POTENCY ASSAY

Abstract

A method for assessing the potency of MSCs to produce anti-inflammatory cytokines in response to a pro-inflammatory stimulus. The method comprises stimulating the MSCs with one or more proinflammatory cytokines, such as TNF-α, for a duration of time and then identifying and quantifying the production of anti-inflammatory cytokines. MSCs that produce potent levels of anti-inflammatory cytokines in response to TNF-α can be used in treatments for aging-related conditions, including aging frailty and Alzheimer's disease, and can also be used to treat corona virus infections. The method shows that TNF-α induced MSCs robustly secrete several anti-inflammatory cytokines, including IL-1 receptor antagonist (IL-1RA), IL-10, and granulocyte colony stimulating factor (G-CSF).

Claims

1. A method for assessing the potency of human mesenchymal stem cells (MSCs), comprising: stimulating a population of MSCs with a pro-inflammatory cytokine or other pro-inflammatory molecule; identifying anti-inflammatory cytokine production from said MSCs; and quantifying levels of the anti-inflammatory cytokine production from said MSCs.

2. The method according to claim 1, wherein the pro-inflammatory cytokine is TNF-α, IL-17a or a combination thereof.

3. The method according to claim 1, wherein the pro-inflammatory cytokine is TNF-α.

4. The method according to claim 1, wherein the stimulation step occurs between 1 hour and 24 hours.

5. The method according to claim 1, wherein the pro-inflammatory cytokine is administered to the MSCs in an amount ranging from 0.1 pg/mL to 1 μg/mL.

6. The method according to claim 1, wherein the MSCs are derived from bone marrow, adipose tissue, peripheral blood, a lung, a heart, amniotic fluid, inner organs, an amniotic membrane, an umbilical cord or a placenta, or other tissue, or differentiated from induced pluripotent stem cells (IPSCs) or other sources.

7. The method according to claim 1, wherein the anti-inflammatory cytokines that can be identified and quantified are selected from the group consisting of IL-1RA, IL-4, IL-7, IL-8, IL-10, IL-13, G-CSF and combinations thereof.

8. The method according to claim 1, wherein the method further comprises a step of checking for the expression of biomarkers on the MSCs before stimulation with the pro-inflammatory cytokine.

9. The method according to claim 8, wherein the biomarkers that are searched for include CD105.sup.+, CD90.sup.+, CD73.sup.+, CD45.sup.−, CD34.sup.−, CD19.sup.−, CD11b.sup.−, IL-17RA.sup.+, HLA-DR.sup.+ or any combination thereof.

10. The method according to claim 1, wherein the method can further comprise a step of seeding the MSCs onto a substrate before stimulation with the pro-inflammatory cytokine.

11. The method according to 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 added substrate coating.

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

13. The method according to claim 1, wherein the MSCs are divided into smaller populations of MSCs before stimulation with the pro-inflammatory cytokine.

14. The method according to claim 1, wherein the method further comprises a step of isolating supernatants of the MSCs after stimulation with the pro-inflammatory cytokine.

15. The method according to claim 14, wherein the supernatants are cryopreserved once they have been isolated from the MSCs.

16. The method according to claim 14, wherein the supernatants are analyzed with a electrochemiluminescence immunoassay or other assays to determine the levels of anti-inflammatory cytokines produced by the MSCs.

17. The method according to claim 1, wherein the method further comprises performing a viability assay on the MSCs after they have been stimulated with the pro-inflammatory cytokine.

18. The method according to 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 cytolysis or membrane leakage assay, a mitochondrial activity or caspase assay, a functional assay, a genomic and proteomic assay or any combination thereof.

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

20. The method according to claim 17, wherein the viability of the MSCs after stimulation with a pro-inflammatory cytokine is greater than 70% when compared to MSC populations treated with a vehicle.

21. The method according to claim 17, further comprising assigning a grade to the potency of the MSCs based on the amount of produced anti-inflammatory molecules.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0022] FIG. 1 depicts the production levels of anti-inflammatory cytokines after stimulation of MSCs with recombinant human TNFα.

