Patent Application
20250222035
Regenerative medicine is an area of medicine that is concerned with the replacement or regeneration of human cells, tissues, or organs, in order to restore or establish normal functions. For example, stem cell therapies can be utilized in order to treat, prevent, or cure a variety of diseases and disorders.
Stem cells are cells that have the ability to divide without limit and that, under certain specific conditions, can differentiate into a variety of different cell types. Totipotent stem cells are stem cells that have the potential to generate all of the cells and tissues that make up an embryo. Pluripotent stem cells are stem cells that give rise to cells of the mesoderm, endoderm, and ectoderm. Multipotent stem cells are stem cells that have the ability to differentiate into two or more cell types, whereas unipotent stem cells are stem cells that differentiate into only one cell type. One type of such stem cells are mesenchymal stem cells. See, for example U.S. Patent Publication No. US20190046576.
However, it is difficult to produce and store live stem cell-based therapies on a clinically relevant scale. (See, Trainor et al., Nature Biotechnology 32(1) (2014)). Moreover, the therapeutic potency and regenerative capacity of such therapies is often variable and the cells can die before or during transplantation. (See, Newell, Seminars in Immunopathology 33(2):91 (2011)). Implanted stem cells are also susceptible to host immune system attack and/or rejection, and it is often difficult to assess potency and/or control “dosing”. Thus, there is a need in the art for additional regenerative therapies that can overcome the cost, storage, and manufacturing quality control limitations that are currently associated with cell-based regenerative medicine therapies, in particular in the context of ocular conditions.
Blast and blunt injuries to the eye can cause a series of mechanical disruptions to the ocular contents including commotio retinae, traumatic cataract, disruption of the zonular attachments to the lens, angle recession, iris dialysis, and rupture of the pupillary sphincter and disruption to optic nerve. Treatment of these injuries has been limited to mechanical repair (when possible) of the iris, replacement of the crystalline lens with plastic lens implants, and repair of retinal detachments. There has been no treatment to repair the cellular architecture of the retina or the anterior chamber as a result of injury, or disease, or inherited genetic conditions such as retinitis pigmentosis. Furthermore, traumatic optic neuropathy and optic nerve avulsion are among the six leading types of ocular injury that required specialized ophthalmic care during Operation Iraqi Freedom (Cho and Savitsky, “Ocular Trauma Chapter 7”, in Combat Casualty Care: Lessons learned from Oef and Oif, by Brian Eastbridge and Eric Savitsky, pp. 299-342, Ft. Detrick, Md.: Borden Institute (US) Government Printing Office, 2012), incorporated herein by reference in its entirety. Sixty percent of traumatic head injuries result in neuro-ophthalmic abnormalities (Van Stavern, et al., J Neuro-Ophthamol 21(2):112-117, 2001) (incorporated herein by reference in its entirety) half of which involve the optic nerves or visual pathways. Traumatic injury to neurons results in axonal damage and irreversible neuronal loss resulting in permanent deficits. While a number of potential neuroprotective therapies have been identified in animals, these single agents have generally failed to translate to therapies in human clinical trials (Turner, et al., J Neurosurg 118(5):1072-1085, 2013, incorporated herein by reference in its entirety). Combination therapies that affect several cellular targets are likely needed to prevent neuronal damage or restore neuronal function.
The cornea serves a protective role as the outermost tissue of the eye, however it is highly vulnerable to severe injury and disease. Its lack of blood vessels enables its transparency but also limits its ability to heal. Corneal injury, due to its potential to cause irreversible blindness, requires prompt intervention and aggressive treatment. The critical need for improved ocular surface healing therapies is particularly apparent for chemical burns and in severe corneal diseases, such as ocular manifestations of acute Chronic Graft v. Host Disease (GvHD), Stevens-Johnson Syndrome, Ocular Mucous Membrane Pemphigoid and other conditions giving rise to persistent corneal epithelial defect, which collectively comprise an incidence of over 100,000 cases per year. (See, Dietrich-Ntoukas et al. Cornea. 2012, 31(3):299-310; Stevenson W, et al., Clin Ophthalmol. 2013, 7:2153-2158. White K D, et al., J Allergy Clin Immunol Pract. 2018; 6(1):38-69; Tauber J. (2002) Autoimmune Diseases Affecting the Ocular Surface. In: Ocular Surface Disease Medical and Surgical Management. Springer, New York, NY.; and Wirostko B, et al., Ocul Surf. 2015 July; 13(3): 204-21; and Haring, R S., et al., JAMA Ophthalmol. 2016 Oct. 1; 134(10):1119-1124.)
