Mesenchymal stem cells and methods of making and using the same

Abstract

This invention relates to the field of biomedical technology, and in particular to mesenchymal stem cells, their preparation methods, and applications. The method for preparing mesenchymal stem cells includes the following steps: culturing the mesenchymal stem cells under hypoxic conditions with an oxygen volume fraction of 2%–4%, and during the hypoxic culture, pre-activating the mesenchymal stem cells by contacting them with an induction system containing 1–3 ng / mL Oncostatin M, 1–10 ng / mL IL-1β, and 2–10 ng / mL HMGB1 for 6–24 hours. Mesenchymal stem cells treated by this method can significantly inhibit lymphocyte proliferation, reduce the proportion of pro-inflammatory T cell subsets such as Th1 and Th17, increase the proportion of Treg cells, and effectively reduce the level of the inflammatory factor TNF-α.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to mesenchymal stem cells, their preparation methods and applications. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell with self-renewal capacity and multi-lineage differentiation potential, derived from various tissues such as bone marrow, adipose tissue, umbilical cord, and placenta. Among these, umbilical cord-derived MSCs have received widespread attention in the field of cell therapy in recent years due to their abundant source, non-invasive extraction process, low immunogenicity, and strong proliferative capacity. Numerous studies have shown that MSCs can not only participate in tissue repair through differentiation but also regulate immune and inflammatory responses by secreting various cytokines, growth factors, and immunomodulatory molecules. Therefore, they have potential therapeutic value in inflammation-related diseases, immune-related diseases, and tissue damage repair.

[0003] In existing technologies, mesenchymal stem cells (MSCs) have been explored for various inflammatory diseases in preclinical research and clinical applications, such as sepsis, acute organ injury, and chronic inflammatory diseases. However, MSCs often exhibit significant differences in immunomodulatory, anti-inflammatory, and paracrine properties depending on the donor source, batch, and culture conditions. These functional differences can further affect the stability and reproducibility of cell therapy. Therefore, in practical applications, it is often necessary to establish a multi-parameter evaluation system, such as detecting cell phenotype, differentiation capacity, immunomodulatory capacity, inflammatory factor secretion levels, and functional performance in in vitro or in vivo disease models, to screen different batches of MSCs and select those with superior functional characteristics for subsequent research or applications.

[0004] To improve the functional performance of mesenchymal stem cells (MSCs), various cell pretreatment or culture optimization strategies have been proposed in existing technologies. For example, some studies have reported that pretreatment of MSCs under hypoxic culture conditions can improve cell viability, paracrine function, and adaptability to the injury microenvironment. Other studies have attempted to induce short-term activation of MSCs through stimulation by inflammatory factors or by simulating an inflammatory microenvironment to express higher levels of immunomodulatory molecules, thereby enhancing their immunomodulatory effects. However, the types and combinations of stimulating factors and culture conditions used in these methods vary considerably across different studies, and their induction effects and stability remain highly uncertain.

[0005] Furthermore, existing methods primarily focus on treating mesenchymal stem cells (MSCs) using single stimuli or combinations of common inflammatory factors, aiming mainly to enhance specific functions, such as promoting cell proliferation or enhancing immunosuppression. However, how to maintain the basic phenotype and multi-lineage differentiation potential of MSCs while leveraging appropriate culture conditions and induction systems to synergistically induce more stable and reproducible functional phenotypes in areas such as immune regulation and anti-inflammation, thereby reducing functional differences between batches and improving the application potential of cell therapy in inflammation-related diseases, remains a pressing technical challenge in this field. Summary of the Invention

[0006] This invention covers the following technical solutions: One aspect of the present invention relates to a method for preparing mesenchymal stem cells, comprising the following steps: The mesenchymal stem cells were cultured under hypoxic conditions with an oxygen volume fraction of 2%–4%, and during the hypoxic culture, the mesenchymal stem cells were pre-activated by contacting an induction system containing 1–3 ng / mL Oncostatin M, 1–10 ng / mL IL-1β, and 2–10 ng / mL HMGB1 for 6–24 hours.

[0007] Another aspect of the present invention relates to mesenchymal stem cells prepared by the method described above.

[0008] Another aspect of the present invention relates to the use of mesenchymal stem cells as described above in the preparation of medicaments for treating inflammation-related diseases.

[0009] This invention provides a method for short-term pre-activation treatment of mesenchymal stem cells (MSCs) using a hypoxic condition combined with an Oncostatin M, IL-1β, and HMGB1 induction system. This method can stably enhance the immunomodulatory and anti-inflammatory functions of MSCs while maintaining their basic phenotype and multi-lineage differentiation potential. MSCs treated with this method significantly inhibit lymphocyte proliferation, reduce the proportion of pro-inflammatory T cell subsets such as Th1 and Th17, increase the proportion of Treg cells, and effectively reduce the level of the inflammatory factor TNF-α. Simultaneously, in an inflammatory injury model, these cells exhibit stronger cytoprotective effects, thereby helping to reduce functional differences between different batches of cells and improving the stability and potential efficacy of cell therapy in inflammation-related diseases. Attached Figure Description

[0010] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0011] Figure 1 Flow cytometry results of cell immunophenotype detection.

[0012] Figure 2 : Chromosome karyotype analysis results.

[0013] Figure 3 : Results of three-lineage differentiation staining.

[0014] Figure 4 : CFSE method for detecting lymphocyte proliferation inhibition ability; A. PBMC resting control group; B. PBMC stimulation group; C. PBMC + MSC of this invention group.

[0015] Figure 5 Flow cytometry of specific lymphocyte subsets of Th1 / Th17; A. PBMC stimulation group; B. PBMC + MSCs of this invention group.

[0016] Figure 6 Flow cytometry of specific lymphocyte subsets of Treg cells; A. PBMC stimulation group; B. PBMC + MSCs of this invention group.

[0017] Figure 7 Effects of different MSC treatments on TNF-α secretion levels (mean ± SEM).

[0018] Figure 8 Effects of different MSC treatments on renal tubular epithelial cell survival (mean ± SEM). Detailed Implementation

[0019] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0020] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) should be understood to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of protection of this invention. Unless the context clearly defines otherwise, the scientific and technical terms used herein, as well as terms and laboratory procedures in related fields such as molecular biology, cell and tissue culture, and biochemistry, are all conventional terms and standard methods well-known and widely used in the art. To facilitate understanding of the technical solutions of this invention, some related terms are further defined and explained below.

[0021] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0022] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0023] Furthermore, in describing representative embodiments of the invention, this specification may present the methods and / or processes of the invention as a specific sequence of steps. However, the method or process should not be limited to the specific order of the steps described herein, to the extent that the method or process does not depend on the specific order of the steps presented herein. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps presented in the specification should not be construed as a limitation of the claims. Additionally, the claims relating to the methods and / or processes of the invention should not be limited to the execution of their steps in the order they are written, and those skilled in the art will readily recognize that the sequence can be changed while still remaining within the spirit and scope of the invention.

[0024] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted.

[0025] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0026] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity greater than or equal to 2.

[0027] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0028] In this invention, unless otherwise stated, the terminology used should be understood as having the common meaning in the relevant technical field for those skilled in the art. To facilitate understanding of the technical solutions of this invention, some terms are explained below.

[0029] The "mesenchymal stem cells" (MSCs) described in this invention refer to a type of adult stem cell derived from mammalian tissues that possesses self-renewal capacity and multi-lineage differentiation potential, capable of differentiating into adipocytes, osteoblasts, and chondrocytes, etc. The mesenchymal stem cells are preferably derived from umbilical cord tissue, but can also be derived from other tissue sources such as bone marrow, adipose tissue, and placenta.

[0030] In this invention, "hypoxic conditions" refers to culture conditions below normoxic levels (approximately 21% oxygen volume fraction). Specifically, in this invention, hypoxic conditions typically refer to a culture environment with an oxygen volume fraction of 2% to 4%. Within this range, mesenchymal stem cells can mimic the hypoxic microenvironment of inflammatory tissue without significantly affecting their survival and basic biological characteristics.

[0031] The "pre-activation treatment" described in this invention refers to the short-term treatment of mesenchymal stem cells under specific induction conditions on the basis of routine cell culture, thereby altering their functional state and enhancing their immunomodulatory, anti-inflammatory, or tissue protective capabilities. In this invention, the pre-activation treatment involves exposing mesenchymal stem cells to an induction system containing Oncostatin M, IL-1β, and HMGB1 under hypoxic conditions for a certain period of time (e.g., 6–24 hours), thereby bringing the cells into a functional state conducive to immunomodulation.

[0032] The “induction system” mentioned in this invention refers to a culture system used to induce the function of mesenchymal stem cells, which contains one or more signaling molecules that can simulate the inflammatory or damaging microenvironment.

[0033] The "Oncostatin M" (OSM) mentioned in this invention refers to a cytokine belonging to the IL-6 family, which can participate in the regulation of inflammatory responses and tissue repair processes. In this invention, it is used as a component of the induction system to regulate the functional state of mesenchymal stem cells.

[0034] In this invention, "IL-1β" refers to interleukin-1β, an important pro-inflammatory cytokine involved in the body's inflammatory response and immune regulation. In this invention, it is used as a component of the induction system to simulate inflammatory signals.

[0035] In this invention, "HMGB1" refers to high mobility group box 1 (HMB1), a molecule belonging to the damage-associated molecular pattern (DAMP) and playing a signaling role in tissue damage and inflammatory responses. In this invention, HMGB1 is added to the induction system in a low dose to simulate tissue damage-related signals.

[0036] The term "inflammation-related disease" as used in this invention refers to diseases that occur or develop with the participation of inflammatory responses, including but not limited to acute inflammatory diseases, chronic inflammatory diseases, and diseases caused by immune imbalance. In a preferred embodiment of this invention, the inflammation-related disease is acute inflammatory tissue damage driven by immune imbalance, such as sepsis-related acute kidney injury.

[0037] The "immunomodulation" described in this invention refers to the process of altering the intensity or nature of an immune response by influencing the activity, differentiation, or proportion of immune cells. In this invention, immune modulation is mainly manifested in inhibiting lymphocyte proliferation, reducing the proportion of pro-inflammatory T cell subsets (such as Th1 and Th17), and increasing the proportion of regulatory T cells (Tregs).

[0038] The "anti-inflammatory effect" described in this invention refers to the ability to reduce the level of inflammatory factors, inhibit the inflammatory response, or alleviate inflammatory damage. In this invention, the anti-inflammatory effect can be evaluated by detecting the secretion levels of inflammatory factors such as TNF-α.

[0039] The "tissue protection effect" described in this invention refers to the protective effect on tissues or cells under inflammatory or injury conditions, achieved by improving cell survival, inhibiting apoptosis, or enhancing cell function. In this invention, the tissue protection effect can be evaluated using an LPS-induced renal tubular epithelial cell injury model.

[0040] This invention relates to a method for preparing mesenchymal stem cells, which includes the following steps: The mesenchymal stem cells were cultured under hypoxic conditions with an oxygen volume fraction of 2%–4%, and during the hypoxic culture, the mesenchymal stem cells were pre-activated by contacting an induction system containing 1–3 ng / mL Oncostatin M, 1–10 ng / mL IL-1β, and 2–10 ng / mL HMGB1 for 6–24 hours.

[0041] According to the experimental results of the embodiments of this application, mesenchymal stem cells treated with the above method showed significantly enhanced lymphocyte proliferation inhibition ability in the in vitro PBMC co-culture system, and its inhibitory effect was significantly better than that of hypoxia treatment alone or inflammatory factor treatment alone. Simultaneously, in the inflammatory factor detection experiment, it could more effectively reduce the secretion levels of inflammatory factors such as TNF-α, and showed stronger cell protection in the inflammatory injury model. The above results indicate that the mesenchymal stem cells prepared by the method of this invention are simultaneously enhanced in multiple functional dimensions, including immune regulation, anti-inflammation, and tissue protection.

[0042] It is worth noting that in the comparative experiments, it was found that treating mesenchymal stem cells with the induction system composed of Oncostatin M, IL-1β, and HMGB1 alone did not exhibit an immunosuppressive enhancement effect comparable to hypoxia treatment. However, the combined use of hypoxia conditions and the induction system produced a significantly enhanced functional output, with effects significantly higher than any single treatment. This indicates that the relationship between hypoxia conditions and the induction system is not a simple additive one, but rather a synergistic effect within a specific parameter range, thereby enabling mesenchymal stem cells to achieve a significantly enhanced comprehensive functional phenotype.

[0043] Therefore, the preparation method provided by this invention can stably induce mesenchymal stem cells to a functional state with stronger immunomodulatory and anti-inflammatory capabilities through the synergistic effect of controllable culture conditions and induction system. This reduces functional differences between different batches of cells while enhancing their application potential in inflammation-related diseases. The significantly stronger functional enhancement effect compared to single-factor treatment demonstrates the unexpected technical effects of the method of this invention.

[0044] In some embodiments, the concentration of Oncostatin M is 1.5–2.0 ng / mL, the concentration of IL-1β is 3–6 ng / mL, and the concentration of HMGB1 is 4–6 ng / mL.

[0045] In some implementations, the pre-activation process takes 8 to 18 hours, for example, 10, 12, 14, or 16 hours.

[0046] In some embodiments, the oxygen volume fraction of the low-oxygen condition is 2.2% to 3.5%, for example 2.5%, 3.0%, or 3.2%.

[0047] In some embodiments, the induction system uses serum-free or low-serum culture medium. By using serum-free or low-serum conditions, the interference of exogenous serum components on the induction system can be reduced, thereby improving the controllability and consistency of the effects of inducing factors such as Oncostatin M, IL-1β, and HMGB1, and helping to obtain more stable functional phenotypes in different batches of cell treatment.

[0048] In some embodiments, the culture medium is one or more of DMEM, α-MEM, or RPMI-1640. These media are all commonly used basic cell culture media in the art, capable of providing mesenchymal stem cells with the necessary nutrients and a suitable growth environment. Among them, DMEM and α-MEM media are effective in supporting the adherent growth of mesenchymal stem cells and maintaining their basic biological characteristics, while RPMI-1640 media has good compatibility in immune cell co-culture systems. Therefore, they can be selected or combined according to specific application scenarios, thereby facilitating pre-activation treatment while ensuring cell viability.

[0049] In some embodiments, the culture medium is α-MEM medium containing 3%–5% fetal bovine serum. Compared to conventional culture conditions containing 10% fetal bovine serum, reducing the serum content can, to some extent, weaken the non-specific effects of growth factors and other components in the serum on cell state, thereby making the effects of various functional factors in the induction system more prominent and promoting the transformation of mesenchymal stem cells to the target functional state. At the same time, appropriately retaining a certain proportion of fetal bovine serum can maintain good adherent growth and survival of cells, avoiding cell stress or decreased activity that may occur under serum-free conditions, thus achieving a balance between induction efficiency and cell activity.

[0050] In some embodiments, the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells.

[0051] Umbilical cord-derived mesenchymal stem cells (MSCs) are characterized by their abundant source, non-invasive acquisition process, low immunogenicity, and strong in vitro expansion capacity, making them advantageous for obtaining sufficient quantities and stable quality cells for subsequent culture and processing. Furthermore, MSCs typically exhibit good functional potential in immunomodulation and anti-inflammation. Combined with the synergistic effect of the hypoxic conditions and induction system described in this invention, it is even more beneficial to obtain cell populations with enhanced immunomodulatory and anti-inflammatory functions, thereby improving their application value in inflammation-related diseases.

[0052] According to another aspect of the present invention, mesenchymal stem cells prepared by the method described above are also provided.

[0053] These cells exhibit significantly enhanced lymphocyte proliferation inhibition in immunomodulation, and can regulate the proportion of T cell subsets, reducing the level of pro-inflammatory T cell subsets while increasing the proportion of regulatory T cells. In terms of inflammatory factor regulation, they can reduce the secretion levels of inflammatory factors such as TNF-α. Furthermore, in inflammatory injury models, they demonstrate a protective effect on damaged cells. These findings indicate that the mesenchymal stem cells prepared by the method of this invention possess relatively stable and enhanced immunomodulatory, anti-inflammatory, and tissue-protective capabilities, which is beneficial in reducing functional differences between different batches of cells and enhancing their application potential in the treatment of related diseases.

[0054] According to another aspect of the invention, the use of mesenchymal stem cells as described above in the preparation of medicaments for treating inflammation-related diseases is also relevant.

[0055] After short-term pre-activation treatment under hypoxic conditions combined with an induction system of Oncostatin M, IL-1β, and HMGB1, the mesenchymal stem cells described above exhibit enhanced immunomodulatory and anti-inflammatory functions while maintaining their basic phenotype and multi-lineage differentiation potential. According to the experimental results in the embodiments of this application, these cells can significantly inhibit lymphocyte proliferation and reduce the proportion of pro-inflammatory T cell subsets such as Th1 and Th17 in an in vitro PBMC co-culture system, while increasing the proportion of Treg cells, thereby contributing to the restoration of the immune system's balance. Furthermore, in inflammatory factor detection experiments, these cells can effectively reduce the secretion levels of inflammatory factors such as TNF-α, indicating a strong anti-inflammatory effect.