[0023] FIG. 2 depicts the viability of MSCs after stimulation with recombinant human TNFα.

[0024] FIG. 3 depicts the production levels of anti-inflammatory cytokines after stimulation of MSCs with recombinant human TNFα over 24 hours.

[0025] FIG. 4 depicts the production levels of IL-8 and IL-13 after MSCs were sensitized to IL-17A stimulation but exposure to recombinant human TNFα for 1 hour.

DETAILED DESCRIPTION

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

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

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

[0029] The MSCs can be stimulated with a pro-inflammatory cytokines or molecules. The 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, the MSCs are stimulated with both 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 aids in the colonization of a niche in a host, immunoevasion or evasion of a host's immune response, immunosuppression or inhibition of a host's immune response, entry into and exit out of cells or obtaining nutrition from a host. One example of a virulence factor is a SARS-CoV-2 spike protein.

[0030] Surprisingly, MSCs have been shown to not produce or produce significantly lower levels of anti-inflammatory cytokines when treated with IL-17A alone. When MSCs are treated with IL-17A and TNF-α together, the MSCs produce significantly higher levels of anti-inflammatory cytokines. The importance of this unexpected discovery stems from the current criteria required to assess the potency of cells, which is to confirm that the cells express certain receptors or biomarkers without assessing the receptors' abilities to promote production of specific molecules. This discovery confirms that even though a cell possesses a receptor known to produce specific molecules, the cell may not produce said molecules at an efficient potency to be useful in following treatments. Furthermore, it shows that MSCs can respond differently to different combinations of pro-inflammatory molecules that are indication- or patient-specific, to best suit particular treatments to specific patients.

[0031] The amount of the pro-inflammatory cytokine or molecule used to stimulate the MSCs can range from 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, under conditions where 500 to 50,000 MSCs are cultured in 50 to 200 microliters of medium. Concentrations are scaled accordingly with changes in cell number and/or volume.

[0032] The MSCs can be stimulated with a pro-inflammatory cytokine or molecule for between 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 more than 120 hours before quantifying the levels of anti-inflammatory cytokine production.

[0033] The anti-inflammatory cytokines that can be examined and quantified after stimulation of the MSCs with a pro-inflammatory cytokine or molecule are IL-1RA, IL-4, IL-7, IL-8, IL-10, IL-13, G-CSF or any combination thereof.

[0034] In other embodiments, stimulation of the MSCs with a pro-inflammatory cytokine or molecule may lead to the production of an anti-inflammatory molecule in 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 every 500 to 50,000 cells cultured in 50 to 200 microliters of medium. Concentrations may be scaled accordingly with changes in cell number and/or volume of medium.

[0035] In some embodiments, the method further comprises checking for the expression of biomarkers on the MSCs before stimulation with a pro-inflammatory cytokine. The biomarkers that can be searched for include CD105.sup.+, CD90.sup.+, CD73.sup.+, CD45.sup.−, CD34.sup.−, CD19.sup.−, CD11b.sup.−, HLA-DR.sup.−, IL-17RA.sup.+ or any combination thereof.

[0036] In other embodiments, the method can further comprise a step of seeding the MSCs onto a substrate before stimulation with a pro-inflammatory cytokine. The substrate can 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 added substrate coating. The duration for seeding the MSCs onto a substrate can be from 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 adhered onto the substrate after the seeding duration has passed.

[0037] The MSCs can be divided into smaller populations of MSCs before stimulation with a pro-inflammatory cytokine. Separation of the MSCs into smaller populations provides a more accurate assessment of the MSCs ability to produce anti-inflammatory cytokines after stimulation.

[0038] In some embodiments, the method can further comprise a step of isolating the supernatants of the MSCs after stimulation with a pro-inflammatory cytokine. The supernatants can be stored at −80° C. once they have been collected. The supernatants can be further analyzed to determine the levels of anti-inflammatory cytokines produced from the MSCs through the use of electrochemiluminescence immunoassays. Methods of detection typically used in potency assays are not as sensitive as electrochemiluminescence immunoassays, so the use of electrochemiluminescence immunoassays allows the detection of cytokines in femtogram concentrations produced by MSCs.