Moreover, topical ophthalmic drug development is impeded by many anatomical constraints including tear turnover and dilution, nasolacrimal drainage, and reflex blinking with often less than 5% of the topically administered dose reaching deeper ocular tissues (Gaudana et al., 2009). In the case of corneal wounds, the initial insult causes rifts in the corneal epithelium thereby enabling the passage of topically applied MSC-S to penetrate the epithelial layers.
Accordingly, there is a large unmet need in the art for ocular therapies that can target the eye and deliver a therapeutic payload to difficult-to-reach sensory tissue which may have degenerated due to inflammation secondary to trauma (such as for example, burns, acute inflammation, age, and/or oxidative stress). The present invention meets this need by providing mesenchymal stem cell secretome compositions for use in such treatments, as well as methods for making such compositions.
In some embodiments, the present invention provides a method for treating limbal stem cell deficiency (LSCD) in a subject in need thereof, the meth-od comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition comprising a bone marrow-derived MSC secretome.
In some embodiments, the MSC secretome comprises: HGF; Pentraxin-3 (TSG-14); VEGF; TIMP-1; Serpin E1; <5 ng/ml IL-8.
In some embodiments, the MSC secretome comprises:
In some embodiments, the MSC secretome further comprises “higher levels” of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and/or Serpin F1, optionally 1 ng/mL-400 ng/mL, or optionally 1 ng/mL-300 ng/ml, or optionally 1 ng/ml-200 ng/mL, or optionally 1 ng/ml-100 ng/mL, or optionally 1 ng/mL-50 ng/mL or optionally 1 ng/mL-10 ng/ml, optionally 1 ng/mL-8 ng/mL of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and/or Serpin F1.
In some embodiments, the MSC secretome further comprises “mid-range” levels of at least one factor selected from the group consisting of Angiopoietin-1, Angiopoietin-2, Amphiregulin, Endostatin, Endothelin-1, Thrombospondin-2, Thrombospondin-1, Angiogenin, DPPIV, IGFBP-3, and/or uPA, optionally 400 pg/mL-3000 pg/mL. In some embodiments, the MSC secretome composition 400 pg/mL, 500 pg/mL, 600 pg/mL, 700 pg/mL, 800 pg/mL, 900 pg/mL, 1000 pg/mL, 1100 pg/mL, 1200 pg/mL, 1300 pg/mL, 1400 pg/mL, 1500 pg/mL, 1600 pg/mL, 1700 pg/mL, 1800 pg/mL, 1900 pg/mL, 2000 pg/mL, 2100 pg/mL, 2200 pg/mL, 2300 pg/mL, 2400 pg/mL, 2500 pg/mL, 2600 pg/mL, 2700 pg/mL, 2800 pg/mL, 2900 pg/mL, or 3000 pg/mL of at least one factor selected from the group consisting of Angiopoietin-1, Angiopoietin-2, Amphiregulin, Endostatin, Endothelin-1, Thrombospondin-2, Thrombospondin-1, Angiogenin, DPPIV, IGFBP-3, and uPA.
In some embodiments, the MSC secretome further comprises at least one factor selected from the group consisting of Apolipoprotein A1, Complement Factor D, Complement factor H, Complement factor I, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, Complement receptor type 1 (CR1), C-reactive protein, Cystatin C, DKK-1, Emmprin, Osteopontin, Vitamin D Binding Protein, MIF, RANTES, uPAR, IL-17a, GDF-15, and/or IFNγ.
In some embodiments, the MSC secretome comprises ratios of anti-angiogenic to pro-angiogenic wherein the ratio is >2, >3, >4, or >5.
In some embodiments, the MSC secretome further comprises “low” levels for VEGF, optionally 0 pg/mL-200 pg/mL or optionally 1 pg/mL-400 pg/mL of VEGF.
In some embodiments, the level of VEGF is 5-10 fold lower than the level of Serpin E1.
In some embodiments, the composition comprises one or more anti-angiogenic factors, and wherein the ratio of the sum of the concentration of the one or more anti-angiogenic factors relative to the concentration of VEGF is >2, >3, >4, or >5.
In some embodiments, the MSC secretome does not comprise and/or comprises very low levels of bFGF, PLGF, and PDGF, optionally less than 1000 pg/mL.
