titer assay

Abstract

A method of assessing the potency of MSCs to produce anti-inflammatory cytokines in response to pro-inflammatory stimuli. The method comprises stimulating MSCs with one or more pro-inflammatory cytokines (e.g., TNF-a) for a period of time, and then identifying and quantifying the production of anti-inflammatory cytokines. MSCs that produce effective levels of anti-inflammatory cytokines in response to TNF-a can be used to treat aging-related disease conditions, including frailty of aging and Alzheimer’s disease, and can also be used to treat coronavirus infections. The method shows that TNF-a-induced MSCs robustly secrete a variety of anti-inflammatory cytokines, including IL-1 receptor antagonist (IL-1RA), IL-10, and granulocyte colony-stimulating factor (G-CSF).

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 012,884, filed April 20, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This article provides a method for evaluating the potency of human mesenchymal stem cells (HMSCs) in responding to exposure to pro-inflammatory cytokines such as TNF-α to produce anti-inflammatory cytokines. Human HMSCs are capable of producing sufficient anti-inflammatory cytokines, which could then be used to treat diseases involving chronic inflammation, such as frailty, Alzheimer's disease, and coronavirus infection. Background Technology

[0004] Frailty due to aging presents a very worrying problem for an individual's overall health and well-being. Frailty due to aging is a syndrome of old age characterized by weakness, reduced physical activity, slowed motor function, exhaustion, and unintentional weight loss. See Yao, X. et al. Clinics in Geriatric Medicine 27(1):79-87 (2011). Furthermore, many studies have shown a direct link between age-related frailty and inflammation. See Hubbard, RE et al., Biogerontology 11(5):635-641 (2010).

[0005] Immunoresaturation is characterized by a low-grade, chronic state of systemic inflammation, known as inflammation. See Franceshi, C. et al. Annals of the New York Academy of Sciences 908:244-254 (2000). This inflammatory state, or exacerbation of chronic inflammation, found in aging and age-related deterioration, leads to immune dysregulation and complex remodeling of innate and adaptive immunity. In immunosenescence, the T-cell and B-cell reservoirs are skewed, resulting in the re-expression of CD45ra (TEMRA) on CD8 cells. + T-effect memory cells and CD19 + Increased late / exhaustion memory B cells and CD8 + Naïve T cells and switching memory B cells (CD27) + (This is a reduction. See Blomberg, BB, et al.) Immunologic Research 57(1-3):354-360 (2013; Colonna-Romano, G. et al.) Mechanisms of Ageing and Development 130(10):681-690(2009); and Koch S. et al., Immunity & Ageing:5:6 (2008). This shift in T-cell and B-cell reservoirs leads to an unresponsive or ineffective immune state. This deterioration of the immune system makes one more susceptible to infectious diseases and reduces the response to vaccination. Optimal B-cell function is crucial for generating an effective antibody response to vaccines and protecting against infectious agents. It is well known that increased age-related systemic inflammation (TNF-α, IL-6, IL-8, INFγ, and CRP) induces impaired B-cell function, resulting in poor antibody responses and reduced vaccine titers.

[0006] Inflammation has received considerable attention because it demonstrates a link between immune changes and many common age-related diseases and conditions, such as age-related frailty. Circulating inflammatory mediators, such as cytokines and acute-phase proteins, are markers of the low-grade inflammation observed to increase with age. These pro-inflammatory cytokines (such as TNF-α and IL-6) impair the ability of B cells to produce protective antibodies against exogenous antigens and vaccines. This impaired B-cell response is measured by reduced class switch recombination (CSR), the ability of immunoglobulins to switch isotypes from IgM to secondary isotypes (IgG, IgA, or IgE). Immunoglobulin isotype switching is crucial for a proper immune response because the effector functions differ for each isotype. A key player in CSR and somatic hypermutation (SHM) is an enzyme encoded by the Aicda gene, namely activation-induced cytidine deaminase (AID). The basic function of AID in CSR and SHM is to initiate DNA breakage by converting cytosine to uracil in the switching and variable regions of immunoglobulins.

[0007] It has also been shown that in humans, the amount of TNF-α produced: (1) depends on the amount of systemic inflammation, and (2) impairs the ability of the same B cells to be stimulated by mitogens or antigens. See Frasca, D. et al. Journal of Immunology 188(1):279-286 (2012). Therefore, the immune response is impaired in individuals with age-related debility for a variety of reasons.

[0008] TNF-α expression is also involved in the initiation, maintenance, and amplification of immune processes that generate neuroinflammation and are associated with the pathogenesis of Alzheimer's disease and related dementias, as well as other forms of inflammation that lead to nerve damage.

[0009] Alzheimer's disease (AD) is a chronic, progressive neurodegenerative brain disease—a syndrome of aging. It is the leading cause of illness and death among nearly 5 million middle-aged and elderly Americans. AD accounts for 70% of all dementia cases. Dementia is a huge public health problem, with a new case diagnosed every 7 seconds worldwide. There is no cure for this disease; it worsens as it progresses, eventually leading to death within 7 years. Less than 3% of people diagnosed live more than 14 years after the diagnosis. People diagnosed with AD are typically over 65 years old and have difficulty completing standard verbal and visual memory tests, in addition to performing decision-making and problem-solving tasks. In 2006, there were 26.6 million people with AD worldwide, 5 million of whom were in the United States. It is projected that by 2050, 1 in 85 people globally will have Alzheimer's disease. Early symptoms are often mistaken for age-related problems or signs of stress.

[0010] In addition to β-amyloid deposition and neurofibrillary tangles, Alzheimer's disease (AD) involves a complex pathology and multiple mechanisms. It is increasingly recognized that pro-inflammatory states can lead to secondary dementia. In this regard, pro-inflammatory cytokines are abundant near amyloid deposits and neurofibrillary tangles, thus linking systemic inflammation and β-amyloid accumulation. Furthermore, individuals may present with abundant amyloid deposits and neurofibrillary tangles at autopsy, consistent with their AD diagnosis, but never exhibit a history of dementia: in these cases, the expression of inflammatory markers is significantly lower than in AD patients.

[0011] AD is also characterized by neurovascular damage leading to adverse outcomes. Of particular note is the hypoperfusion and impairment of the blood-brain barrier (BBB). As a result, BBB damage can impair transendothelial exchange. Due to the direct inhibition of endothelial cell proliferation and migration by AβP, this transendothelial exchange is partially impaired. Ultimately, AβP clearance from the BBB is inefficient, leading to AβP accumulation in the brain parenchyma. Therefore, damaged neurovascular systems are another important therapeutic target for AD.

[0012] Coronavirus infection has proven to be a significant threat to humans. Specifically, patients infected with COVID-19 who require advanced respiratory support have particularly poor outcomes. The mortality rate for these patients is approximately 54%. Clinical deterioration typically occurs 7–10 days after symptom onset and is associated with a decline in viral titers, indicating that the pathology is driven by inflammation rather than direct viral damage. In patients with severe COVID-19, inflammatory markers are often significantly elevated, leading to a hyperinflammatory syndrome, which may contribute to morbidity and mortality. Hyperinflammatory syndrome typically involves uncontrolled, self-perpetuating, and tissue-destructive inflammatory activity.

[0013] The aforementioned diseases, or similar diseases, are typically treated with therapeutic agents such as small molecules, proteins, vaccines, or antibodies. The use of cell therapy to treat these diseases is not well documented or explored in this field. Cell therapy represents a new and exciting therapeutic modality spanning a wide range of therapeutic indications.

[0014] Mesenchymal stem cells (MSCs) are pluripotent stem cells capable of migrating to injury sites, immune-privileged due to the undetectable expression of major histocompatibility complex class II (MHC-II) molecules, and expressing low levels of MHC-I molecules. See LeBlanc, K. et al. Lancet 371(9624):1579-1586 (2008) and Klyushnenkova E. et al., J. Biomed. Sci. 12(1):47-57 (2005). Therefore, allogeneic mesenchymal stem cells hold great promise for therapeutic and regenerative medicine, and have been repeatedly demonstrated to have high safety and efficacy in clinical trials across various disease processes. See Hare, JM et al., Journal of the American College of Cardiology 54(24):2277-2286 (2009); Hare, JM et al. Tex. Heart Inst. J. 36(2):145-147 (2009); and Lalu, MM et al., PloS One 7(10):e47559 (2012). It was also demonstrated that they did not undergo malignant transformation after transplantation into patients. See Togel F. et al., American Journal of Physiology Renal Physiology 289(1): F31-F42 (2005). It has been demonstrated that treatment with mesenchymal stem cells can improve severe graft-versus-host disease, prevent ischemic acute renal failure, aid in the repair of pancreatic islets and glomeruli in diabetic patients, reverse severe liver failure, regenerate damaged lung tissue, alleviate sepsis, reverse remodeling after myocardial infarction, and improve cardiac function. See Le Blanc K. et al., Lancet 371(9624):1579-1586 (2008); Hare, JM et al. Journal of the American College of Cardiology 54(24):2277-2286 (2009); Togel F. et al., American Journal of Physiology Renal Physiology 289(1):F31-F42(2005); Lee RH et al., PNAS 103(46):17438-17442 (2006); Parekkadan, B. et al. PloS One2(9):e941 (2007); Ishizawa K. et al., FEBS Letters 556(1-3):249-252(2004); Nemeth K. et al. Nature Medicine 15(1):42-49 (2009); Iso Y. et al., Biochem. Biophys. Res. Comm. 354(3):700-706 (2007); Schuleri KH et al., Eur. Hearth J. 30(22):2722-2732 (2009); and Heldman AW et al., JAMA 311(1):62-73 (2014). Furthermore, mesenchymal stem cells are also a potential source of various cell types for tissue engineering. See Gong Z. et al., Methods in Mol. Bio. 698:279-294 (2011); Price, AP et al. Tissue Engineering Part A 16(8):2581-2591 (2010); and Togel F. et al., Organogenesis 7(2):96-100 (2011).

[0015] Mesenchymal stem cells possess immunomodulatory capabilities. They control inflammation and the production of cytokines by lymphocytes and myeloid-derived immune cells, show no evidence of immunosuppressive toxicity, and exhibit low immunogenicity. See Bernardo ME et al. Cell Stem Cell 13(4):392-402 (2013).

[0016] In vivo studies have shown that when transplanted into fetal sheep, human mesenchymal stem cells undergo site-specific differentiation into various cell types, including myocytes and cardiomyocytes. See Airey JA et al. Circulation 109(11):1401-1407 (2004). These mesenchymal stem cells persisted in multiple tissues for up to 13 months after transplantation into non-immunosuppressive, immunocompetent hosts. Other in vivo studies using rodents, dogs, goats, and baboons have also shown that human mesenchymal stem cell xenografts do not induce lymphocyte proliferation or systemic alloantibody production in recipients. See Klyushnenkova E. et al., J. Biomed. Sci. 12(1):47-57 (2005); Aggarwal S. et al. Blood 105(4):1815-22 (2005); Augello A. et al., Arthritis and Rheumatism56(4):1175-86 (2007); Bartholomew A. et al., Exp Hematol. 30(1):42-48. (2002); Dokic J. et al., European Journal of Immunology 43(7):1862-72 (2013); Gerdoni E. et al., Annals of Neurology 61(3):219-227 (2007); Lee SH et al., Respiratory Research 11:16 (2010); Urban vs. others, Stem Cells 26(1): 244-253 (2008); Yang H. et al., PloS One 8(7):e69129 (2013); Zappia E. et al., Blood 106(5):1755-1761 (2005); Bonfield TL et al., American Journal of Physiology Lung Cellular and Molecular Physiology 299(6):L760-70 (2010); Glenn JD et al., World Journal of Stem Cells. 6(5):526-39 (2014); Guo K. et al., Frontiers in Cell and Developmental Biology 2:8 (2014); Puissant B. et al., British Journal of Haematology 129(1):118-129 (2005); and Sun L. et al., Stem Cells 27(6):1421-32(2009). Overall, these repeated findings on the safety and efficacy of allogeneic grafts reinforce the concept of successful tissue regeneration using mesenchymal stem cells as allogeneic grafts.