[0056] Furthermore, in an inflammatory injury model, the mesenchymal stem cells prepared by the method of this invention significantly improved the survival rate of renal tubular epithelial cells under LPS-induced injury conditions, suggesting that they have a certain protective effect on tissue cells in an inflammatory environment. Based on the above experimental results regarding immunomodulation, inhibition of inflammatory factors, and cell protection, it can be inferred that the mesenchymal stem cells prepared by the method of this invention are suitable for treating various diseases caused by or involved in the development of inflammatory responses. In particular, for acute inflammatory tissue injury driven by immune imbalance, the mesenchymal stem cells described in this invention can exert beneficial effects by regulating immune responses, inhibiting the release of inflammatory factors, and improving the inflammatory microenvironment.

[0057] In some preferred embodiments, the inflammation-related disease is an acute inflammatory tissue injury driven by immune imbalance, such as sepsis-associated acute kidney injury. Since sepsis-associated acute kidney injury is usually accompanied by significant uncontrolled inflammatory responses, immune dysregulation, and damage to renal tubular epithelial cells, the mesenchymal stem cells prepared by the method of this invention, through their enhanced immunomodulatory and anti-inflammatory capabilities, are beneficial in alleviating inflammatory responses and improving tissue damage, thus possessing potential application value in the treatment of such diseases.

[0058] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these embodiments are only used to illustrate the technical content of the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the specific experimental conditions in the following embodiments are given priority reference to the guidelines provided in this specification, or may be carried out according to generally accepted experimental manuals or conventional experimental conditions, or other experimental methods known in the art, or according to the conditions recommended by the relevant reagent or instrument manufacturers. In specific embodiments, unless otherwise specified, minor deviations within the weighing accuracy range are allowed for the measurement parameters involving raw material components; reasonable deviations due to instrument detection accuracy or operational accuracy are also allowed for parameters such as temperature and time.

[0059] Example 1 This embodiment provides a method for isolating, culturing, establishing a library, and assessing the basic quality of umbilical cord-derived mesenchymal stem cells.

[0060] Take a fresh human umbilical cord obtained from a healthy full-term delivery, cut off the surgical suture ligation sites and obvious bruising on both sides of the umbilical cord, and under aseptic conditions, make a longitudinal incision along the umbilical vein, remove the arteries and veins, and then cut the remaining tissue into 1-3 mm pieces. 3 Tissue blocks of appropriate size were prepared. These blocks were attached to the bottom of T175 cell culture flasks at an appropriate density, inverted, and incubated statically at 37°C with 5% CO2 for 4 hours. Then, 5 mL of α-MEM medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin) was added, followed by 10 mL of medium the next day. The medium was completely replaced every 3 days thereafter, and the cell migration and growth around the attached blocks were continuously observed. Cells grew in a spindle-shaped or fibroblast-like manner. When the confluence of the migrated cells reached over 80%, primary cell digestion and further passage were performed. After passage to P2, the cells were cryopreserved as seed bank cells. P2 cells were thawed and passaged to P4 for use as working bank cells.

[0061] Basic quality assessments were performed on the established seed bank and working bank cells. Flow cytometry was used to detect the expression of cell surface markers, and the results are as follows: Figure 1 As shown, the cells highly expressed CD73, CD90, and CD105, with positive rates not less than 95%, while CD34, CD45, CD11b, CD19, and HLA-DR were expressed at low or negative rates, with negative rates not exceeding 2%, indicating that the obtained cells conformed to the phenotypic characteristics of mesenchymal stem cells. Further STR mapping analysis was used to identify the cell origin, and the results showed that the obtained cells were from a single source and no contamination from other human-derived cells was observed. Chromosome karyotype analysis was used to evaluate the genetic stability of the cells, and the results are as follows. Figure 2The cells showed normal chromosome number and structure, with no obvious chromosome deletions, translocations, mosaicism, or other clonal abnormalities.

[0062] Further analysis of the trilineage differentiation capacity of the above-mentioned working bank cells was performed. The cells were cultured in adipogenic induction medium, osteogenic induction medium, and chondrogenic induction medium for 14–21 days, respectively. For example... Figure 3 As shown, after adipogenic induction, a large number of red lipid droplets were observed to form after Oil Red O staining. After osteogenic induction, obvious red calcium nodules were observed after Alizarin Red staining. After chondrogenic induction, the cells formed chondrocytes. After sectioning and Alsin Blue staining, a large number of blue collagen fibers were observed inside the spheres, indicating that the obtained cells have good differentiation potential for adipogenesis, osteoogenesis and chondrogenesis.

[0063] The above results indicate that the umbilical cord-derived mesenchymal stem cells obtained in this embodiment not only possess the basic phenotype and trilineage differentiation potential of typical mesenchymal stem cells, but can also serve as starting cells for subsequent pre-activation treatment and functional enhancement studies.

[0064] Example 2 This embodiment provides a method for preparing mesenchymal stem cells pre-activated by a hypoxia-induced induction system.

[0065] The umbilical cord-derived mesenchymal stem cells obtained in Example 1 were seeded in T175 culture flasks and cultured under normoxic conditions until approximately 75% confluence. The culture medium was then replaced with α-MEM induction medium containing 3% fetal bovine serum. The final concentrations of Oncostatin M, IL-1β, and HMGB1 in the induction medium were 1.8 ng / mL, 4 ng / mL, and 5 ng / mL, respectively. The cells were immediately transferred to a hypoxic incubator with 3% oxygen and cultured at 37°C for 12 hours to complete the pre-activation treatment. After treatment, the induction medium was discarded, and the cells were washed 1-2 times with conventional medium free of the induction factors. The collected cells were designated as the pre-treatment group and used for in vitro functional evaluation, in vivo efficacy verification, or further scale-up culture in subsequent examples.

[0066] In this embodiment, the introduction of hypoxia simulates the relatively hypoxic microenvironment in inflamed tissues. The induction system composed of Oncostatin M, IL-1β, and HMGB1 simulates the microenvironment where multiple signals interact during tissue damage and inflammatory responses, thereby allowing mesenchymal stem cells to pre-enter a functional state more conducive to immune regulation and anti-inflammatory responses while maintaining their basic phenotype and activity. The mesenchymal stem cells treated in this embodiment can be used as test cells for subsequent in vitro immunomodulation experiments, inflammation model validation experiments, and sepsis-related acute kidney injury animal model experiments.

[0067] To examine the feasibility of pre-activation processing under different parameter conditions, this embodiment further sets different processing conditions.

[0068] Firstly, under hypoxic conditions with an oxygen volume fraction of 2.5%, the medium was used to treat the patient for 8 hours with induction medium containing Oncostatin M, IL-1β, and HMGB1 at final concentrations of 1.5 ng / mL, 3 ng / mL, and 4 ng / mL, respectively. Secondly, under hypoxic conditions with an oxygen volume fraction of 3.5%, the medium was used to treat the patient for 18 hours with induction medium containing Oncostatin M, IL-1β, and HMGB1 at final concentrations of 2.0 ng / mL, 6 ng / mL, and 6 ng / mL, respectively. Third, under hypoxic conditions with an oxygen volume fraction of 4%, the medium was used to treat the patients for 24 hours with induction medium containing Oncostatin M, IL-1β, and HMGB1 at final concentrations of 3.0 ng / mL, 10 ng / mL, and 10 ng / mL, respectively.

[0069] After treatment, cell morphology, adhesion status and survival were detected. The results showed that the cells treated within the above parameter range still maintained a fibroblast-like adherent growth morphology, and no obvious cell fragmentation, shrinkage and shedding or large-area death were observed, indicating that the hypoxia combined induction system can be used for short-term pre-activation treatment of mesenchymal stem cells.

[0070] Example 3 This embodiment is used to verify the in vitro immunomodulatory capacity of the pretreated mesenchymal stem cells prepared by the method described in Example 2. This embodiment uses a peripheral blood mononuclear cell (PBMC) co-culture system, and evaluates the immunomodulatory effect of the mesenchymal stem cells prepared by the method of this invention by detecting lymphocyte proliferation, changes in T cell subsets, and the secretion level of inflammatory factors.

[0071] 1. First, the inhibitory effect of mesenchymal stem cells (MSCs) on lymphocyte proliferation in each group was examined. Peripheral blood samples were collected from healthy donors, and peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation. PBMCs were then fluorescently labeled with 5 μM CFSE. Specifically, PBMCs were centrifuged at approximately 1 × 10⁻⁶ m³ / h. 6 The cells were resuspended at a density of cells / mL in serum-free PBS solution, and CFSE was added to a final concentration of 5 μM. After incubation at 37°C in the dark for about 10 minutes, the staining reaction was terminated with RPMI-1640 medium containing 10% fetal bovine serum. The cells were washed twice with this medium, and finally the PBMCs were resuspended in RPMI-1640 medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) for later use.

[0072] The pretreated mesenchymal stem cells obtained in Example 2 were pre-seeded in 24-well culture plates and cultured at 37°C and 5% CO2 to ensure stable adhesion. Subsequently, CFSE-labeled PBMCs were added to the culture plates and co-cultured with mesenchymal stem cells at a ratio of approximately MSC:PBMC 1:5. As a lymphocyte proliferation stimulant, phytohemagglutinin (PHA) was added to the corresponding experimental systems to a final concentration of approximately 5 μg / mL. Cells in each group were co-cultured for approximately 3 days at 37°C and 5% CO2 to induce PBMC proliferation and evaluate the inhibitory effect of mesenchymal stem cells on its proliferation.

[0073] The experiment was conducted in three groups: a PBMC resting control group, containing only PBMCs without any lymphocyte proliferation stimulant; a PBMC stimulation group, in which PBMCs were treated with phytohemagglutinin (PHA) to a final concentration of 5 μg / mL to induce lymphocyte proliferation; and a PBMC + MSC group, in which PBMCs were treated with phytohemagglutinin (PHA) to a final concentration of 5 μg / mL and co-cultured with the pretreated mesenchymal stem cells obtained in Example 2. After co-culture, PBMCs were collected and flow cytometry was used to detect changes in CFSE fluorescence intensity to evaluate PBMC proliferation.

[0074] Experimental results are as follows Figure 4 As shown, the proliferation of lymphocytes was significantly enhanced after the addition of a proliferation stimulant to PBMCs; however, when mesenchymal stem cells prepared by the method of this invention were added for co-culture, the proliferation of PBMCs was significantly inhibited, and its proliferation level was close to that of the resting PBMC control group. This indicates that the immunosuppressive ability of mesenchymal stem cells was enhanced after pre-activation treatment with a hypoxia combined with the Oncostatin M, IL-1β, and HMGB1 induction system.

[0075] 2. Further analysis of T cell subset changes. PBMCs were co-cultured with pretreated mesenchymal stem cells obtained in Example 2, and a lymphocyte proliferation stimulator was added. After culture, the proportion of T cell subsets was detected by flow cytometry. The expression of CD3 was also analyzed. + / CD8 - / IFN-γ + Th1 cells and CD3 expression + / CD8 - / IL-17A + The proportion of Th17 cells was measured to evaluate changes in pro-inflammatory T cell subsets. The experimental results are as follows: Figure 5 As shown, compared with the PBMC stimulation group, the proportion of Th1 and Th17 cells was significantly reduced after the addition of mesenchymal stem cells prepared by the method of the present invention, indicating that the mesenchymal stem cells prepared by the method of the present invention can inhibit the expansion of pro-inflammatory T cell subsets.

[0076] 3. Simultaneously, changes in the proportion of regulatory T cells (Tregs) were detected. PBMCs were co-cultured with pretreated mesenchymal stem cells obtained in Example 2, and a lymphocyte proliferation stimulator was added. After culture, flow cytometry was used to detect CD4 expression. + / CD25 + / CD127^low and / or CD4 + / CD25 + / Foxp3 + The proportion of Treg cells. Experimental results are as follows: Figure 6 As shown, compared with the PBMC stimulation group, the proportion of Treg cells increased significantly after the addition of mesenchymal stem cells prepared by the method of the present invention, indicating that the mesenchymal stem cells prepared by the method of the present invention can promote the production of regulatory T cells, thereby helping to restore immune homeostasis.

