gel → PVDF membrane → filter paper → positive electrode" (ensure that there are no air bubbles in each layer and that the protein sides of the gel and membrane are facing each other). Place the transfer clamp in the transfer tank and fix it in place. Place the transfer clamp in the transfer tank (the transfer tank should be placed in an ice bath to avoid excessive temperature during the transfer process). Add the pre-cooled transfer buffer, turn on the power, set the transfer parameters to a constant voltage of 100V, and transfer for 75 minutes. After the transfer, remove the PVDF membrane and wash it twice with TBST buffer (5 min each time). Then, place the membrane in blocking buffer (5% skim milk or BSA solution, prepared with TBST) and block it on a shaker at room temperature for 1-2 h. After blocking, discard the blocking buffer, wash the membrane three times with TBST (5 min each time), add the primary antibody diluted with blocking buffer according to the instructions, and incubate overnight on a shaker at 4°C. After the primary antibody incubation, discard the primary antibody solution, wash the membrane three times with TBST (10 min each time), add HRP-labeled secondary antibody diluted with blocking buffer, and incubate on a shaker at room temperature for 1 h. After the secondary antibody incubation, discard the secondary antibody solution, wash the membrane three times with TBST (10 min each time).

[0088] Imaging: The membrane was immersed in ECL chemiluminescence developing solution (mixed in a 1:1 ratio of solution A to solution B) for 1-2 minutes, then exposed and imaged using a chemiluminescence imager. The target protein bands were analyzed for grayscale using software such as ImageJ. See [link to image analysis] for detailed results. Figure 6 .

[0089] Western blot results showed that the expression level of integrin ITGA2 was upregulated by 1.454-fold after COL21 treatment, suggesting that ITGA2 is the receptor for COL21 on the cell surface.

[0090] Literature review reveals that the stem cell pathway closely related to integrins is the integrin-FAK-Src pathway. This pathway is a key signaling axis for cells to sense extracellular matrix (ECM) signals and transmit them into the cell. Integrin, as a transmembrane receptor, activates intracellular focal adhesion kinase (FAK) by recognizing components such as collagen and fibronectin in the ECM. Phosphorylated FAK (such as phosphorylation at Y397 site) further recruits and activates Src family kinases. The resulting FAK-Src complex can regulate basic biological processes such as cell adhesion, migration, proliferation, and survival by phosphorylating downstream substrates (such as p130Cas and peg proteins). This pathway is closely related to cell stemness (referring to the cell's self-renewal capacity and multi-directional differentiation potential): on the one hand, the pathway can maintain the expression of stemness-related transcription factors (such as Oct4, Sox2, and Nanog) by activating downstream signals such as PI3K / Akt and MAPK, thus ensuring stemness homeostasis of stem cells in the niche environment; on the other hand, ECM can regulate the adhesion properties of stem cells through integrin-mediated pathway signals, affecting their symmetric / asymmetric division selection, thereby maintaining stemness or inducing differentiation. When cell stemness is enhanced (e.g., through stem cell-directed induction of stemness enhancement or acquisition of stemness from tumor stem cells), this pathway exhibits significant activation characteristics: specifically, the expression level of integrins is upregulated, enhancing the cell's ability to bind to the ECM; the phosphorylation level of FAK is increased, promoting its binding to Src and the recruitment of downstream signaling molecules; simultaneously, the activity of Src kinase is enhanced, further upregulating the expression of stemness transcription factors by amplifying signal transduction efficiency and inhibiting the activation of differentiation-related genes, ultimately forming a positive regulatory cycle of "pathway activation - stemness maintenance," supporting the enhancement of cell stemness.

[0091] To verify whether COL21 regulates the stemness of human mesenchymal stem cells (MSCs) through the aforementioned signaling pathway, researchers further used Western blotting to analyze the expression levels of marker proteins closely related to this pathway and to preliminarily elucidate its potential molecular mechanism.

[0092] Prepare SDS-PAGE gels and mix a 12% separating gel according to the formula. Assemble the electrophoresis apparatus and add Tris-glycine electrophoresis buffer. Add an equal mass (20 μg) of denatured protein sample to each well, and reserve 1-2 wells for pre-stained protein molecular weight standards (markers). Connect the power supply and set the initial voltage to 80V for stacking gel electrophoresis. After the bromophenol blue indicator enters the separating gel, adjust the voltage to 120V and continue electrophoresis until the bromophenol blue reaches about 1 cm from the bottom of the separating gel.

[0093] Transfer: Beforehand, soak the PVDF membrane in methanol for 10-30 seconds to activate the active groups on the membrane. At the same time, prepare the transfer buffer (containing 20% ​​methanol) and pre-cool it. Stack the gel, PVDF membrane, and filter paper after electrophoresis in the following order: "negative electrode → filter paper → gel → PVDF membrane → filter paper → positive electrode" (ensure that there are no air bubbles in each layer and that the protein sides of the gel and membrane are facing each other). Place the transfer clamp in the transfer tank and fix it in place. Place the transfer clamp in the transfer tank (the transfer tank should be placed in an ice bath to avoid excessive temperature during the transfer process). Add the pre-cooled transfer buffer, turn on the power, set the transfer parameters to a constant voltage of 100V, and transfer for 75 minutes. After the transfer, remove the PVDF membrane and wash it twice with TBST buffer (5 min each time). Then, place the membrane in blocking buffer (5% skim milk or BSA solution, prepared with TBST) and block it on a shaker at room temperature for 1-2 h. After blocking, discard the blocking buffer, wash the membrane three times with TBST (5 min each time), add the primary antibody diluted with blocking buffer according to the instructions, and incubate overnight on a shaker at 4°C. After the primary antibody incubation, discard the primary antibody solution, wash the membrane three times with TBST (10 min each time), add HRP-labeled secondary antibody diluted with blocking buffer, and incubate on a shaker at room temperature for 1 h. After the secondary antibody incubation, discard the secondary antibody solution, wash the membrane three times with TBST (10 min each time).

[0094] Imaging: The membrane was immersed in ECL chemiluminescence developing solution (mixed in a 1:1 ratio of solution A to solution B) for 1-2 minutes, then exposed and imaged using a chemiluminescence imager. The target protein bands were analyzed for grayscale using software such as ImageJ. See [link to image analysis] for detailed results. Figure 7 .

[0095] This expression pattern is highly consistent with the activation pattern of the integrin-FAK-Src stemness regulation pathway. Specifically, the upregulation of ITGA2 as a transmembrane receptor can enhance the binding ability of MSCs to the extracellular matrix, thereby initiating intracellular signal transduction. The results of no significant upregulation of total FAK expression and significant increase of p-FAK confirm that COL21 does not play a role by increasing FAK protein synthesis, but directly induces FAK phosphorylation through ITGA2-mediated receptor activation. The activated FAK can further recruit Src to form a functional complex, which initiates signaling pathways such as PI3K / Akt and MAPK by phosphorylating downstream substrates. On the one hand, it maintains the expression of stemness transcription factors such as Oct4 and Sox2 to ensure the self-renewal capacity of MSCs, and on the other hand, it stabilizes cell adhesion properties and regulates cell division direction to inhibit differentiation, ultimately achieving the enhancement of MSC stemness.

[0096] Further, prepare SDS-PAGE gels. Prepare a 12% separating gel according to the formula. Load the polymerized gel into the electrophoresis tank, add Tris-glycine electrophoresis buffer to both the upper and lower wells, carefully remove the comb, and pipette 20 μg of the denatured protein sample. Simultaneously, add protein molecular weight standards (markers) to 1-2 wells. Turn on the power and perform electrophoresis at a constant voltage of 80V (adjust the voltage to 120V after the sample enters the separating gel). Observe the migration of the bromophenol blue indicator during electrophoresis. When the indicator reaches 1-2 cm from the bottom of the separating gel, turn off the power and stop electrophoresis. Then, perform membrane transfer. Beforehand, soak the PVDF membrane in methanol for 10-30 seconds to activate the active groups on the membrane. Prepare transfer buffer (containing 20% ​​methanol) and pre-cool it. Transfer the gel, PVDF membrane, and filter paper in the following order: negative electrode → filter paper → gel → PVDF. Stack the membrane, filter paper, and positive electrode in the following order (ensuring no air bubbles in each layer and that the protein surfaces of the gel and membrane are facing each other). Place the membrane in the transfer clamp and secure it. Place the transfer clamp in the transfer tank (the transfer tank should be placed in an ice bath to avoid excessive temperature during transfer). Add pre-cooled transfer buffer, turn on the power, and select the transfer parameters (usually constant voltage 100V, transfer for 75 minutes). After transfer, remove the PVDF membrane and wash it twice with TBST buffer (5 minutes each time). Then, place the membrane in blocking buffer (5% skim milk or BSA solution, prepared with TBST) and block on a shaker at room temperature for 1-2 hours. After blocking, discard the blocking buffer, wash the membrane three times with TBST (5 minutes each time), add the primary antibody diluted with the blocking buffer according to the instructions, and incubate overnight on a shaker at 4°C. After primary antibody incubation, discard the primary antibody solution, wash the membrane three times with TBST (10 minutes each time), and add HRP diluted with the blocking buffer according to the instructions. Secondary antibody was labeled and incubated on a shaker at room temperature for 1 hour. After incubation, the secondary antibody solution was discarded, and the membrane was washed three times with TBST (10 minutes each time). The membrane was then immersed in ECL chemiluminescence developing solution (mixed in a 1:1 ratio of solution A to solution B) for 1-2 minutes, followed by exposure imaging in a chemiluminescence imager (adjusting the exposure time according to the signal intensity to avoid overexposure or underexposure). Phosphorylated Src protein was exposed first, and after the phosphorylated Src protein exposure was complete, the secondary antibody on the membrane surface was peeled off using commercial antibody stripping solution. The total Src protein was then exposed again following the blocking, primary antibody labeling, and secondary antibody labeling procedure. After imaging, grayscale analysis of the target protein bands was performed; see [link to relevant documentation] for details. Figure 8 .

[0097] The Western blot results showed that FAK expression was slightly upregulated after COL21 treatment, with an overall upregulation of about 1.12-fold, and phosphorylated FAK was upregulated by 1.97-fold. The protein expression characteristics presented by this Western blot are highly consistent with the activation pattern of the integrin-FAK-Src stemness regulatory pathway. After COL21 treatment, there was no significant difference in total SRC compared to the control group, but phosphorylated activated P416-SRC was significantly enhanced. This result forms a continuous link with the previous signal transduction logic of "ITGA2 upregulation and FAK phosphorylation activation"—that is, COL21 enhances the binding of MSCs to the extracellular matrix by upregulating the cytokine ITGA2, thereby inducing FAK phosphorylation activation. Activated FAK further recruits SRCs and causes them to phosphorylate. Phosphorylated activated SRCs can form a functional complex with FAK, which initiates stemness-related signaling pathways such as PI3K / Akt and MAPK through phosphorylation of downstream substrates, ultimately maintaining the expression of stemness transcription factors such as Oct4 and Sox2, while stabilizing cell adhesion properties and regulating division direction to inhibit differentiation, thereby enhancing MSC stemness.

[0098] Prepare SDS-PAGE gels. Prepare a 12% separating gel according to the formula. Load the polymerized gel into the electrophoresis tank. Add Tris-glycine electrophoresis buffer to both the upper and lower wells. Carefully remove the comb and pipette 20 μg of the denatured protein sample. Simultaneously, add protein molecular weight standards (markers) to 1-2 wells. Turn on the power and perform electrophoresis at a constant voltage of 80V (adjust the voltage to 120V after the sample enters the separating gel). Observe the migration of the bromophenol blue indicator during electrophoresis. When the indicator reaches 1-2 cm from the bottom of the separating gel, turn off the power and stop electrophoresis. Then perform membrane transfer. Beforehand, soak the PVDF membrane in methanol for 10-30 seconds to activate the active groups on the membrane. Prepare transfer buffer (containing 20% ​​methanol) and pre-cool it. Transfer the gel, PVDF membrane, and filter paper in the following order: negative electrode → filter paper → gel → PVDF membrane → filter paper → positive electrode. Stack the gel and membrane layers in the following order (ensuring no air bubbles in each layer, with the protein sides of the gel and membrane facing each other). Place the gel in the transfer clamp and secure it. Place the transfer clamp in the transfer tank (the transfer tank should be placed in an ice bath to avoid excessive temperature during transfer). Add pre-cooled transfer buffer, turn on the power, and select the transfer parameters (usually constant voltage 100V, transfer for 75 min). After transfer, remove the PVDF membrane and wash it twice with TBST buffer (5 min each time). Then, place the membrane in blocking buffer (5% skim milk or BSA solution, prepared with TBST) and block on a shaker at room temperature for 1-2 hours. After blocking, discard the blocking buffer, wash the membrane three times with TBST (5 min each time), add the primary antibody diluted with the blocking buffer according to the instructions, and incubate overnight on a shaker at 4°C. After primary antibody incubation, discard the primary antibody solution, wash the membrane three times with TBST (10 min each time), and add HRP diluted with the blocking buffer according to the instructions. Secondary antibody was labeled and incubated on a shaker at room temperature for 1 hour. After incubation, the secondary antibody solution was discarded, and the membrane was washed three times with TBST (10 minutes each time). The membrane was then immersed in ECL chemiluminescence developing solution (mixed in a 1:1 ratio of solution A to solution B) for 1-2 minutes, followed by exposure imaging in a chemiluminescence imager (adjusting the exposure time according to the signal intensity to avoid overexposure or underexposure). Phosphorylated AKT protein was exposed first, and after the exposure of phosphorylated AKT protein was complete, the secondary antibody on the membrane surface was peeled off using commercial antibody stripping solution. Then, the total AKT protein was exposed again following the blocking, primary antibody labeling, and secondary antibody labeling procedure. After imaging, grayscale analysis of the target protein bands was performed; see [link to relevant documentation] for details. Figure 9 .

[0099] Western blot (WB) results showed that the total expression level of AKT remained essentially unchanged after COL21 treatment, while phosphorylated AKT was upregulated by 2.39-fold. These WB results further clarified the signal transduction network by which COL21 regulates MSC stemness. The signaling logic of "COL21 inducing FAK phosphorylation through upregulation of ITGA2, thereby activating SRC" forms a continuous link. The activated FAK-SRC functional complex can initiate the PI3K / Akt pathway through phosphorylation of downstream substrates. The significant upregulation of p-AKT is a direct marker of this pathway activation, confirming the activation effect of COL21 on stemness-related signaling axes and providing key molecular evidence for its functional conclusion of maintaining the expression of stemness transcription factors such as Oct4 and Sox2 and inhibiting MSC differentiation.

[0100] Those skilled in the art will also recognize, or be able to identify, many equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. These equivalents are also included in the appended claims.

Claims

1. A method for improving the stem cell stemness of mesenchymal stem cells, characterized in that, The method includes treating the mesenchymal stem cells with an active substance, activating the integrin-FAK-Src pathway to enhance FAK and Src phosphorylation, thereby activating the downstream PI3K / Akt pathway and improving the stem cell activity of the mesenchymal stem cells. The active substance is recombinant humanized collagen type 21, the amino acid sequence of which is SEQ ID No. 1, and the concentration of which is 1-50 μg / mL.

2. The method according to claim 1, characterized in that, The method upregulates four stem cell stemness genes—Oct-4, Klf4, Nanog, and SOX2—in mesenchymal stem cells, thereby improving the stemness of the mesenchymal stem cells.

3. A method for promoting the upregulation of four stem cell genes—Oct-4, Klf4, Nanog, and SOX2—in mesenchymal stem cells, characterized in that, The method includes treating the mesenchymal stem cells with an active substance, the active substance being recombinant humanized collagen type 21, the amino acid sequence of which is SEQ ID No. 1, and the concentration of which is 1-50 μg / mL.

4. The application of the compound in the preparation of a stem cell therapy agent that enhances the stemness of mesenchymal stem cells, characterized in that, The compound is type 21 recombinant humanized collagen, the amino acid sequence of which is SEQ ID No. 1, and the concentration of which is 1-50 μg / mL.

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