Potency Assay

Abstract

A method for assessing the potency of MSCs to produce anti-inflammatory cytokines in response to proinflammatory stimuli includes stimulating MSCs with one or more proinflammatory cytokines, such as TNF-α, for a period of time, and then confirming and quantifying the production of anti-inflammatory cytokines. MSCs that produce high levels of anti-inflammatory cytokines in response to TNF-α may be used to treat age-related conditions, such as age-related frailty and Alzheimer's disease, and may also be used to treat coronavirus infections. This method demonstrates that TNF-α-induced MSCs reliably secrete several anti-inflammatory cytokines, including IL-1 receptor antagonist (IL-1RA), IL-10, and granulocyte colony-stimulating factor (G-CSF).

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 012884, filed April 20, 2020, the contents of which are incorporated herein in their entirety.

[0002] [Field]

[0002] Provided herein are methods for evaluating the potency of human mesenchymal stem cells to produce anti-inflammatory cytokines in response to exposure to pro-inflammatory cytokines, such as TNF-α. Human mesenchymal stem cells that exhibit appropriate anti-inflammatory cytokine production can then be used in methods for treating diseases involving long-term inflammation, such as age-related frailty, Alzheimer's disease, and coronavirus infection.

[0003] [background]

[0003] Age-related frailty poses significant challenges to an individual's overall health and well-being. Age-related frailty is a geriatric syndrome characterized by weakness, low physical activity, slow motor activity, extreme fatigue, and unintentional weight loss. See Yao, X. et al., Clinics in Geriatric Medicine 27(1):79-87 (2011). Furthermore, there are many studies showing a direct correlation between age-related frailty and inflammation. See Hubbard, RE et al., Biogerontology 11(5):635-641 (2010).

[0004]

[0004] Immunosenescence is characterized by a state of low-grade chronic systemic inflammation known as inflammasenesis. See Franceshi, C. et al., Annals of the New York Academy of Sciences 908:244-254 (2000). This state of high inflammation or chronic inflammation seen in aging and age-related frailty leads to immune dysregulation and complex remodeling of both innate and adaptive immunity. In immunosenescence, the T cell and B cell repertoire becomes skewed, with CD8 re-expressing CD45ra (TEMRA) and CD8+ Effector memory T cells and CD19 + Increase in late / exhausted memory B cells and CD8 + Naive T cells and switched memory B cells (CD27 + (See Blomberg, B.B. 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 repertoire leads to a refractory or inefficient immune state. This immune system deterioration contributes to higher susceptibility to infectious diseases and a lower response to vaccinations. Optimal B cell function is critical for providing effective antibody responses to vaccines and protection from infectious pathogens. It is well known that age-related increases in systemic inflammation (TNF-α, IL-6, IL-8, INF-γ, and CRP) cause a decline in B cell function, leading to inadequate antibody responses and reduced vaccine efficacy.

[0005] Inflammatory aging has attracted considerable attention because it suggests a link between immune changes and several diseases and conditions common in older adults, such as age-related frailty. Circulating inflammatory mediators, such as cytokines and acute-phase proteins, are markers of low-grade inflammation that have been shown to increase with age. These proinflammatory cytokines (e.g., TNF-α, IL-6) reduce the ability of B cells to form protective antibodies against foreign antigens and vaccines. This reduced B cell response is measured by a decrease in class switch recombination (CSR), the ability of immunoglobulins to switch isotype from IgM to a secondary isotype (IgG, IgA, or IgE). IgI isotype switching is crucial for appropriate immune responses because effector functions differ between each isotype. A key player in CSR and somatic hypermutation (SHM) is the enzyme activation-induced cytidine deaminase (AID), encoded by the Aicda gene. The essential function of AID in CSR and SHM is to initiate DNA cleavage by converting cytosines to uracils in immunoglobulin switch and variable regions.

[0006]

[0006] It has also been shown in humans that the amount of TNF-α formed (1) depends on the amount of inflammation in the system and (2) reduces 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). Thus, the immune response in subjects suffering from aging frailty is impaired for several reasons.

[0007]

[0007] Expression of TNF-α is also involved in the initiation, maintenance, and amplification of immune processes that lead to neurological inflammation, and is closely linked to the pathogenesis of Alzheimer's disease and related dementias, as well as other forms of inflammation that lead to neurological damage.