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

[0040] The viability of the MSCs after stimulation with a pro-inflammatory cytokine may be greater than 70% when compared to MSC populations treated with a vehicle.

[0041] In other embodiments, the method further comprises assigning a grade to the potency of the MSCs based on the amount of produced anti-inflammatory molecules. The grades assigned to the potency of the MSCs include thresholds grades wherein the MSCs may possess a potency grade of 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 every 500 to 50,000 cells cultured in 50 to 200 microliters of medium.

EXAMPLES

Example 1

[0042] A population of human MSCs derived from bone marrow aspirates and subsequently cryopreserved were thawed. Upon thaw, an aliquot of the MSCs were taken 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.

[0043] 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, media in the 96-well plate was replaced with fresh culture medium and either vehicle (PBS, Gibco) or concentrations of pro-inflammatory cytokines (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 immunoassays. The supernatants were incubated on appropriate MSD plates overnight at 4° C., before detection the following day.

[0044] FIG. 1 depicts the concentration levels of the immunomodulatory cytokine produced from the MSCs in the supernatants after stimulation with TNF-α for 24 hours. Data shown is mean±standard deviation of a representative experiment from 3 individual lots of MSCs. The MSCs showed robust production of multiple immunomodulatory cytokines including IL-1RA, IL-4, IL-7, IL-8, IL-10 and IL-13 within 24 hours of stimulation with TNF-α in a dose-dependent manner.

[0045] FIG. 2 depicts the cell viability of the MSCs after incubation with TNF-α for 24 hours. Supernatants were collected and a cell titer glo reagent was added to the MSCs. The reagent was allowed to incubate for 10 minutes at room temperature. After 10 minutes, luminescence readings were taken on a SpectraMax plate reader. Cell viability was determined by normalizing values to cells treated with vehicle only. All MSCs treated with TNF-α, including those stimulated with the highest concentration of 100 ng/ml, showed mean cell viability of above 80%.

[0046] To measure the production of immunomodulatory cytokines by MSCs over time, 10,000 MSCs from Example 1 were seeded per well into a 96 well plate in culture medium and allowed to adhere overnight. The following day, media was replaced with fresh culture medium, and cells were stimulated for the indicated amount of time with either vehicle (PBS, Gibco) or 10 pg/ml of recombinant human TNF-α (R&D Systems). Supernatants were collected and analyzed for anti-inflammatory cytokine production through MSD electrochemiluminescence immunoassays.

[0047] FIG. 3 shows the anti-inflammatory cytokine production of the MSCs after exposure to 10 pg/mL of TNF-α at various time points. Data shown as mean fold change ±SD of a representative experiment from 3 individual lots of MSCs. The cells showed sustained production of IL-1RA, IL-4, IL-7, IL-8, IL-10 and IL-13 over the 24 hour timecourse.

Example 3

[0048] To measure the production of immunomodulatory cytokines by MSCs in response to IL-17A, 10,000 LMSCs were seeded per well into a 96 well plate in culture medium and allowed to adhere overnight. The following day, media was replaced with fresh culture medium, and cells were stimulated for one hour with either vehicle (PBS, Gibco) or 1 pg/ml of recombinant human TNF-α (R&D Systems) prior to the addition of the indicated concentrations of IL-17A for 24 hours. Supernatants were collected and analyzed for anti-inflammatory cytokine production.

[0049] FIG. 4 depicts that production of anti-inflammatory cytokines IL-8 and IL-13 after exposure of IL-17A alone or IL-17A and TNF-α. The cells showed no or minimal production of IL-8 and IL-13 when stimulated with IL-17A alone (FIG. 4a), but when exposed to IL-17A and TNF-α, IL-8 and IL-13 production significantly increased in a dose dependent response to IL-17A, suggesting TNF-α sensitizes MSCs to IL-17A. These results were also discovered during examination of IL-13 production (FIG. 4b).

[0050] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the subject matter provided herein, in addition to those 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.

[0051] Various publications, patents and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties.