Composition for enhancing immunomodulatory function of stem cells

Abstract

The invention relates to the technical field of biology, and discloses a composition for enhancing the immunomodulatory function of stem cells, and the composition comprises a ferrous ion source, a ferroptosis inhibitor and L-arginine in a basic culture medium. According to the method, a substrate (L-arginine) synthesized by nitric oxide (NO), a necessary cofactor (divalent iron ions) for activating a key enzyme iNOS and a ferroptosis inhibitor are connected in parallel to avoid the toxicity of iron ions and guarantee cell survival, the three components have a synergistic effect, an optimized intracellular environment is constructed, and the method can be used for preparing an anti-inflammatory drug under the condition of not depending on the stimulation of inflammatory factors. The immunoregulation function of the stem cells is safely and efficiently enhanced. By using the composition disclosed by the invention, the treatment efficacy of the stem cells can be remarkably improved, meanwhile, the immunogenicity of the stem cells is effectively prevented from being improved, and a new technical path is provided for developing safer cell treatment products.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a composition for enhancing the immunomodulatory function of stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) have become one of the most promising seed cells in cell therapy and regenerative medicine due to their self-renewal, multi-lineage differentiation potential, and strong immunomodulatory functions. In particular, MSCs have demonstrated enormous clinical potential in the treatment of graft-versus-host disease, autoimmune diseases, and other inflammatory dysregulation-related diseases by inhibiting the excessive activation and proliferation of immune cells such as T lymphocytes.

[0003] However, mesenchymal stem cells (MSCs) in a resting state have relatively limited natural immunosuppressive capabilities, and their therapeutic effects often fall short of expectations when directly applied to the complex in vivo microenvironment. Therefore, pre-treating or functionally enhancing MSCs in vitro before clinical application to stimulate their immunomodulatory potential has become a common technical strategy in this field to improve the efficacy of cell therapy products.

[0004] Currently, the widely adopted technique in the industry is to pretreat mesenchymal stem cells with pro-inflammatory cytokines, especially interferon-γ (IFN-γ). This method can indeed effectively activate intracellular signaling pathways and significantly upregulate the expression of key effector molecules such as inducible nitric oxide synthase (iNOS), thereby greatly enhancing their ability to suppress immune responses.

[0005] Nevertheless, this traditional approach suffers from a profound and unavoidable inherent flaw. While IFN-γ-activated JAK / STAT signaling enhances immunosuppressive function, it inevitably leads to a dramatic increase in the expression of major histocompatibility complex II (MHC-II, primarily HLA-DR in humans) molecules on the surface of mesenchymal stem cells. MHC-II molecules are core molecules in triggering adaptive immune responses; their increased expression fundamentally alters the immunological phenotype of stem cells, causing them to lose their valuable "low immunogenicity" characteristic. This alteration makes functionally enhanced stem cells more susceptible to recognition as "foreign substances" and elimination by the host immune system, severely weakening their survival time and therapeutic efficacy in vivo, and posing serious safety risks for clinical applications. Therefore, existing technologies are generally caught in a dilemma: functional enhancement and increased immunogenicity are closely linked and difficult to separate, which greatly limits the development of highly effective, safe, and universal stem cell therapy products. Summary of the Invention

[0006] The technical problem to be solved by this invention is that, in order to enhance the immunomodulatory function of mesenchymal stem cells, the existing technology generally leads to a corresponding increase in cellular immunogenicity, which in turn brings safety risks in clinical applications.

[0007] To address the above problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a composition for enhancing the immunomodulatory function of stem cells, employing the following technical solution: A composition for enhancing the immunomodulatory function of stem cells, said composition comprising the following components in a basal culture medium: Source of ferrous ions; Ferrocyte inhibitors; L-arginine.

[0008] By employing the above-described technical solution, the composition provided by this invention can provide complete biochemical support for the efficient synthesis and secretion of nitric oxide (NO) by stem cells at both the post-translational and metabolic levels, thereby enhancing their immunomodulatory function without relying on traditional inflammatory factor stimulation. Its mechanism of action lies in the synergistic effect of the three components in the composition against key rate-limiting steps in the nitric oxide synthesis pathway: 1. Providing a substrate for synthesis: L-arginine is the only physiological substrate for the synthesis of nitric oxide catalyzed by inducible nitric oxide synthase (iNOS). This composition ensures a sufficient supply of exogenous L-arginine for the NO synthesis pathway, which is fundamental to increasing NO production.

[0009] 2. Activation of key enzymes: Inducible nitric oxide synthase is a heme protein whose catalytic activity is highly dependent on the valence state and saturation of iron ions in the heme cofactor. Ferrous ions (Fe²⁺) 2+ ) is an essential cofactor for maintaining the activity of this enzyme. This composition, by providing an exogenous source of ferrous ions, can effectively increase intracellular Fe... 2+ The bioavailability of the pre-expressed iNOS enzyme protein is directly applied to the iNOS protein, saturating its cofactor requirements and thus maximizing the catalytic potential of the pre-expressed iNOS enzyme protein.

[0010] 3. Ensuring Cell Survival: Exogenous supplementation with high concentrations of ferrous ions can be cytotoxic, primarily through the Fenton reaction catalyzing the production of reactive oxygen species, which in turn trigger lipid peroxidation, ultimately leading to programmed cell death known as ferroptosis. This composition, by introducing a ferroptosis inhibitor, specifically blocks the ferric-mediated cell death pathway, effectively avoiding the cytotoxic side effects of ferrous ions. This component ensures that stem cells maintain their cellular structural integrity and biological activity during intensive metabolic remodeling to synthesize NO.

[0011] Therefore, this composition constructs an optimized intracellular environment through a synergistic mechanism of providing substrate, activating enzymes, and ensuring survival, enabling stem cells to safely and efficiently convert L-arginine into nitric oxide, ultimately achieving a significant enhancement of immunomodulatory function.

[0012] Preferably, the ferrous ions are derived from ferrous ascorbate.

[0013] By adopting the above technical solution, ferrous ascorbate is a stable divalent iron ion complex. The ascorbic acid it contains has reducing properties, which helps to maintain iron ions in a divalent state that is easily utilized by cells, thereby improving the bioavailability of iron ions.

[0014] Preferably, the ferroptosis inhibitor is Ferrostatin-1.

[0015] By adopting the above technical solution, Ferrostatin-1 is a highly specific and efficient inhibitor of ferroptosis that can precisely act on the target site and achieve effective protection of cells at a low concentration.

[0016] Preferably, the composition comprises ferrous ascorbate at a final concentration of 10–100 µM, ferrostatin-1 at a final concentration of 0.1–5 µM, and L-arginine at a final concentration of 1–4 mM.

[0017] By employing the above-described technical solution, a balance between function and safety can be achieved among the components within the specified concentration range. This ratio range ensures that the iNOS enzyme receives sufficient substrate and cofactors to achieve maximum catalytic activity, while the concentration of the ferroptosis inhibitor is sufficient to completely neutralize the cytotoxicity that may result from the corresponding concentration of iron ions, ultimately leading to a stable and reproducible functional enhancement effect.

[0018] Secondly, the present invention provides a method for enhancing the immune regulatory function of stem cells, employing the following technical solution: A method for enhancing the immunomodulatory function of stem cells includes the following steps: a) Instantaneous hypoxia treatment of stem cells; b) Culture the stem cells treated in step a) using the composition described in the first aspect.

[0019] By employing the above-described technical solution, the method provided in this application establishes a dual, synergistic regulatory system that separates the upstream transcriptional activation and downstream post-translational function of inducible nitric oxide synthase (iNOS) in terms of both time and function, thereby efficiently and safely enhancing the immunomodulatory function of stem cells. The innovative mechanism of action of this method is as follows: 1. Step a) Pre-activation at the transcriptional level: Transient hypoxia treatment of stem cells simulates an ischemic microenvironment. This treatment activates the hypoxia-inducible factor (HIF) signaling pathway within the cell. HIF, as a key transcription factor, can directly or indirectly initiate the transcription of downstream iNOS genes, thereby accumulating large amounts of iNOS messenger RNA (mRNA) within the cell. This step completes the transcriptional preparation for iNOS synthesis without introducing exogenous inflammatory factors, laying the molecular foundation for the subsequent efficient synthesis of NO. Importantly, this pathway bypasses the classical IFN-γ / JAK / STAT signaling pathway, thus avoiding the synchronous upregulation of major histocompatibility complex class II (MHC-II) molecules, fundamentally preventing increased cellular immunogenicity.

[0020] 2. Step b) Post-translational and metabolic activation and protection: After transcription preparation, cells are treated with the composition described in the first aspect of this application. This step follows the mRNA accumulation in the previous step, enabling efficient NO synthesis at the post-translational and metabolic levels. Cells translate accumulated iNOS mRNA into iNOS enzyme protein.

[0021] The ferrous ion source in the composition (such as ferrous ascorbate) serves as an essential cofactor, saturating the active site of the iNOS enzyme protein and enabling it to possess maximum catalytic activity.

[0022] The L-arginine in the composition serves as a substrate, providing ample raw materials for the activated iNOS enzyme.

[0023] The ferroptosis inhibitors in the composition (such as Ferrostatin-1) neutralize the cytotoxicity that may be caused by high concentrations of ferrous ions, ensuring the survival and stability of cells when performing their functions.

[0024] This two-step method overcomes the limitations of single treatments by first upregulating transcription and then activating function, resulting in a final NO production that is far greater than the sum of the effects of either step alone, thereby significantly enhancing the immunosuppressive function of stem cells.

[0025] Preferably, the stem cells are mesenchymal stem cells, and more preferably human umbilical cord mesenchymal master stem cells.

[0026] By adopting the above technical solution, human umbilical cord mesenchymal stem cells have the characteristics of abundant source, easy acquisition, strong proliferation capacity and low natural immunogenicity, making them an ideal application carrier for this technical solution.

[0027] Preferably, the conditions for the instantaneous hypoxia treatment are: treatment for 2 to 6 hours in an environment with an oxygen concentration of 1% to 5%.

[0028] By adopting the above technical solution, the treatment conditions are transient and non-lethal hypoxia stimulation, which is sufficient to effectively activate the HIF signaling pathway and initiate iNOS transcription, while avoiding irreversible damage to cells caused by prolonged or extreme hypoxia, thus ensuring the biological activity of cells after treatment.

[0029] Preferably, the culture process described in step b) lasts for 12 to 24 hours.

[0030] By employing the above-described technical approach, this culture duration provides cells with ample time to complete the translation of the iNOS protein, activation of enzymes, and synthesis and secretion of NO, thereby achieving the goal of enhanced function. Simultaneously, this limited treatment duration controls the interaction time between chemicals and cells, reducing potential long-term effects.

[0031] Thirdly, the present invention provides applications of the composition and functionally enhanced stem cells prepared by the method, employing the following technical solutions: The use of the composition described in the first aspect in the preparation of functionally enhanced stem cells is provided, wherein the functional enhancement is specifically manifested as enhanced immunomodulatory function without a significant increase in immunogenicity.

[0032] By employing the above-described technical solution, the application value of the composition described in this application lies in providing a novel technical pathway for enhancing stem cell function that is independent of traditional inflammatory factor stimulation. This application, by providing stem cells with a precise combination of metabolic regulatory molecules, can directionally and efficiently amplify the endogenous nitric oxide synthesis pathway, thereby enhancing their immunomodulatory function. Simultaneously, because the target of this composition is located at the metabolic level and does not involve the widespread upregulation of cell surface immunophenotype-related genes (such as HLA-DR), it successfully decouples the processes of functional enhancement and increased immunogenicity, which are usually intertwined in existing technologies. This allows stem cell products prepared through this application to maintain or enhance therapeutic efficacy while possessing higher biological safety.

[0033] Furthermore, the present invention also provides a functionally enhanced stem cell, which is prepared by the method described in the second aspect.

[0034] By employing the aforementioned technical approach, the obtained stem cells, as a technological product, exhibit beneficial alterations in both their internal state and external function. Compared to the original stem cells, this cell product demonstrates pre-upregulated iNOS gene expression at the transcriptomic level and possesses the ability to efficiently synthesize and secrete nitric oxide at the functional protein level. Ultimately, this intrinsic functional remodeling manifests as a potent inhibitory effect on the proliferation of immune cells such as T lymphocytes. Simultaneously, this cell product maintains the low levels of MHC-II molecule expression before the original intervention, preserving its advantage of low immunogenicity as a universal cell therapy product. Therefore, this functionally enhanced stem cell is a cell therapy product that combines high efficacy and high safety.

[0035] In summary, the present invention has at least one of the following beneficial technical effects: 1. This invention enhances the immunomodulatory function of stem cells while effectively avoiding increased immunogenicity, significantly improving the biological safety of the product. This invention employs transient hypoxia treatment to pre-regulate iNOS gene transcription. This process bypasses the JAK / STAT signaling pathway dependent on traditional IFN-γ stimulation, thus avoiding simultaneous upregulation of MHC-II molecule expression. Therefore, this invention fundamentally solves the key problem of increased immunogenicity often accompanying functional enhancement in existing technologies.

[0036] 2. This invention achieves highly efficient enhancement of stem cell immunomodulatory function through a two-step synergistic approach, with effects far superior to single-treatment methods. The method first pre-deploys nitric oxide at the transcriptional level through transient hypoxia, followed by functional activation at the post-translational and metabolic levels using a composition containing a ferrous iron source and L-arginine. This strategy of first upregulating genes and then activating proteins results in a superadditive increase in the yield of the final functional product (nitric oxide), leading to a significant enhancement effect.

[0037] 3. The technical solution adopted in this invention has high biological safety, maintaining the survival rate and biological activity of stem cells during high-intensity metabolic remodeling. Its core lies in the inclusion of a ferroptosis inhibitor as a key component in the composition of this invention. This component effectively neutralizes the cytotoxicity caused by ferrous ions introduced to activate the iNOS enzyme, ensuring that cells can survive and stably perform their biological functions, thus ensuring the stable achievement of the final technical effect. Attached Figure Description

[0038] Figure 1 Bar chart of cell viability of hUC-MSCs after treatment in the embodiments and comparative examples of the present invention; Figure 2 Bar chart of relative expression levels of iNOS mRNA in hUC-MSCs cells after treatment in the embodiments and comparative examples of this invention; Figure 3 Bar chart of nitrite concentration in hUC-MSCs culture medium after treatment in the embodiments and comparative examples of this invention; Figure 4 A bar chart showing the inhibition rate of T lymphocyte proliferation by hUC-MSCs after treatment in the embodiments and comparative examples of this invention. Detailed Implementation

[0039] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0040] Human umbilical cord mesenchymal stem cells (hUC-MSCs): Cells from passage 3 to passage 6 purchased from commercial cell banks. Flow cytometry analysis showed that the expression rates of cell surface markers CD73, CD90, and CD105 were not less than 95%, while the expression rates of CD34, CD45, CD11b, CD19, and HLA-DR were not higher than 2%.

[0041] Human peripheral blood mononuclear cells: isolated from the peripheral blood of healthy volunteers.

[0042] Example 1: This embodiment provides a method for enhancing the immunomodulatory function of mesenchymal stem cells, including the following steps: 1. Fourth-generation human umbilical cord mesenchymal stem cells were introduced at a concentration of 1.5 × 10⁻⁶. 4 cells / cm 2 Cells were seeded at a density in cell culture dishes and cultured in DMEM / F-12 medium containing 10% fetal bovine serum at 37°C in a normoxic incubator with 5% CO2 until the cell confluence reached 70%–80%.

[0043] 2. Place the above-mentioned petri dishes directly in a three-gas incubator at 37℃, 5% CO2, and 3% O2 ​​for a continuous 4-hour low-oxygen pretreatment.

[0044] 3. After the hypoxia treatment, discard the culture medium and wash once with PBS. Then add functional enhancement medium, which is prepared from serum-free DMEM / F-12 basal medium containing 50 µM ferrous ascorbate, 1 µM ferrostatin-1, and 2 mM L-arginine. Incubate the cells in a normoxic incubator at 37°C and 5% CO2 for 18 hours.

[0045] 4. After culture, discard the functional enhancement medium, wash the cells three times with PBS, digest them with trypsin and collect the cells for subsequent tests.

[0046] Example 2: This embodiment provides a method for enhancing the immunomodulatory function of mesenchymal stem cells, including the following steps: 1. Fourth-generation human umbilical cord mesenchymal stem cells were injected at a concentration of 1.0 × 10⁻⁶. 4 cells / cm 2 Cells were seeded at a density in cell culture dishes and cultured in DMEM / F-12 medium containing 10% fetal bovine serum at 37°C in a normoxic incubator with 5% CO2 until the cell confluence reached 70%–80%.

[0047] 2. Place the above-mentioned petri dishes directly in a three-gas incubator at 37℃, 5% CO2, and 5% O2 for a continuous low-oxygen pretreatment for 2 hours.

[0048] 3. After the hypoxia treatment, discard the culture medium and wash once with PBS. Then add functional enhancement medium, which is prepared from serum-free DMEM / F-12 basal medium containing 10 µM ferrous ascorbate, 0.1 µM ferrostatin-1, and 1 mM L-arginine. Incubate the cells in a normoxic incubator at 37°C and 5% CO2 for another 12 hours.

[0049] 4. After culture, discard the functional enhancement medium, wash the cells three times with PBS, digest them with trypsin and collect the cells for subsequent tests.

[0050] Example 3: This embodiment provides a method for enhancing the immunomodulatory function of mesenchymal stem cells, including the following steps: 1. Fourth-generation human umbilical cord mesenchymal stem cells were used at a concentration of 2.0 × 10⁻⁶. 4 cells / cm 2 Cells were seeded at a density in cell culture dishes and cultured in DMEM / F-12 medium containing 10% fetal bovine serum at 37°C in a normoxic incubator with 5% CO2 until the cell confluence reached 70%–80%.

[0051] 2. Place the above-mentioned petri dishes directly in a three-gas incubator at 37℃, 5% CO2, and 1% O2 for a continuous 6-hour low-oxygen pretreatment.

[0052] 3. After the hypoxia treatment, discard the culture medium and wash once with PBS. Then add functional enhancement medium, which is prepared from serum-free DMEM / F-12 basal medium containing 100 µM ferrous ascorbate, 5 µM Ferrostatin-1, and 4 mM L-arginine. Incubate the cells in a 37°C, 5% CO2 incubator for 24 hours.

[0053] 4. After the culture is completed, discard the functional enhancement medium, wash the cells three times with PBS, digest them with trypsin and collect the cells for subsequent tests.

[0054] Example 4: This embodiment provides a method for enhancing the immunomodulatory function of mesenchymal stem cells. The steps are basically the same as in Example 1, except that the hypoxia pretreatment condition in step 2 is 4 hours at a concentration of 1% O2. The remaining steps are the same as in Example 1.

[0055] Example 5: This embodiment provides a method for enhancing the immunomodulatory function of mesenchymal stem cells. The steps are basically the same as in Example 1, except that the concentrations of each component in the functional enhancement culture medium in step 3 are as follows: ferrous ascorbate final concentration is 100 µM, ferrostatin-1 final concentration is 2 µM, and L-arginine final concentration is 4 mM. The remaining steps are the same as in Example 1.

[0056] Comparative Example 1: This comparative example is a blank control group, in which human umbilical cord mesenchymal stem cells are routinely cultured in standard growth medium without any of the hypoxia pretreatment or functional enhancement medium treatments described in the examples.

[0057] Comparative Example 2: This comparative example is a control group of the prior art. The difference from Example 1 is that: no hypoxia pretreatment and functional enhancement culture medium treatment were performed. Instead, recombinant human interferon-γ with a final concentration of 10 ng / mL was added to the standard growth medium and treated in a normoxic incubator for 48 hours. All other aspects are the same.

[0058] Comparative Example 3: Compared with Example 1, the difference is that only the hypoxic pretreatment in step 2 is performed. After the treatment, the medium is replaced with serum-free DMEM / F-12 basal medium without additional components and cultured under normoxic conditions for 18 hours. That is, no chemical metabolic reprogramming treatment is performed. All other aspects are the same.

[0059] Comparative Example 4: Compared with Example 1, the difference is that the hypoxia pretreatment in step 2 is not performed. After the cells are conventionally cultured to confluence, they are directly replaced with the functional enhancement medium described in Example 1 for culture. All other aspects are the same.

[0060] Comparative Example 5: The difference from Example 1 is that ferrous ascorbate is not added to the functional enhancement culture medium used in step 3, but all other aspects are the same.

[0061] Comparative Example 6: The difference from Example 1 is that Ferrostatin-1 was not added to the functional enhancement medium used in step 3, but all other aspects were the same.

[0062] Comparative Example 7: The difference from Example 1 is that the functional enhancement medium used in step 3 does not contain any additional L-arginine (i.e., only the basic concentration contained in the medium itself is maintained), while the rest are the same.

[0063] Test Example 1: To evaluate the impact of the method of the present invention on the biological safety of human umbilical cord mesenchymal stem cells (hUC-MSCs), the viability and subsequent vasodilation capacity of cells treated in Examples 1-5 and Comparative Examples 1-7 were detected by trypan blue staining and CCK-8 assay, respectively.

[0064] The experimental steps are as follows: 1. Cell viability assay: Cells from each group were collected after treatment, digested with 0.25% trypsin, and centrifuged to collect the cell pellet after digestion was terminated. The cells were resuspended in an appropriate amount of PBS. 10 µL of the cell suspension was mixed with an equal volume of 10 µL of 0.4% trypan blue dye, and gently mixed. 10 µL of this mixture was then added to the counting chamber of a cell counting chamber. Under a light microscope, the total number of cells and the number of non-viable cells stained blue were counted. Each group was counted three times. Cell viability (%) was calculated using the following formula: Cell viability = (Total number of cells - Number of blue cells) / Total number of cells × 100%.

[0065] 2. Cell proliferation assay: Live cells collected from each group were reseeded into 96-well plates at a density of 2000 cells / well, with 5 replicates per group. The 96-well plates were incubated at 37°C in a 5% CO2 incubator. At 24, 48, and 72 hours post-seeding, 10 µL of CCK-8 solution was added to each well, and incubation was continued for 2 hours. The absorbance (OD) value of each well was measured at 450 nm using a microplate reader.

[0066] Experimental data are shown in Table 1 and Figure 1 As shown.

[0067] Table 1. Results of cell viability and subsequent proliferation capacity of hUC-MSCs after treatment in each group:

[0068] From Table 1 and Figure 1The data, obtained through statistical analysis, showed that after treatment of hUC-MSCs using the methods employed in Examples 1-5, the cell viability remained above 92%, with no significant difference compared to the untreated Comparative Example 1 (p > 0.05). Subsequent proliferation curves indicated that the cells maintained good proliferative capacity after treatment, with OD values ​​steadily increasing over time. This demonstrates that the proposed method of transient hypoxia combined with chemical metabolic reprogramming possesses high biological safety and does not cause irreversible damage to the cells' basic survival and proliferative potential.

[0069] The key comparison lies between Example 1 and Comparative Example 6. In Comparative Example 6, the cell viability significantly decreased to 68.3% (p < 0.01) in the absence of Ferrostatin-1, and subsequent proliferation essentially ceased. This data confirms that the ferrous ions (ferrous ascorbate) actively introduced in this scheme do indeed have a strong toxic effect on cells without the presence of a specific protective agent; while the addition of Ferrostatin-1 can effectively inhibit the iron-mediated cell death pathway, ensuring the integrity of cells during high-intensity metabolic remodeling. Therefore, ferroptosis inhibitors are essential components in the compositions of this invention to achieve a balance between enhanced function and maintenance of cell viability.

[0070] Compared with the prior art Comparative Example 2 (IFN-γ treatment), the cells treated in Examples 1-5 showed an advantage in subsequent proliferation capacity, with a significantly higher proliferation rate than the IFN-γ treatment group (p < 0.05). This indicates that the method of the present invention is superior to the traditional IFN-γ stimulation protocol in terms of safety, especially in maintaining long-term cell biological activity.

[0071] Test Example 2: To verify the activation effect of transient hypoxia treatment on the transcription of inducible nitric oxide synthase (iNOS) gene in the method of the present invention, the relative expression level of iNOS mRNA in hUC-MSCs treated in Examples 1-5 and Comparative Examples 1-7 was quantitatively detected by real-time quantitative PCR (qRT-PCR).

[0072] The experimental steps are as follows: 1. Total RNA extraction and quantification: Cells from each group were collected after treatment, and total RNA was extracted using Trizol reagent. The concentration and purity (A260 / A280 ratio) of the RNA samples were then determined using a micro-spectrophotometer. Samples with an A260 / A280 ratio between 1.8 and 2.0 were selected for subsequent experiments.

[0073] 2. Reverse transcription reaction: Take 1µg of total RNA and use a commercial reverse transcription kit to reverse transcribe it into the first strand of cDNA according to its instructions.

[0074] 3. qRT-PCR reaction: Using the synthesized cDNA as a template, amplification was performed using SYBR Green qPCR premix. The reaction system (20 µL) included: 10 µL SYBR Green premix, 0.8 µL forward primer (10 µM), 0.8 µL reverse primer (10 µM), 2 µL cDNA template, and nuclease-free water to a final volume of 20 µL. GAPDH was used as an internal control gene. The primer sequences used were: Human iNOS - Positive: 5'-AGGGACAAGCCTACCCCTC-3' Human iNOS-reverse: 5'-TCCTCAACCTGCTCCTCACT-3' Human GAPDH - Forward: 5'-GGAGCGAGATCCCTCCAAAAT-3' Human GAPDH - Reverse: 5'-GGCTGTTGTCATACTTCTCATGG-3' 4. Amplification Program and Data Analysis: PCR reactions were performed on a real-time PCR instrument. The standard amplification program was: 95℃ pre-denaturation for 3 minutes; followed by 40 cycles of 95℃ denaturation for 10 seconds and 60℃ annealing extension for 30 seconds. Three technical replicates were used for each sample. After amplification, 2... - The relative quantitative analysis was performed using the ΔΔCt method, with Comparative Example 1 (blank control group) as the reference group, and the relative expression fold of iNOS mRNA in each group was calculated.

[0075] Experimental data are shown in Table 2 and Figure 2 As shown.

[0076] Table 2. Results of relative expression levels of iNOS mRNA after hUC-MSCs treatment in each group:

[0077] Table 2 and Figure 2 Data showed that, according to statistical analysis, the relative expression levels of iNOS mRNA in all groups subjected to transient hypoxia treatment (Examples 1-5 and Comparative Example 3) were significantly higher than those in Comparative Example 1 (without hypoxia treatment) and Comparative Examples 4-7 (received only chemical treatment) (p < 0.01). The iNOS mRNA expression level in Example 1 was more than 15 times that in Comparative Example 1, while the expression level in Comparative Example 4 showed no significant difference compared to Comparative Example 1 (p > 0.05). This data indicates that transient hypoxia treatment in the method is the main factor causing the upregulation of iNOS gene transcription.

[0078] The iNOS mRNA expression level in Comparative Example 3 (hypoxia treatment only) was on a similar order of magnitude to that in Example 1 (hypoxia combined with chemical treatment), with no significant difference between the two (p > 0.05). This result suggests that subsequent chemical treatment does not directly affect the transcription process of the iNOS gene. Comparative Example 2 (IFN-γ treatment), as a positive control, showed the highest iNOS mRNA expression level, consistent with existing knowledge.

[0079] Comprehensive analysis shows that the test data supports the design of the method of this invention, namely, that the expression of the iNOS gene can be pre-regulated at the transcriptional level through the transient hypoxia treatment step, which provides a transcriptome basis for the efficient synthesis of nitric oxide in the subsequent chemical metabolic reprogramming step.

[0080] Test Example 3: To directly evaluate the effect of the method of this invention on the production of nitric oxide (NO), a key effector molecule in the immunomodulatory function of hUC-MSCs, the Griess method was used to measure the nitrite (NO2) content in the cell culture supernatant of each group. - The concentration of stable metabolites of NO in aqueous solution was quantitatively detected.

[0081] The experimental steps are as follows: 1. Sample collection and preparation: Collect the supernatant of the culture medium from each group of cells during the functional enhancement culture stage (or the corresponding time point), centrifuge at 4℃ and 1000×g for 5 minutes, and take the supernatant to remove cell debris.

[0082] 2. Preparation of standard curve: Sodium nitrite (NaNO2) was used as a standard and diluted with serum-free DMEM / F-12 basal medium to a series of concentration gradients of 0, 1, 2.5, 5, 10, 25, 50, and 100 µM.

[0083] 3. Griess reaction: Add 100 µL of the sample or standard to each well of a 96-well plate. Then, add 100 µL of Griess reagent (prepared fresh from equal volumes of Griess reagent I and Griess reagent II) to each well.

[0084] 4. Incubation and detection: Incubate at room temperature and in the dark for 15 minutes until the color reaction is complete. Measure the absorbance (OD) value of each well at 540 nm using a microplate reader.

[0085] 5. Data Analysis: The concentration of nitrite in each sample was calculated based on the linear regression equation of the standard curve. Three technical replicates were set for each sample.

[0086] Experimental data are shown in Table 3 and Figure 3 As shown.

[0087] Table 3. Results of nitrite concentration detection in culture medium after hUC-MSCs treatment in each group:

[0088] Table 3 and Figure 3 Data showed that, according to statistical analysis, the nitrite concentration in the culture medium of cells treated in Examples 1-5 was significantly higher than that in Comparative Example 1 (blank control group) (p<0.01), proving that the method of the present invention can effectively promote the generation of NO.

[0089] Comparing Example 1 with Comparative Example 3 (hypoxia treatment only) and Comparative Example 4 (chemical treatment only), the nitrite concentration in Example 1 (24.87 µM) was significantly higher than that in Comparative Example 3 (7.91 µM) and Comparative Example 4 (11.68 µM) (p < 0.01), and its value was also higher than the sum of the concentrations of the latter two. This data indicates that there is a synergistic effect between transient hypoxia treatment and chemical metabolic reprogramming treatment, rather than a simple additive effect.

[0090] The nitrite concentrations in Comparative Examples 5 (lacking an iron source) and 7 (lacking L-arginine substrate) were not significantly different from those in Comparative Example 1 (p > 0.05), confirming that iron cofactors and L-arginine substrates are necessary conditions for NO synthesis. The nitrite concentration in Comparative Example 6 (lacking an ferroptosis inhibitor) was even lower than that in the blank control, consistent with the extremely low cell viability in this group in Test Example 1, indicating that cell survival is a prerequisite for performing biological functions.

[0091] Compared with Comparative Example 2 (IFN-γ treatment), the NO production levels of Examples 1, 3, 4, and 5 all reached or exceeded the levels achievable by the prior art, with no significant differences between groups (p > 0.05).

[0092] Comprehensive analysis shows that the data from this test case confirms the technical mechanism of the method of the present invention at the level of final functional products: by upregulating iNOS expression at the transcriptional level through transient hypoxia, and then by providing the necessary cofactors and substrates at the post-translational level through chemical composition and ensuring cell survival, the two work synergistically to ultimately lead to a significant increase in NO secretion.

[0093] Test Example 4: To evaluate the immunomodulatory function of hUC-MSCs treated with the method of this invention, the CFSE labeling method was used to detect their inhibitory ability on phytohemagglutinin (PHA)-induced T lymphocyte proliferation, and the function was evaluated.

[0094] The experimental steps are as follows: 1. Cell preparation: hUC-MSCs treated in each example and comparative example were collected and irradiated with a cobalt-60 source at a dose of 30 Gy to inhibit their self-proliferation. Simultaneously, peripheral blood mononuclear cells (PBMCs) from healthy individuals were isolated and fluorescently labeled with 5 µM CFSE at 37°C for 15 minutes. Labeling was then terminated with serum-containing culture medium and the cells were washed.

[0095] 2. Cell co-culture: In 96-well round-bottom plates, irradiated hUC-MSCs were cultured at a ratio of 2 × 10⁶ cells / well. 4 A density of 10 cells / hole was achieved in the plating. Subsequently, 2 × 10⁻⁶ 5 CFSE-labeled PBMCs were added per well to form a 1:10 co-culture ratio of MSCs and PBMCs.

[0096] 3. Proliferation Stimulation: PHA was added to the co-culture system at a final concentration of 5 µg / mL to stimulate T cell proliferation. The following control groups were also established: a) PBMCs cultured alone (negative control); b) PBMCs co-cultured with PHA (positive control).

[0097] 4. Incubation and detection: The 96-well plate was incubated at 37°C and 5% CO2 for 72 hours. After incubation, the suspended cells (mainly PBMCs) in each well were collected, washed with PBS, and the fluorescence intensity of CFSE was detected by flow cytometry.

[0098] 5. Data Analysis: Using the cell population that proliferated (i.e., CFSE fluorescence intensity decreased) in the positive control group (PBMCs+PHA) as a benchmark, the percentage of proliferating cells in each experimental group was calculated. The T lymphocyte proliferation inhibition rate (%) was calculated using the following formula: Inhibition rate = [1 - (Percentage of proliferating cells in the experimental group / Percentage of proliferating cells in the positive control group)] × 100%.

[0099] Experimental data are shown in Table 4 and Figure 4 As shown.

[0100] Table 4. Inhibition rate of hUC-MSCs on T lymphocyte proliferation at a 1:10 co-culture ratio:

[0101] Table 4 and Figure 4Data showed that, according to statistical analysis, hUC-MSCs treated in Examples 1-5 all exhibited high levels of T cell proliferation inhibition, with inhibition rates significantly higher than those in Comparative Example 1 (blank control group) (p < 0.01). The inhibition rate of Example 1 (75.3%) was significantly higher than that of Comparative Example 3 (hypoxia treatment only, 47.9%) and Comparative Example 4 (chemical treatment only, 58.2%) (p < 0.05), and also higher than the sum of the two. This data indicates that transient hypoxia treatment and chemical metabolic reprogramming have a synergistic effect in enhancing cellular immunosuppressive function.

[0102] The inhibition rates of Comparative Examples 5 (lacking iron source), 6 (lacking ferroptosis inhibitor), and 7 (lacking L-arginine) were all at extremely low levels, showing no significant difference compared to Comparative Example 1 (p > 0.05). This result is directly related to the extremely low NO production in these groups in Test Example 3, confirming that iron ions, L-arginine, and Ferrostatin-1, which ensures cell survival, are essential components for achieving the final immunosuppressive function.

[0103] When comparing the Example Group with Comparative Example 2 (IFN-γ treatment), the T cell proliferation inhibition rates shown in Examples 1, 3, 4, and 5 all reached levels comparable to or higher than those of IFN-γ stimulation in the prior art, with no significant difference between the groups (p > 0.05).

[0104] Comprehensive analysis shows that the experimental data confirms the effectiveness of the method of this invention at the level of cellular function. The method effectively increases NO production through a dual synergistic mechanism, ultimately translating into a potent inhibitory effect on T lymphocyte proliferation, achieving a functional enhancement level comparable to existing technologies.

[0105] Test Example 5: To evaluate the effect of the method of this invention on the immunogenicity of hUC-MSCs, flow cytometry was used to detect the expression level of HLA-DR, a key subtype of major histocompatibility complex class II (MHC-II) molecules on the cell surface. Upregulation of HLA-DR expression is a major marker of enhanced cellular immunogenicity.

[0106] The experimental steps are as follows: 1. Cell Collection and Preparation: hUC-MSCs treated in each example and comparative example were collected separately. After digestion with trypsin, the cells were collected by centrifugation at 300×g for 5 minutes. The cells were washed twice with pre-cooled flow cytometry buffer (PBS containing 1% BSA).

[0107] 2. Cell counting and resuspending: Resuspend the cells in flow cytometry buffer and adjust the cell concentration to 1×10⁻⁶. 6 per mL.

[0108] 3. Antibody incubation: Take 100 µL of cell suspension (approximately 1 × 10⁻⁶) per tube. 5 (1 cell), and add PE-labeled anti-human HLA-DR antibody or isotype control antibody (PE-labeled mouse IgG2a, κ isotype control). Incubate at 4°C in the dark for 30 minutes.

[0109] 4. Washing and Detection: After incubation, wash the cells with 1 mL of flow cytometry buffer, centrifuge, and discard the supernatant. Resuspend the cell pellet in 500 µL of flow cytometry buffer and immediately perform detection using a flow cytometer.

[0110] 5. Data analysis: Using isotype control tubes to set a negative gate, the percentage of HLA-DR positive cells in each group and the mean fluorescence intensity (MFI) of the positive cell population were analyzed and recorded.

[0111] The experimental data are shown in Table 5.

[0112] Table 5. Results of HLA-DR expression levels on the surface of each group after hUC-MSCs treatment:

[0113] Data showed that, according to statistical analysis, the proportion of HLA-DR positive cells and the average fluorescence intensity of cells treated in Examples 1-5 remained at extremely low baseline levels, with no significant difference compared to the untreated control example 1 (p>0.05). Similarly, the HLA-DR expression of controls 3-7, which underwent only single treatment, was not statistically significantly upregulated (p>0.05).

[0114] In contrast, in control group 2 (IFN-γ treatment), the proportion of HLA-DR positive cells was significantly increased to 91.7% (p < 0.001), and the average fluorescence intensity was more than 40 times that of control group 1, a difference with extremely high statistical significance (p < 0.001). This result indicates that IFN-γ greatly enhances the immunogenicity of cells by activating the intracellular JAK / STAT signaling pathway.

[0115] The results of this test case confirm that the transient hypoxia combined with chemical metabolic reprogramming method used in this invention bypasses the classic IFN-γ / STAT1 signaling pathway, thus effectively avoiding the upregulation of MHC-II molecules while enhancing cellular immunosuppressive function (as shown in Test Case 4). This method fundamentally solves the key safety issue of increased cellular immunogenicity caused by existing IFN-γ stimulation protocols.

[0116] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A composition for enhancing the immunomodulatory function of stem cells, characterized in that, The composition is contained in the basal culture medium: Source of ferrous ions; Ferrocyte inhibitors; L-arginine.

2. The composition for enhancing the immunomodulatory function of stem cells according to claim 1, characterized in that, The ferrous ions are derived from ferrous ascorbate.

3. The composition for enhancing the immunomodulatory function of stem cells according to claim 1, characterized in that, The ferroptosis inhibitor is Ferrostatin-1.

4. The composition for enhancing the immunomodulatory function of stem cells according to claim 1, characterized in that, The composition comprises ferrous ascorbate at a final concentration of 10–100 µM, ferrostatin-1 at a final concentration of 0.1–5 µM, and L-arginine at a final concentration of 1–4 mM.

5. A method for enhancing the immunomodulatory function of stem cells, characterized in that, Includes the following steps: a) Instantaneous hypoxia treatment of stem cells; b) Culture the stem cells treated in step a) using the composition for enhancing the immunomodulatory function of stem cells according to any one of claims 1-4.

6. The method for enhancing the immunomodulatory function of stem cells according to claim 5, characterized in that, The stem cells mentioned are mesenchymal stem cells.

7. The method for enhancing the immune regulatory function of stem cells according to claim 6, characterized in that, The stem cells mentioned are human umbilical cord mesenchymal stem cells.

8. The method for enhancing the immune regulatory function of stem cells according to claim 5, characterized in that, The conditions for the instantaneous hypoxia treatment are as follows: Treat for 2 to 6 hours in an environment with an oxygen concentration of 1% to 5%.

9. The method for enhancing the immune regulatory function of stem cells according to claim 5, characterized in that, The cultivation process described in step b) lasts for 12 to 24 hours.

10. A type of enhanced stem cell, characterized in that, It is prepared by the method for enhancing the immune regulatory function of stem cells as described in any one of claims 5-8.

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