[0008] Alzheimer's disease (AD) is a chronic, progressive neurodegenerative brain disorder, or aging syndrome. It is the leading cause of morbidity and mortality among approximately 5 million elderly Americans. AD accounts for 70% of all dementia cases. Dementia is a major public health concern, with a new case diagnosed somewhere around the world every 7 seconds. There is no cure for the disease, and it worsens as the disease progresses, ultimately leading to death within 7 years. Fewer than 3% of individuals survive 14 years or more after diagnosis. People diagnosed with AD are usually over 65 years old and have difficulty completing standard verbal and visual memory tests, as well as decision-making and problem-solving challenges. In 2006, there were 26.6 million patients worldwide, 5 million of whom were in the United States. Alzheimer's disease is predicted to affect 1 in 85 people worldwide by 2050. Early symptoms are often mistaken for "age-related" problems or signs of stress.

[0009]

[0009] Alzheimer's disease (AD) involves complex pathology, involving diverse mechanisms in addition to β-amyloid deposits and neurofibrillary tangles. There is growing recognition that a proinflammatory state contributes to subsequent dementia. In this regard, proinflammatory cytokines are abundant in the vicinity of amyloid deposits and neurofibrillary tangles, and a link exists between systemic inflammation and β-amyloid accumulation. Furthermore, individuals may have significant amyloid deposits and neurofibrillary tangles at autopsy, thereby qualifying for a diagnosis of AD, yet demonstrate no history of dementia; in these cases, the expression of inflammatory markers is dramatically lower than in AD patients.

[0010]

[0010] AD is also characterized by impaired neurovascular systems, which contribute to adverse events. Notable among these are hypoperfusion and compromised blood-brain barrier (BBB). Compromised BBB can impair transendothelial exchange. Impaired transendothelial exchange is manifested in part by AβP's direct inhibition of endothelial cell proliferation and migration. Ultimately, inefficient clearance of AβP across the BBB results in its accumulation in the brain parenchyma. Therefore, compromised neurovascular systems are another important therapeutic target in AD.

[0011] Coronavirus infections have proven to be a significant threat to humanity. Patients infected with COVID-19, particularly those requiring advanced respiratory support, suffer from a particularly poor prognosis. The mortality rate for these patients reaches approximately 54%. Clinical deterioration is associated with a decline in viral titers and often occurs 7–10 days after symptom onset, suggesting that pathology is driven by inflammation rather than direct viral damage. Inflammatory markers are often significantly elevated in patients with severe COVID-19, leading to a hyperinflammatory syndrome that may contribute to the morbidity and mortality of the infection. Hyperinflammatory syndromes typically involve uncontrolled, self-perpetuating, and tissue-damaging inflammatory activity.

[0012]

[0012] Diseases similar to or listed above are typically treated using therapeutic agents such as small molecules, proteins, vaccines, or antibodies. The use of cell therapy to treat the above diseases is not well documented or considered in the art. Cell therapy is a novel and exciting treatment for a wide range of therapeutic indications.

[0013]

[0013] Mesenchymal stem cells are pluripotent cells capable of migrating to sites of injury and are also immune privileged by expressing undetectable major histocompatibility complex class II (MHC-II) molecules and low levels of MHC-I molecules. See Le Blanc, K. et al., Lancet 371(9624):1579-1586 (2008) and Klyushnenkova E. et al., J. Biomed. Sci. 12(1):47-57 (2005). Thus, allogeneic mesenchymal stem cells hold great promise for therapeutic and regenerative medicine and have repeatedly been shown to have a high safety and efficacy profile in clinical trials for multiple 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). Allogeneic mesenchymal stem cells have also been shown not to undergo malignant transformation after transplantation into patients. See Togel F. et al., American Journal of Physiology Renal Physiology 289(1):F31-F42 (2005). Treatment with mesenchymal stem cells has been shown to reverse severe graft-versus-host disease, prevent acute ischemic renal failure, contribute to pancreatic islet and renal glomerular repair in diabetes, reverse fulminant liver failure, regenerate damaged lung tissue, alleviate sepsis, reverse remodeling, and improve cardiac function after myocardial infarction.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 One 2(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 See 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 a potential source of multiple cell types for use in tissue engineering. See Gong Z. et al., Methods in Mol. Biol. 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).

[0014]

[0014] Mesenchymal stem cells have immunomodulatory capabilities. Mesenchymal stem cells control inflammation and cytokine production in lymphocytes and myeloid-derived immune cells without evidence of immunosuppressive toxicity, and are low immunogenic. See Bernardo ME et al., Cell Stem Cell 13(4):392-402 (2013).

[0015]

[0015] In vivo studies have shown that human mesenchymal stem cells, when transplanted into fetal sheep, 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 can persist for as long as 13 months in multiple tissues after transplantation into non-immunosuppressed, immunocompetent hosts. Other in vivo studies using rodents, dogs, goats, and baboons have similarly shown that human mesenchymal stem cell xenografts do not induce lymphocyte proliferation or systemic alloantibody production in recipients.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 Rheumatism 56(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 etc., 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 of allogeneic safety and efficacy strengthen the notion of using mesenchymal stem cells as allografts for successful tissue regeneration.

[0016]

[0016] Studies in AD animal models also support the clinical potential of MSCs. See Neves AF et al., Exp. Neurol. 2021:113706. Beneficial effects include reduced inflammation, increased Aβ degrading factors and Aβ clearance, reduced hyperphosphorylated tau, and elevated levels of alternatively activated (M2) microglial markers. These benefits are thought to be due, at least in part, to the release of chemoattractants by Aβ-induced MSCs that recruit alternative microglia to the brain and reduce Aβ deposition. See Lee JK et al., Stem Cells 2012;30(7):1544-55. MSCs are effective in young AD model mice before Aβ accumulation, causing a significant reduction in brain Aβ deposition and a significant increase in the expression of presynaptic proteins. See Bae JS et al., Curr Alzheimer Res. 2013;10(5):524-31. Impressively, these effects persisted for at least 2 months, suggesting that MSCs may be useful as an interventional treatment for prodromal AD. In summary, preclinical studies of AD have shown that MSCs can cross the BBB, inhibit neuroinflammation, promote neurogenesis, inhibit β-amyloid deposition and promote its clearance, reduce apoptosis, promote hippocampal neurogenesis, improve dendritic morphology, and improve behavioral and spatial memory performance.

[0017] [overview]

[0017] The property of mesenchymal stem cells (MSCs) to produce immunomodulatory cytokines in response to proinflammatory stimuli is an important therapeutic mechanism of action employed by MSCs.

[0018]

[0018] Accurate, reproducible and suitable assays for assessing the potency of cells used in cell therapy are important for quality control purposes, for example, to ensure the stability and consistency of cell-based therapeutic products.

[0019]

[0019] Current assays used in the art to assess cell potency focus on confirming the expression of specific biomarkers or cell surface receptors. These assays are expected to provide an indirect measurement of cell potency (e.g., MSCs expressing TNFR1 are expected to inhibit PBMC proliferation). Therefore, the "potency assays" used in the art are homogeneous assays that measure the expression of cell receptors or biomarkers, but cannot accurately measure the ability or potency of cells to express or produce important macromolecules such as anti-inflammatory cytokines.

[0020]

[0020] While MSC potency assays have been developed in which MSCs are stimulated with LPS, these potency assays generate "irrelevant" stimuli (e.g., LPS stimulation mimics bacterial infection and is irrelevant because MSCs are not used as antimicrobial agents). These assays are further irrelevant because MSCs generally do not express TLR4 and CD14, both of which are required for LPS stimulation and signaling. Therefore, the goal of this application is to develop a potency assay that accurately determines the ability of mesenchymal stem cells (MSCs) to produce immunomodulatory cytokines in response to proinflammatory cytokines such as TNF-α. Ideally, measurements are performed on physiologically relevant components.

[0021]

[0021] Provided herein are methods for assessing MSC potency, e.g., in a cell preparation (e.g., a preparation of MSCs belonging to a number of cells intended for therapeutic use). Compared to standard cell potency assays used in the art, which only involve detecting the presence of a cell surface receptor or biomarker and fail to assess whether the cells can express molecules associated with stimulation of the receptor or biomarker, the methods provided herein utilize a TNF-α stimulation step to assess whether and to what level MSCs produce anti-inflammatory cytokines prior to assessing the potency of a cell or cell lot. The incorporation of a TNF-α stimulation step has been shown to result in potency assays with increased reliability and reduced variability across MSC preparations harvested from the same cell lot and across MSC preparations containing the same cell type but harvested from different cell lots. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 shows the production levels of anti-inflammatory cytokines after stimulation of MSCs with recombinant human TNFα. [Figure 2] FIG. 1 shows the viability of MSCs after stimulation with recombinant human TNFα. [Figure 3] FIG. 1 shows the production levels of anti-inflammatory cytokines after stimulation of MSCs with recombinant human TNFα for 24 hours. [Figure 4] FIG. 1 shows the production levels of IL-8 and IL-13 after MSCs were sensitized to IL-17A stimulation and exposed to recombinant human TNFα for 1 hour.

[0023] [Detailed explanation]

[0026] One aspect of the present application relates to a method for assessing the potency of MSCs to produce anti-inflammatory cytokines.

[0024]

[0027] In one embodiment, the method comprises stimulating the MSCs with a pro-inflammatory cytokine or molecule for a period of time before determining and quantifying the level of anti-inflammatory cytokine production.

[0025]

[0028] MSCs may be derived from bone marrow, adipose tissue, peripheral blood, lung, heart, amniotic fluid, inner organs, amniotic membrane, umbilical cord or placenta, or other tissues, or may be differentiated from induced pluripotent stem cells (IPSCs) or other sources.

[0026]

[0029] MSCs can be stimulated with proinflammatory cytokines or molecules. Proinflammatory 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 both TNF-α and IL-17A, or other combinations. Other proinflammatory molecules include C-reactive protein (CRP) or virulence factors. A virulence factor can be any viral molecule that aids in colonization of a host's biological niche, immune evasion or evasion of a host's immune response, immunosuppression or inhibition of a host's immune response, cellular entry and exit, or nutrient acquisition from the host. An example of a virulence factor is the SARS-CoV-2 spike protein.

[0027]

[0030] Surprisingly, when treated with IL-17A alone, MSCs were shown to produce no or very low levels of anti-inflammatory cytokines. When MSCs were treated with IL-17A and TNF-α together, MSCs produced very high levels of anti-inflammatory cytokines. This unexpected finding is significant because the current standard for assessing cell potency is to confirm that the cells express a specific receptor or biomarker without assessing the receptor's ability to promote the production of that specific molecule. This finding confirms that even if cells possess a receptor known to produce a specific molecule, they may not produce that molecule at an efficient potency useful for subsequent treatment. Furthermore, it demonstrates that MSCs can respond differently to various combinations of indication- or patient-specific pro-inflammatory molecules to optimize a particular treatment for a particular patient.

[0028]

[0031] The amount of proinflammatory cytokine or molecule used to stimulate MSCs may be in the range of 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 when culturing 500 to 50,000 MSCs in 50 to 200 microliters of medium. The concentration is adjusted depending on the cell number and / or volume.

[0029]

[0032] Prior to quantifying the level of anti-inflammatory cytokine production, MSCs can be stimulated with pro-inflammatory cytokines or molecules for 1 hour to 24 hours, 1 hour to 12 hours, 2 hours to 6 hours, or 1 hour to 4 hours, 24 hours to 120 hours, 24 hours to 72 hours, or for more than 120 hours.

[0030]

[0033] Anti-inflammatory cytokines that can be investigated and quantified after stimulating MSCs with pro-inflammatory cytokines or molecules are IL-1RA, IL-4, IL-7, IL-8, IL-10, IL-13, G-CSF, or any combination thereof.

[0031]

[0034] 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 to 50,000 cells cultured in 50 to 200 microliters of medium. Concentrations may be adjusted depending on cell number and / or medium volume.

[0032]

[0035] In some embodiments, the method further comprises checking the expression of a biomarker on the MSCs before stimulating with a proinflammatory cytokine. Biomarkers that may be explored include CD105 + , CD90 + , CD73 + , CD45 - , CD34 - , CD19 - , CD11b - , HLA-DR - , IL-17RA + , or any combination thereof.

[0033]

[0036] In other embodiments, the method further comprises seeding the MSCs onto a substrate prior to stimulation with the proinflammatory cytokines. The substrate may be a membrane, a plastic surface, a glass surface, or a cell culture well plate such as a 96-well plate, with or without an additional substrate coating. The period for seeding the MSCs 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. The MSCs should be properly attached to the substrate after the seeding period has elapsed.

[0034]

[0037] MSCs can be separated into smaller populations before stimulation with pro-inflammatory cytokines. Separating MSCs into smaller populations provides a more accurate assessment of their ability to produce anti-inflammatory cytokines after stimulation.

[0035]

[0038] In some embodiments, the method may further include isolating the supernatant of the MSCs after stimulation with proinflammatory cytokines. Once collected, the supernatant can be stored at -80°C. The supernatant can be further analyzed using an electrochemiluminescence immunoassay to determine the level of anti-inflammatory cytokines produced by the MSCs. Because detection methods typically used in potency assays are not as sensitive as electrochemiluminescence immunoassays, electrochemiluminescence immunoassays allow for the detection of femtogram concentrations of cytokines produced by MSCs.

[0036]

[0039] In other embodiments, the method further comprises performing a viability assay on the MSCs after they have been stimulated with proinflammatory cytokines for a period of time. The viability assay may be an ATP detection assay such as the CellTiter-Glo assay (Promega), a tetrazolium reduction assay, a resazurin reduction assay, a protease viability marker assay, a sodium-potassium ratio assay, a cell lysis or membrane leakage assay, a mitochondrial activity or caspase assay, a functional assay, a genomic and proteomic assay, or any combination thereof. MSC viability may also be assessed using flow cytometry.

[0037]

[0040] The viability of MSCs after stimulation with proinflammatory cytokines can exceed 70% when compared to vehicle-treated MSC populations.

[0038]

[0041] In other embodiments, the method further includes assigning a grade to the potency of the MSCs based on the amount of anti-inflammatory molecule produced. Grades assigned to the potency of the MSCs include threshold grades, where the MSCs can possess a potency grade that produces 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 to 50,000 cells cultured in 50 to 200 microliters of medium.

[0039] [Example]

[0042] Example 1

[0043] A population of human MSCs obtained from bone marrow aspirates and subsequently cryopreserved was thawed. Upon thawing, an aliquot of MSCs was collected for immunophenotyping to confirm cell identity. This included confirming that MSCs expressed CD105, CD90, and CD73, but not CD45, CD34, CD19, CD11b, or HLA-DR.

[0040]

[0044] From the remaining cells, 10,000 MSCs were seeded into wells of a 96-well plate and allowed to adhere overnight in culture medium. The following day, the medium in the 96-well plate was replaced with fresh culture medium and either vehicle (PBS, Gibco) or proinflammatory cytokine concentrate (R&D Systems). After 24 hours, supernatants were collected and cell viability was assessed using a Cell-titer glo assay. Supernatants were analyzed for immunomodulatory cytokine production by MSD electrochemiluminescence immunoassay. Supernatants were incubated overnight at 4°C on the appropriate MSD plate before detection the following day.

[0041]

[0045] Figure 1 shows the concentration levels of immunomodulatory cytokines produced by MSCs in the supernatant after 24 hours of stimulation with TNF-α. Data shown are the mean ± standard deviation of a representative experiment from three individual lots of MSCs. MSCs demonstrated potent production of multiple 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 stimulation with TNF-α.

[0042]

[0046] Figure 2 shows the cell viability of MSCs after 24 hours of incubation with TNF-α. The supernatant was collected and cell titer glo reagent was added to the MSCs. The reagent was allowed to incubate at room temperature for 10 minutes. After 10 minutes, luminescence was read on a SpectraMax plate reader. Cell viability was determined by normalizing values ​​to cells treated with vehicle alone. All MSCs treated with TNF-α, including those stimulated with the highest concentration of 100 ng / ml, showed an average cell viability of over 80%.

[0043]

[0047] Example 2

[0048] To measure immunomodulatory cytokine production by MSCs over time, 10,000 MSCs from Example 1 were seeded into each well of a 96-well plate in culture medium and allowed to adhere overnight. The following day, the medium was replaced with fresh culture medium, and the cells were stimulated with either vehicle (PBS, Gibco) or 10 pg / ml recombinant human TNF-α (R&D Systems) for the indicated times. Supernatants were collected and analyzed for anti-inflammatory cytokine production by MSD electrochemiluminescence immunoassay.

[0044]

[0049] Figure 3 shows anti-inflammatory cytokine production by MSCs at various time points after exposure to 10 pg / mL TNF-α. Data shown as the mean fold change ± standard deviation of a representative experiment from three individual lots of MSCs. Cells demonstrated sustained production of IL-1RA, IL-4, IL-7, IL-8, IL-10, and IL-13 over a 24-hour time course.