[0077] As described above, after short-term pre-activation treatment of mesenchymal stem cells using the hypoxic conditions combined with the Oncostatin M, IL-1β and HMGB1 induction system described in Example 2, the resulting mesenchymal stem cells can significantly inhibit lymphocyte proliferation, reduce the proportion of pro-inflammatory T cell subsets, and increase the proportion of regulatory T cells, thereby exhibiting good immunomodulatory ability and anti-inflammatory function, providing experimental evidence for their application in inflammation-related diseases.

[0078] Example 4 This embodiment is used to further verify the enhancing effect of the pre-activation treatment process described in Example 2 on the immunomodulatory function of mesenchymal stem cells, and to evaluate the effect of different treatment conditions on cell function through multiple comparative experiments.

[0079] The umbilical cord-derived mesenchymal stem cells obtained in Example 1 were used as the starting cells, and the following experimental groups were set up according to different treatment conditions: conventional MSC group, hypoxia treatment group, inflammatory factor treatment group, and the pretreatment group of the present invention.

[0080] (1) The conventional MSC group was cultured under normoxic conditions (oxygen volume fraction of about 21%) without any induction treatment; (2) The hypoxia treatment group was cultured for 24 hours under hypoxia conditions with an oxygen volume fraction of 3% without the addition of inducing factors; (3) The inflammatory factor treatment group was cultured under normoxic conditions (oxygen volume fraction of about 21%), and 1.8 ng / mL Oncostatin M, 4 ng / mL IL-1β and 5 ng / mL HMGB1 were added to the culture system for 12 hours. (4) The pretreatment group of the present invention was cultured under hypoxic conditions with an oxygen volume fraction of 3% according to the method described in Example 2, and simultaneously 1.8 ng / mL Oncostatin M, 4 ng / mL IL-1β and 5 ng / mL HMGB1 induction system were added for 12 hours of pre-activation treatment.

[0081] After treatment, the cells in each group were washed with PBS, digested, and collected for subsequent experiments.

[0082] All experiments were independently repeated n = 5 times. Experimental data are expressed as mean ± standard error (mean ± SEM). One-way ANOVA was used for comparisons between groups, followed by Tukey's post-hoc multiple comparison test. P A difference of <0.05 is considered statistically significant.

[0083] I. PBMC Proliferation Inhibition Experiment Peripheral blood samples were collected from healthy donors, and peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation and fluorescently labeled with 5 μM CFSE. The PBMCs were then centrifuged at 1 × 10⁻⁶ ppm. 6 Cells / mL were seeded in RPMI-1640 medium (containing 10% fetal bovine serum) with PHA (5 μg / mL) added as a lymphocyte proliferation stimulant. PBMCs were co-cultured with MSCs from each group at a ratio of MSC:PBMC = 1:5 and cultured at 37℃ and 5% CO2 for 5 days before flow cytometry analysis.

[0084] After co-culture, suspended PBMCs were collected and analyzed by flow cytometry. First, the main PBMC cell population was delineated based on forward and side-scatter light parameters, excluding debris and abnormal events, and further excluding cell clumps if necessary. Then, CFSE fluorescence signals were detected, and the high fluorescence peak of CFSE in the resting control group of PBMCs that had not undergone division was used as the threshold for non-proliferating cells. Cell populations exhibiting a stepwise decrease in CFSE fluorescence intensity compared to this threshold were defined as proliferating PBMCs, and their proportion of the total PBMC population was recorded as the proliferation ratio. Using the proliferation ratio of the PBMC stimulation group as a positive control, the inhibition rate of PBMC proliferation in each MSC treatment group was calculated using the following formula: PBMC proliferation inhibition rate (%) = [1 - (proliferating PBMC ratio in the MSC co-culture group / proliferating PBMC ratio in the PBMC stimulation group)] × 100%. Each experiment was independently repeated n times, and the results are expressed as mean ± standard error, and statistical comparisons were performed using one-way ANOVA.

[0085] The results of PBMC proliferation inhibition rate are shown in Table 1.

[0086] Table 1: Effects of different treatments on the proliferation inhibition ability of MSCs on PBMCs (n = 5)

[0087] Note: P <0.05 vs. regular MSC group P <0.01 vs. hypoxia treatment group and inflammatory factor treatment group Compared with the conventional MSC group, the hypoxia treatment group showed a significantly enhanced inhibitory effect on PBMC proliferation, with the inhibition rate increasing from 21.3% to 34.7%, indicating that hypoxia culture conditions can enhance the immunosuppressive ability of MSCs to a certain extent. In contrast, treatment with only an inflammatory cytokine system consisting of Oncostatin M, IL-1β, and HMGB1 showed a limited improvement in the inhibitory effect of MSCs on PBMC proliferation, with an inhibition rate of 25.8%, showing only a slight improvement compared to the conventional MSC group and significantly lower than the hypoxia treatment group. This suggests that stimulation with inflammatory cytokines alone is insufficient to stably induce stronger immunosuppressive function in MSCs. This may be because Oncostatin M is more inclined towards repair and paracrine regulation, while IL-1β and HMGB1 are more inclined towards damage alarm and inflammation activation. Without the pre-establishment of a basal environment adapted to the inflammatory microenvironment by hypoxia, the simultaneous input of these signals may lead to competition, restraint, or even partial cancellation among different pathways within MSCs, ultimately resulting in a lack of concentrated functional output, manifested as limited effects of individual treatments.

[0088] However, when hypoxia was combined with the aforementioned inflammatory factor induction system, i.e., the pretreatment group of this invention, the inhibitory effect on PBMC proliferation was significantly enhanced, with an inhibition rate of 79.6%, which was significantly higher than that of the hypoxia treatment group and the inflammatory factor treatment group, and the difference was statistically significant (P<0.01). This result indicates that short-term pre-activation treatment of MSCs in a hypoxic microenvironment combined with Oncostatin M, IL-1β, and HMGB1 can give MSCs a much stronger immunosuppressive ability than single-factor treatments.

[0089] Mechanistically, hypoxic culture conditions may first alter the metabolic state and stress response threshold of MSCs, allowing them to adapt to a microenvironment closer to that of inflammatory and damaged tissues, thereby enhancing their responsiveness to inflammatory and damage signals. Based on this, the inflammation and damage-related signals provided by Oncostatin M, IL-1β, and HMGB1 can further induce MSCs to express immunomodulatory molecules and enhance their anti-inflammatory paracrine function. Since the inflammatory factor system did not produce a significant enhancing effect when used alone, but significantly improved the immunosuppressive capacity of MSCs when used in combination under hypoxic conditions, this indicates a significant synergistic effect between hypoxia and the induction system, resulting in significantly enhanced immunomodulatory function of MSCs. This significantly stronger functional enhancement than single-factor treatment demonstrates that the technical solution of this invention can stably obtain MSCs with stronger immunosuppressive capabilities.

[0090] II. Detection of the inflammatory factor TNF-α The supernatant of the PBMC-MSC co-culture system was collected, and the TNF-α content was detected by ELISA.

[0091] Test results are shown Figure 7 The results showed that, compared with the conventional MSC group, both the hypoxia treatment group and the inflammatory factor treatment group could reduce the TNF-α level in the PBMC co-culture system. However, the TNF-α level in the pretreatment group of this invention was significantly reduced, and its inflammatory factor inhibition effect was significantly better than that of the hypoxia or inflammatory factor treatment groups alone.

[0092] III. LPS-induced model of renal tubular epithelial cell injury Human renal tubular epithelial cells were seeded into culture plates, and LPS (1 μg / mL) was added when the cell confluence reached approximately 70% to induce inflammatory damage. Subsequently, MSCs with different treatments were added for co-culture, and cell viability was assessed after 24 hours of culture.

[0093] Cell viability was determined using the CCK-8 assay; the results are shown below. Figure 8 The results showed that in the LPS-induced inflammatory injury model, all MSC treatment groups could improve the survival rate of renal tubular epithelial cells to varying degrees, with the pretreatment group of this invention showing the most significant improvement in cell damage.

[0094] In summary, pre-activation treatment of mesenchymal stem cells (MSCs) under hypoxic conditions in combination with the Oncostatin M, IL-1β, and HMGB1 induction system can significantly enhance the immunosuppressive capacity, inflammatory factor inhibition capacity, and protective effect against inflammatory damage of MSCs. The effect is significantly better than hypoxia treatment or inflammatory factor treatment alone, indicating that the technical solution of the present invention has a significant synergistic enhancement effect.

[0095] Furthermore, the results of different single-factor treatment groups showed different trends in the three experiments, which is related to the different functional focuses of each treatment on MSCs. Hypoxia treatment is more likely to first improve the metabolic state, stress tolerance, and immune regulatory readiness of MSCs, thus showing a more significant advantage in the PBMC proliferation inhibition experiment. In contrast, inflammatory factor treatment is more likely to preferentially enhance the ability of MSCs to sense inflammatory signals and anti-inflammatory and repair-related paracrine responses, thus showing better effects than hypoxia treatment alone in the TNF-α secretion inhibition and LPS-induced renal tubular epithelial cell injury models. In comparison, the pretreatment group of this invention, which combines Oncostatin M, IL-1β, and HMGB1 for short-term pre-activation under hypoxic conditions, enables MSCs to simultaneously possess a higher baseline immune regulatory state and stronger inflammatory response and tissue protection capabilities, thus showing the best effects in PBMC proliferation inhibition, inflammatory factor regulation, and damaged cell protection. This indicates that hypoxia conditions and the inflammatory factor induction system in this invention are not simply superimposed, but rather form a synergistic effect through different pathways, ultimately enabling MSCs to achieve a significantly enhanced comprehensive functional phenotype.

[0096] Example 5 This embodiment is used to further verify the combined effect of each component in the induction system composed of Oncostatin M, IL-1β and HMGB1, and to evaluate the promoting effect of hypoxia on the functional enhancement of the induction system, so as to illustrate the advantages of the hypoxia combined with the three factors in inducing mesenchymal stem cells to obtain enhanced immune regulation-related paracrine function.

[0097] Umbilical cord-derived mesenchymal stem cells obtained in Example 1 were used as starting cells and pretreated according to the culture conditions described in Example 2. The following experimental groups were established: conventional MSC group, hypoxia + Oncostatin M + IL-1β group, hypoxia + Oncostatin M + HMGB1 group, hypoxia + IL-1β + HMGB1 group, normoxic + Oncostatin M + IL-1β + HMGB1 group, and hypoxia + Oncostatin M + IL-1β + HMGB1 group. The conventional MSC group was cultured under normoxic conditions without induction treatment; the normoxic + Oncostatin M + IL-1β + HMGB1 group was treated with the three inducing factors under normoxic conditions; the remaining groups were cultured under hypoxic conditions with an oxygen volume fraction of 3%, and the corresponding induction systems were added during the culture process. The concentrations of Oncostatin M, IL-1β, and HMGB1 in each group were 1.8 ng / mL, 4 ng / mL, and 5 ng / mL, respectively, and the treatment time was 12 hours. After the treatment, the induction medium was discarded, and α-MEM medium (containing 3% fetal bovine serum) without induction factors was added and cultured for another 24 hours. Then, the conditioned culture supernatant of each group of cells was collected for subsequent detection.

[0098] The secretion levels of prostaglandin E2 (PGE2) and tumor necrosis factor-stimulated gene-6 (TSG-6), immunomodulatory molecules, in the supernatant of conditioned MSC culture in each group were detected using ELISA. PGE2 and TSG-6 are classic functionally relevant secretory molecules in the immunomodulatory and anti-inflammatory functions of mesenchymal stem cells (MSCs). Changes in their secretion levels can more directly characterize whether MSCs have formed a stronger immunomodulatory paracrine state after pretreatment. Therefore, PGE2 and TSG-6 were selected as detection indicators in this study, which is more conducive to comparing the advantages and disadvantages of different induction combinations from the perspective of mechanism of action and intrinsic cellular functional state, and further demonstrates that hypoxia combined with Oncostatin M, IL-1β, and HMGB1 treatment can promote enhanced immunomodulatory capacity of MSCs. All experiments were independently repeated n = 5 times. Experimental data are expressed as mean ± standard error (mean ± SEM). One-way ANOVA was used for comparisons between groups, followed by Tukey's post-hoc multiple comparison test. A p-value < 0.05 was considered statistically significant.

[0099] The test results are shown in Table 2.

[0100] Table 2: Secretion levels of PGE2 and TSG-6 in MSC culture supernatant after different induction systems (n = 5)

[0101] Note: P <0.05, compared to the conventional MSC group; # P <0.05, compared with the hypoxia + OSM + HMGB1 group; P <0.01, compared with any hypoxia dual-factor combination group; ## P <0.01, compared with the normoxia + OSM + IL-1β + HMGB1 group.

[0102] The results showed that, compared with the conventional MSC group, the secretion levels of PGE2 and TSG-6 in the conditioned culture supernatant of MSCs were increased after treatment with different dual-factor combinations under hypoxic conditions, indicating that the combination of inflammation-related factors under hypoxic conditions can enhance the immunomodulatory paracrine function of MSCs. Specifically, the secretion levels of PGE2 and TSG-6 in the hypoxia + IL-1β + HMGB1 group were higher than those in the hypoxia + OSM + HMGB1 group, and overall higher than those in the hypoxia + OSM + IL-1β group. This suggests that even in the absence of Oncostatin M, the combination of IL-1β and HMGB1 can still enhance the secretion of immunomodulatory molecules by MSCs to some extent, but its enhancing effect is still lower than that of the complete three-factor system.

[0103] Furthermore, compared with each dual-factor combination under hypoxic conditions, the secretion levels of PGE2 and TSG-6 were significantly increased in the hypoxia + Oncostatin M + IL-1β + HMGB1 group, indicating that the combined treatment of the three factors had a superior induction effect than any combination lacking a single factor. This result demonstrates that the combined application of Oncostatin M, IL-1β, and HMGB1 is more conducive to promoting the functional phenotype transformation of MSCs towards enhanced immunomodulatory capabilities. In particular, the hypoxia + Oncostatin M + IL-1β + HMGB1 group showed further increased PGE2 and TSG-6 secretion levels compared to the hypoxia + Oncostatin M + IL-1β group, indicating that the introduction of HMGB1 can further enhance the promoting effect of the induction system on the paracrine function of MSCs, thus demonstrating the positive role of HMGB1 in this induction system.

[0104] Furthermore, comparisons between the normoxic + Oncostatin M + IL-1β + HMGB1 group and the hypoxic + Oncostatin M + IL-1β + HMGB1 group showed that, under the same three-factor induction conditions, the hypoxic treatment group exhibited further increases in PGE2 and TSG-6 secretion levels. This indicates that hypoxia amplifies the enhancing effect of the three-factor induction system on the immunomodulatory paracrine function of MSCs. Therefore, hypoxia is not merely a background factor but can work in conjunction with the induction system composed of Oncostatin M, IL-1β, and HMGB1 to optimize the functional status of MSCs.

[0105] In summary, this embodiment demonstrates that the combined application of Oncostatin M, IL-1β, and HMGB1 can increase the secretion levels of PGE2 and TSG-6 in the supernatant of MSC culture under normoxic or hypoxic conditions, with the enhanced effect being more significant under hypoxic conditions. Compared to any single hypoxic dual-factor combination, the hypoxia + Oncostatin M + IL-1β + HMGB1 group exhibited higher PGE2 and TSG-6 secretion levels, suggesting that the complete three-factor combination is more conducive to inducing enhanced immunomodulatory paracrine function in MSCs; simultaneously, the addition of HMGB1 further enhances the induction effect, indicating its important promoting role in this system.

[0106] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing mesenchymal stem cells, characterized in that, Includes the following steps: The mesenchymal stem cells were cultured under hypoxic conditions with an oxygen volume fraction of 2%–4%, and during the hypoxic culture, the mesenchymal stem cells were pre-activated by contacting an induction system containing 1–3 ng / mL Oncostatin M, 1–10 ng / mL IL-1β, and 2–10 ng / mL HMGB1 for 6–24 hours.

2. The method according to claim 1, characterized in that, The concentration of Oncostatin M is 1.5–2.0 ng / mL, the concentration of IL-1β is 3–6 ng / mL, and the concentration of HMGB1 is 4–6 ng / mL.

3. The method according to claim 1, characterized in that, The pre-activation process takes 8 to 18 hours.

4. The method according to claim 1, characterized in that, The oxygen volume fraction under the hypoxia conditions is 2.2% to 3.5%.

5. The method according to any one of claims 1 to 4, characterized in that, The induction system uses serum-free or low-serum culture medium.

6. The method according to claim 5, characterized in that, The culture medium is one or more of DMEM, α-MEM, or RPMI-1640.

7. The method according to claim 6, characterized in that, The culture medium is α-MEM medium containing 3% to 5% fetal bovine serum.

8. The method according to any one of claims 1 to 4, 6, and 7, characterized in that, The mesenchymal stem cells mentioned are umbilical cord-derived mesenchymal stem cells.

9. Mesenchymal stem cells prepared by the method according to any one of claims 1 to 8.

10. The use of the mesenchymal stem cells of claim 9 in the preparation of a medicament for treating inflammation-related diseases.

Images