[0017] Animal model studies of Alzheimer's disease (AD) also support the clinical potential of mesenchymal stem cells (MSCs). See Neves AF et al. Exp. Neurol. 2021:113706. Beneficial effects include reduced inflammation, increased Aβ degradation factors and Aβ clearance, decreased hyperphosphorylated tau, and enhanced selectively activated (M2) microglial markers. These benefits appear to be at least partly due to Aβ-induced release of chemical attractants from MSCs, which recruit alternative microglia into the brain to reduce Aβ deposition. See Lee JK et al., Stem Cells 2012; 30(7):1544-55. MSCs were effective in young AD model mice prior to Aβ accumulation, leading to a significant reduction in brain Aβ deposition and a significant increase in presynaptic protein expression. See Bae JS et al.,Curr Alzheimer Res. 2013;10(5):524-31. Impressively, these effects lasted for at least 2 months, suggesting that MSCs can be used as an interventional treatment for pre-AD. In summary, preclinical studies of AD have shown that MSCs can cross the BBB, inhibit neuroinflammation, promote neurogenesis, inhibit β-amyloid deposition, promote clearance, reduce apoptosis, promote hippocampal neurogenesis, improve dendritic morphology, and enhance behavioral and spatial memory performance. Summary of the Invention

[0018] The ability of mesenchymal stem cells (MSCs) to produce immunomodulatory cytokines in response to pro-inflammatory stimuli is an important therapeutic mechanism employed by MSCs.

[0019] Accurate, reproducible, and relevant assays for evaluating cell titers used in cell therapy are essential for quality control purposes, such as ensuring the stability and consistency of cell-based therapeutic products.

[0020] General assays used in the art to assess cell titer focus on identifying the expression of specific biomarkers or cell surface receptors. These assays are intended to provide an indirect measurement of cell titer (e.g., MSCs expressing TNFR1 are expected to inhibit PBMC proliferation). Therefore, the “titer assay” used in the art is an identity analysis that measures the expression of cell receptors or biomarkers and cannot accurately measure the ability or titer of cells to express or produce key macromolecules (e.g., anti-inflammatory cytokines).

[0021] MSC potency assays have been developed in which MSCs are stimulated with LPS; however, these potency assays produce “irrelevant” stimulation (e.g., irrelevant because LPS stimulation is a mimic of bacterial infection, and MSCs are not used as antibacterial agents). Since MSCs typically do not express TLR4 or CD14, both of which are essential for LPS stimulation and signal transduction, these assays are further irrelevant. Therefore, the object of this application is to provide a potency assay that accurately measures the ability of mesenchymal stem cells (MSCs) to produce immunomodulatory cytokines in response to pro-inflammatory cytokines such as TNF-α. Ideally, the measurement should target physiologically significant components.

[0022] This article provides a method for evaluating MSC potency, for example, evaluating the potency of MSCs in cell preparation (e.g., preparing MSC formulations belonging to a variety of cells for therapeutic use). The method provided herein utilizes a TNF-α stimulation step to assess whether MSCs produce anti-inflammatory cytokines and, compared to standard cell potency assays used in the art, prior to evaluating cell or cell batch potency. Standard cell potency assays only involve detecting the presence of cell surface receptors or biomarkers and cannot assess whether cells are able to express molecules associated with stimulation of said receptors or biomarkers. It has been determined that adding a TNF-α stimulation step can improve the reliability of potency assays and reduce variability between MSC formulations derived from the same cell batch and MSC formulations containing the same cell type but derived from different cell batches. Attached Figure Description

[0023] Figure 1 The levels of anti-inflammatory cytokines produced after MSC stimulation with recombinant human TNFα were described.

[0024] Figure 2 The activity of MSCs after stimulation with recombinant human TNFα was described.

[0025] Figure 3 The levels of anti-inflammatory cytokines produced after MSCs were stimulated with recombinant human TNFα for more than 24 hours were described.

[0026] Figure 4 The levels of IL-8 and IL-13 produced were depicted after MSCs were sensitive to IL-17A stimulation but exposed to recombinant human TNFα1. Detailed Implementation

[0027] One aspect of this application relates to a method for evaluating the potency of anti-inflammatory cytokines produced by MSCs.

[0028] In one implementation, the method includes stimulating MSCs with pro-inflammatory cytokines or molecules for a period of time before identifying and quantifying the levels of anti-inflammatory cytokine production.

[0029] MSCs can originate from bone marrow, adipose tissue, peripheral blood, lungs, heart, amniotic fluid, internal organs, amnion, umbilical cord or placenta or other tissues, or they can differentiate from induced pluripotent stem cells (IPSCs) or other sources.

[0030] MSCs can be stimulated with pro-inflammatory cytokines or molecules. Pro-inflammatory cytokines can be selected from TNF-α, IL-1, IL-2, IL-6, IL-12, IL-17A, IL-18, IFN-γ, or any combination thereof. In some embodiments, MSCs are stimulated with TNF-α and IL-17A or other combinations. Other pro-inflammatory molecules include C-reactive protein (CRP) or virulence factors. Virulence factors can be any viral molecule that contributes to: colonization in the host microenvironment, immune evasion or escape from the host immune response, immunosuppression or suppression of the host immune response, entry into or exit from cells, or nutrient acquisition from the host. An example of a virulence factor is the SARS-CoV-2 spike protein.

[0031] Surprisingly, it has been demonstrated that MSCs treated with IL-17A alone do not produce or produce significantly low levels of anti-inflammatory cytokines. However, when MSCs are treated with both IL-17A and TNF-α, they produce significantly higher levels of anti-inflammatory cytokines. The importance of this unexpected finding stems from the current standard required to assess cell titer, which involves confirming that cells express certain receptors or biomarkers without assessing the ability of these receptors to promote the production of specific molecules. This finding confirms that even if cells possess receptors known to produce specific molecules, they may not produce those molecules at a titer that is effective for subsequent treatment. Furthermore, it suggests that MSCs respond differently to different combinations of pro-inflammatory molecules specific to an indication or patient, to be best suited to a particular treatment for a particular patient.

[0032] When culturing 500-50,000 mesenchymal stem cells in 50-200 μL of medium, the amount of pro-inflammatory cytokines or molecules used to stimulate MSCs can be 10 fg / mL to 10 µg / mL, 1 pg / mL to 10 µg / mL, 1 µg / mL to 10 µg / mL, 1 fg / mL to 1 pg / mL, 1 fg / mL to 10 µg / mL, or 1 pg / mL to 5 µg / mL. The concentration should be adjusted accordingly based on changes in cell number and / or volume.

[0033] Before quantifying the level of anti-inflammatory cytokine production, MSCs can be stimulated with pro-inflammatory cytokines or molecules for 1 to 24 hours, 1 to 12 hours, 2 to 6 hours, 1 to 4 hours, 24 to 120 hours, 24 to 72 hours, or more than 120 hours.

[0034] After MSCs are stimulated with pro-inflammatory cytokines or molecules, the anti-inflammatory cytokines that can be examined and quantified are IL-1RA, IL-4, IL-7, IL-8, IL-10, IL-13, G-CSF or any combination thereof.

[0035] In other embodiments, stimulation of MSCs with pro-inflammatory cytokines or molecules can result in the production of anti-inflammatory molecules at concentrations ranging from 1 fg / mL to 100 ng / mL, 1 fg / mL to 10 µg / mL, 1 fg / mL to 10 pg / mL, 1 fg / mL to 10 fg / mL, 10 fg / mL to 10 pg / mL, 10 pg / mL to 10 µg / mL, 10 µg / mL to 1 mg / mL, 1 pg / mL to 10 pg / mL, 1 µg / mL to 10 µg / mL, or 10 pg / mL to 1 µg / mL, per 500-50,000 cells cultured in 50-200 µL of medium. The concentrations can be adjusted accordingly with variations in cell number and / or medium volume.

[0036] In some implementations, the method further includes examining the expression of biomarkers on MSCs prior to stimulation with pro-inflammatory cytokines. Biomarkers that can be explored include CD105. + CD90 + CD73 + CD45 - CD34 - CD19 - CD11b - HLA-DR - IL-17RA + Or any combination thereof.

[0037] In other embodiments, the method may further include a step of seeding MSCs onto a substrate prior to stimulation with pro-inflammatory cytokines. The substrate may be a membrane, plastic surface, glass surface, or cell culture plate, with or without a substrate coating, such as a 96-well plate. The duration of MSC seeding onto the substrate may be 1 hour to 24 hours, 1 hour to 12 hours, 2 hours to 6 hours, or 1 hour to 4 hours. After the seeding period, the MSCs should be properly adhered to the substrate.

[0038] Before stimulation with pro-inflammatory cytokines, MSCs can be divided into smaller MSC populations. Separating MSCs into smaller populations allows for a more accurate assessment of their ability to produce anti-inflammatory cytokines after stimulation.

[0039] In some implementations, the method may further include a step of separating the MSC supernatant after stimulation with pro-inflammatory cytokines. Once the supernatant is collected, it can be stored at -80°C. The supernatant can be further analyzed using electrochemiluminescence immunoassay to determine the levels of anti-inflammatory cytokines produced by MSCs. Since the detection methods commonly used in potency assays are less sensitive than electrochemiluminescence immunoassay, it can be used to detect femtogram concentrations of cytokines produced by MSCs.

[0040] In other embodiments, the method further includes analyzing the viability of MSCs after stimulation with pro-inflammatory cytokines for a period of time. Viability analysis can be ATP assays, such as CellTiter Glo assay (Promega), tetrazolium reduction assay, resazurin reduction assay, protease activity labeling assay, sodium-potassium ratio assay, cell lysis or membrane leakage assay, mitochondrial activity or cysteine-aspartate enzyme assay, functional analysis, genomic and proteomic analysis, or any combination thereof. MSC viability can also be assessed by flow cytometry.

[0041] Compared with MSCs treated with mediators, the activity of MSCs stimulated with pro-inflammatory cytokines can be increased by 70%.

[0042] In other embodiments, the method further includes assigning a potency grade to the MSCs based on the amount of anti-inflammatory molecules produced. The potency grade assigned to the MSCs includes a threshold grade, wherein the MSCs may have the following potency grades: producing at least 1 fg / mL to 100 ng / mL, 1 fg / mL to 10 µg / mL, 1 fg / mL to 10 pg / mL, 1 fg / mL to 10 fg / mL, 10 fg / mL to 10 pg / mL, 10 pg / mL to 10 µg / mL, 10 µg / mL to 1 mg / mL, 1 pg / mL to 10 pg / mL, 1 µg / mL to 10 µg / mL, or 10 pg / mL to 1 µg / mL of anti-inflammatory cytokines per 500-50,000 cells cultured in 50-200 µL of medium.

[0043] Example

[0044] Example 1

[0045] Human MSC populations obtained from bone marrow aspirates and subsequently cryopreserved were thawed. After thawing, an aliquot of the MSCs was used for immunophenotyping to confirm cell identity. This included confirming that the MSCs expressed CD105, CD90, and CD73, but lacked expression of CD45, CD34, CD19, CD11b, and HLA-DR.

[0046] From the remaining cells, 10,000 MSCs were seeded into the wells of a 96-well plate and allowed to adhere overnight in the medium. The next day, the medium in the 96-well plate was replaced with fresh medium and mediators (PBS, Gibco) or various concentrations of pro-inflammatory cytokines (R&D Systems). After 24 hours, the supernatant was collected, and cell viability was assessed using a cell-titer glo assay. Immunomodulatory cytokine production in the supernatant was analyzed by an MSD electrochemiluminescence immunoassay. The supernatant was incubated overnight at 4°C on an appropriate MSD plate before detection the following day.

[0047] Figure 1 The concentration levels of immunomodulatory cytokines produced by MSCs in the supernatant 24 hours after TNF-α stimulation were depicted. The data presented are the mean ± standard deviation of representative experiments from three individual batches of MSCs. MSCs robustly produced a variety of immunomodulatory cytokines, including IL-1RA, IL-4, IL-7, IL-8, IL-10, and IL-13, in a dose-dependent manner within 24 hours of TNF-α stimulation.

[0048] Figure 2 Cell viability of MSCs was described after 24 hours of incubation with TNF-α. Supernatant was collected, and a cell titer of glo reagent was added to the MSCs. Incubation was allowed at room temperature for 10 minutes. After 10 minutes, luminescent readings were taken on a SpectraMax plate reader. Cell viability was determined by normalizing the values ​​to those treated only with the carrier. All MSCs treated with TNF-α, including those stimulated at the highest concentration of 100 ng / ml, exhibited a mean cell viability greater than 80%.

[0049] Example 2

[0050] To measure the immunomodulatory cytokines produced by MSCs over time, 10,000 MSCs from Example 1 were seeded into each well of a 96-well plate and allowed to adhere overnight. The next day, the medium was replaced with fresh medium, and the cells were stimulated for a specified amount of time with either PBS (Gibco) or 10 pg / ml recombinant human TNF-α (R&D Systems). The supernatant was collected, and the production of anti-inflammatory cytokines was analyzed by MSD electrochemiluminescence immunoassay.

[0051] Figure 3 This study demonstrates the production of anti-inflammatory cytokines in MSCs after exposure to 10 pg / mL TNF-α at different time points. Data are presented as mean fold change ± SD for representative experiments from three individual batches of MSCs. Cells continuously produced IL-1RA, IL-4, IL-7, IL-8, IL-10, and IL-13 over 24 hours.

[0052] Example 3

[0053] To measure the production of immunomodulatory cytokines by MSCs in response to IL-17A, 10,000 LMSCs were seeded into each well of a 96-well plate and allowed to adhere overnight. The next day, the medium was replaced with fresh medium, and the cells were stimulated for 1 hour with a medium (PBS, Gibco) or 1 pg / ml recombinant human TNF-α (R&D Systems), followed by the addition of IL-17A at the indicated concentration for 24 hours. The supernatant was collected and the production of anti-inflammatory cytokines was analyzed.

[0054] Figure 4 The production of anti-inflammatory cytokines IL-8 and IL-13 was depicted after exposure to IL-17A alone or IL-17A and TNF-α. When stimulated with IL-17A alone, cells showed no or very little production of IL-8 and IL-13. Figure 4 A), but when exposed to IL-17A and TNF-α, the production of IL-8 and IL-13 increased significantly in a dose-dependent manner in response to IL-17A, indicating that TNF-α sensitizes MSCs to IL-17A. These results were also observed during the examination of IL-13 production. Figure 4 B).

[0055] The scope of this disclosure is not limited to the specific embodiments described herein. In fact, various modifications to the subject matter provided herein, in addition to those described, will become apparent to those skilled in the art based on the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0056] This article cites various publications, patents, and patent applications, the disclosures of which are incorporated in their entirety through citation.

Claims

1. Methods for evaluating the potency of human mesenchymal stem cells, including: Human mesenchymal stem cell populations were stimulated with pro-inflammatory cytokines containing IL-17A and TNF-α. Anti-inflammatory cytokines are generated from the human mesenchymal stem cells, wherein the anti-inflammatory cytokines are selected from the group consisting of IL-1RA, IL-4, IL-7, IL-8, IL-10, IL-13, and combinations thereof; and Quantify anti-inflammatory cytokines derived from the aforementioned human mesenchymal stem cells; The human mesenchymal stem cells were administered IL-17A at a rate of 0.1 pg / mL to 10 µg / mL, and TNF-α at a rate of 1 pg / mL to 10 µg / mL.

2. The method of claim 1, wherein the stimulation step is performed for 1 hour to 24 hours.

3. The method according to claim 1 or 2, wherein the human mesenchymal stem cells are derived from bone marrow, adipose tissue, peripheral blood, lung, heart, amniotic fluid, amnion, umbilical cord or placenta, or differentiated from induced pluripotent stem cells.

4. The method according to claim 1 or 2, wherein the anti-inflammatory cytokine is selected from the group consisting of IL-8, IL-13 and combinations thereof.

5. The method according to claim 1 or 2, wherein the method further comprises examining the expression of biomarkers on the human mesenchymal stem cells prior to stimulation with the pro-inflammatory cytokines.

6. The method of claim 5, wherein the biomarker is selected from CD105. + CD90 + CD73 + CD45 - CD34 - CD19 - CD11b - IL-17RA + HLA-DR - Groups composed of its components.

7. The method according to claim 1 or 2, further comprising seeding the human mesenchymal stem cells onto a matrix prior to stimulation with the pro-inflammatory cytokine.

8. The method according to claim 7, wherein the substrate is a membrane, a plastic surface, a glass surface, or a cell culture plate.

9. The method of claim 7, wherein the human mesenchymal stem cells are seeded onto the matrix for 1 hour to 24 hours.

10. The method according to claim 1 or 2, further comprising dividing the human mesenchymal stem cells into smaller human mesenchymal stem cell populations before stimulation with the pro-inflammatory cytokine.

11. The method according to claim 1 or 2, further comprising separating the supernatant of the human mesenchymal stem cells after stimulation with the pro-inflammatory cytokine.

12. The method of claim 11, wherein once the supernatant is isolated from the human mesenchymal stem cells, it is cryopreserved.

13. The method of claim 11, wherein the supernatant is analyzed by electrochemiluminescence immunoassay to determine the level of anti-inflammatory cytokines produced by the human mesenchymal stem cells.

14. The method according to claim 1 or 2, further comprising performing a viability analysis on the human mesenchymal stem cells after stimulating them with the pro-inflammatory cytokines.

15. The method of claim 14, wherein the activity analysis is ATP detection analysis, tetrazolium reduction analysis, resazurite reduction analysis, protease activity labeling analysis, sodium-potassium ratio analysis, cell lysis or membrane leakage analysis, mitochondrial activity or cysteine ​​aspartate enzyme analysis, genomics and proteomics analysis, or any combination thereof.

16. The method of claim 14, wherein the viability analysis comprises using flow cytometry.

17. The method according to claim 14, wherein, Compared with the same human mesenchymal stem cell population treated with the medium, the human mesenchymal stem cells stimulated with the pro-inflammatory cytokines showed 70% greater viability.

18. The method of claim 14, further comprising assigning a titer to the human mesenchymal stem cells based on the amount of anti-inflammatory molecules produced.

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