[0045]

[0050] Example 3

[0051] To measure immunomodulatory cytokine production by MSCs in response to IL-17A, 10,000 LMSCs were seeded in culture medium in each well of a 96-well plate and allowed to adhere overnight. The following day, the medium was replaced with fresh culture medium, and the cells were stimulated with either vehicle (PBS, Gibco) or 1 pg / ml recombinant human TNF-α (R&D Systems) for 1 hour before adding the indicated concentrations of IL-17A for 24 hours. Supernatants were collected and analyzed for anti-inflammatory cytokine production.

[0046]

[0052] Figure 4 shows the production of anti-inflammatory cytokines IL-8 and IL-13 after exposure to IL-17A alone or IL-17A and TNF-α. When stimulated with IL-17A alone, cells showed no or little production of IL-8 and IL-13 (Figure 4a). However, when exposed to IL-17A and TNF-α, IL-8 and IL-13 production increased significantly in a dose-dependent manner in response to IL-17A, suggesting that TNF-α sensitizes MSCs to IL-17A. These results were also found when examining IL-13 production (Figure 4b).

[0047]

[0053] The present disclosure is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the subject matter provided herein in addition to the specific embodiments described will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0048]

[0054] Various publications, patents, and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties. The present invention may have the following aspects. Item 1. 1. A method for assessing the potency of human mesenchymal stem cells (MSCs), comprising: Stimulating the population of MSCs with a pro-inflammatory cytokine or other pro-inflammatory molecule; confirming the production of anti-inflammatory cytokines from the MSCs; Quantifying the level of anti-inflammatory cytokine production from the MSCs. A method comprising: Item 2. 2. The method of claim 1, wherein the proinflammatory cytokine is TNF-α, IL-17a, or a combination thereof. Item 3. 2. The method of claim 1, wherein the proinflammatory cytokine is TNF-α. Item 4. Item 1, wherein the stimulation step is carried out for 1 hour to 24 hours. Item 5. 2. The method of item 1, wherein the proinflammatory cytokine is administered to the MSCs in an amount ranging from 0.1 pg / mL to 1 μg / mL. Item 6. 2. The method of claim 1, wherein the MSCs are derived from bone marrow, adipose tissue, peripheral blood, lung, heart, amniotic fluid, viscera, amniotic membrane, umbilical cord or placenta, or other tissue, or are differentiated from induced pluripotent stem cells (IPSCs) or other sources. Item 7. 2. The method of claim 1, wherein the anti-inflammatory cytokine that can be identified and quantified is selected from the group consisting of IL-1RA, IL-4, IL-7, IL-8, IL-10, IL-13, G-CSF, and combinations thereof. Item 8. 2. The method of claim 1, further comprising checking the expression of a biomarker on the MSCs prior to stimulation with the proinflammatory cytokine. Item 9. The biomarker being explored is CD105 + , CD90 + , CD73 + , CD45 - , CD34 - , CD19 - , CD11b - , IL-17RA + , HLA-DR - 9. The method according to item 8, comprising: Item 10. 2. The method of claim 1, further comprising the step of seeding the MSCs onto a substrate prior to stimulation with the proinflammatory cytokines. Item 11. 11. The method of claim 10, wherein the substrate is a membrane, a plastic surface, a glass surface, or a cell culture well plate such as a 96-well plate, with or without an additional substrate coating. Item 12. Item 11. The method of item 10, wherein the seeding of the MSCs onto the substrate lasts for 1 hour to 24 hours. Item 13. 2. The method of claim 1, wherein the MSCs are divided into smaller populations of MSCs prior to stimulation with the proinflammatory cytokines. Item 14. 2. The method of claim 1, further comprising isolating the supernatant of the MSCs after stimulation with the proinflammatory cytokine. Item 15. 15. The method of claim 14, wherein once the supernatant is isolated from the MSCs, the supernatant is stored frozen. Item 16. 15. The method of claim 14, wherein the supernatant is analyzed by electrochemiluminescence immunoassay or other assay to determine the level of anti-inflammatory cytokines produced by the MSCs. Item 17. 2. The method of claim 1, further comprising the step of performing a viability assay on the MSCs after stimulation with the proinflammatory cytokines. Item 18. 18. The method of item 17, wherein the viability assay is an ATP detection assay, a tetrazolium reduction assay, a resazurin reduction assay, a protease viability marker assay, a sodium-potassium ratio assay, a cell lysis or membrane leakage assay, a mitochondrial activity or caspase assay, a functional assay, a genomic and proteomic assay, or any combination thereof. Item 19. 18. The method of claim 17, wherein the viability assay comprises the use of flow cytometry. Item 20. 18. The method of claim 17, wherein the survival rate of the MSCs after stimulation with a proinflammatory cytokine is greater than 70% when compared to a vehicle-treated MSC population. Item 21. 18. The method of claim 17, further comprising assigning a grade to the potency of the MSCs based on the amount of anti-inflammatory molecules produced.

Claims

1. 1. A method for assessing human mesenchymal stem cells (MSCs), comprising: (a) stimulating a first population of MSCs with IL-17a; (b) producing an anti-inflammatory cytokine by the first population of MSCs, wherein the anti-inflammatory cytokine is selected from the group consisting of IL-1RA, IL-4, IL-7, IL-8, IL-10, IL-13, and combinations thereof; (c) quantifying the anti-inflammatory cytokine in step (b), thereby obtaining a first measure of the anti-inflammatory cytokine produced by the first population of MSCs; (d) stimulating the second population of MSCs with IL-17a and TNF-α; (e) producing the anti-inflammatory cytokine by the second population of MSCs; (f) quantifying the anti-inflammatory cytokine in step (e), thereby obtaining a second measure of the anti-inflammatory cytokine produced by the second population of MSCs; A method comprising:

2. The method of claim 1, further comprising a step of comparing the first measurement value with the second measurement value.

3. The method described in claim 2, further comprising a step of identifying an MSC having the second measurement value greater than the first measurement value.

4. 4. The method of any one of claims 1 to 3, wherein the stimulating step (a) or (d) is carried out for 1 hour to 24 hours.

5. The method of any one of claims 1 to 4, wherein the IL-17a or TNF-α is administered to the MSCs in an amount ranging from 0.1 pg / mL to 1 μg / mL.

6. 6. The method of any one of claims 1 to 5, wherein the MSCs are derived from bone marrow, adipose tissue, peripheral blood, lung, heart, amniotic fluid, viscera, amniotic membrane, umbilical cord or placenta, or are differentiated from induced pluripotent stem cells (IPSCs).

7. A method according to any one of claims 1 to 6, wherein the anti-inflammatory cytokine is selected from the group consisting of IL-8, IL-13, and combinations thereof.

8. A method described in any one of claims 1 to 7, further comprising a step of checking the expression of a biomarker on the MSCs before step (a).

9. The biomarker is CD105 ＋ , CD90 ＋ , CD73 ＋ , CD45 － , CD34 － , CD19 － , CD11b － , IL-17RA ＋ , HLA-DR － 9. The method of claim 8, wherein the hydroxyl group is selected from the group consisting of:

10. The method of claim 1, further comprising the step of seeding the MSCs onto a substrate prior to step (a).

11. The method of claim 10, wherein the substrate is a membrane, a plastic surface, a glass surface, or a cell culture well plate.

12. The method of claim 10 or 11, wherein the seeding of the MSCs onto the substrate lasts for 1 hour to 24 hours.

13. The method of claim 1, further comprising, prior to step (a), dividing the MSCs into the first and second populations.

14. The method of claim 1, further comprising, after step (b) or (e), isolating the supernatant of the first or second population.

15. 15. The method of claim 14, wherein once the supernatant is isolated, the supernatant is stored frozen.

16. 16. The method of claim 14 or 15, wherein the supernatant is analyzed in step (c) or (f) by electrochemiluminescence immunoassay.

17. The method of any one of claims 1 to 16, further comprising the step of performing a viability assay on the first or second population of MSCs after stimulation.

18. 18. The method of claim 17, wherein the viability assay is an ATP detection assay, a tetrazolium reduction assay, a resazurin reduction assay, a protease viability marker assay, a sodium-potassium ratio assay, a cell lysis or membrane leakage assay, a mitochondrial activity or caspase assay, a functional assay, a genomic and proteomic assay, or any combination thereof.

19. 19. The method of claim 17 or 18, wherein the viability assay comprises the use of flow cytometry.

20. 20. The method of any one of claims 17 to 19, wherein the survival rate of the first or second population of MSCs after stimulation is greater than 70% when compared to the same population treated with vehicle.

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