In some embodiments, the MSC secretome composition has a pH of about 4.7 to about 7.5.
In some embodiments, the MSC secretome is formulated in a buffer system selected from the group consisting of di/mono sodium phosphate, sodium citrate/citric acid, boric acid/sodium citrate, boric acid/sodium tetraborate, and citric acid/disodium phosphate.
In some embodiments, the MSC secretome composition further comprises a tonicity modifying agent.
In some embodiments, the tonicity modifying agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.
In some embodiments, the MSC secretome further comprises mono/di-sodium phosphate, mannitol, and trehalose, wherein the composition has a pH of about pH 7.4. In some embodiments, the MSC secretome further comprises divalent cations.
In some embodiments, the divalent cations are selected from the group consisting of Mg2+, Ca2+, and Zn2+.
In some embodiments, the MSC secretome further comprises di-sodium phosphate/citric acid, mannitol, and trehalose, wherein the composition has a pH of about pH 6.4.
In some embodiments, the composition further comprises an adhesive agent.
In some embodiments, the adhesive agent is selected from the group consisting of hypromellose, Poloxamer 407, Poloxamer 188, Poloxomer 237, Poloxomer 338, Hypromellose, polycarbophil, polyvinylpyrrolidone (PVP), PVA (polyvinyl alcohol), polyimide, sodium hyaluronate, gellan gum, poly(lactic acid-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), hydroxypropyl methylcellulose (HPMC), hydroxy methyl cellulose, hydroxy ethyl cellulose, sodium carboxy methyl cellulose, fibrin glue, polyethyelene glycol, and GelCORE.
In some embodiments, the MSC secretome is characterized by:
In some embodiments, the biopotency and/or stability of the MSC secretome is characterized by:
In some embodiments, the results in (ii) from a physical component characterization identify an anti-angiogenic MSC secretome in accordance with the embodiments provided herein.
In some embodiments, the results in (ii) from safety analyses provide for a MSC secretome that exhibits blood compatibility, and low and/or no pyrogens and/or endotoxins.
In some embodiments, the results in (ii) from a stability assay provides for a MSC secretome that exhibits stability at −20° C., 4° C., and/or 20° C. (for example, room temperature) for at least 7 days or for at least 14 days.
In some embodiments, the results in (ii) from a proliferation assay provides for a MSC secretome that induces proliferation.
In some embodiments, the results in (ii) from a migration assay pro-vides for a MSC secretome that induces migration.
In some embodiments, the results in (ii) from a neovascularization as-say provides for a MSC secretome that inhibits or does not promote neovascularization.
In some embodiments, the results in (ii) from a differentiation/scarring assay provides for a MSC secretome that inhibits differentiation and/or scarring.
In some embodiments, the results in (ii) from an inflammation assay provides for a MSC secretome that inhibits inflammation.
In some embodiments, the method further comprises:
In some embodiments, MSC secretome composition comprises:
In some embodiments, the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition comprising:
In some embodiments, the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition comprising:
In some embodiments, 1 U/mL of the MSC secretome composition comprises:
In some embodiments, 1 U/mL of the MSC secretome composition comprises:
In some embodiments, 1 U/mL of the MSC secretome composition comprises:
In some embodiments, 1 U/mL of the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.
In some embodiments, the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition comprising:
In some embodiments, the MSC secretome composition comprising:
In some embodiments, the MSC secretome composition comprising:
In some embodiments, the MSC secretome composition comprising:
In some embodiments, the MSC secretome composition comprising:
In some embodiments, the MSC secretome composition comprising:
In some embodiments, the MSC secretome composition comprising:
In some embodiments, the MSC secretome composition does not comprise hypromellose.
In some embodiments, the MSC secretome composition is administered topically to the affected eye(s) of the subject.
In some embodiments, the MSC secretome composition comprises about 0.1 U/mL, 0.5 U/mL, 1 U/mL, 2 U/mL, 3 U/mL, 4 U/mL, 5 U/mL, 6 U/mL, 7 U/mL, 8 U/mL, 9 U/mL, 10 U/mL, or more, of the MSC secretome.
In some embodiments, the MSC secretome composition comprises about 1 U/mL or 3 U/mL of the MSC secretome.
In some embodiments, about one, two, three, or more, drops of the MSC secretome composition is administered.
In some embodiments, a dosage of about 0.1 U, 0.5 U, 1 U, 1.5 U, 2 U, 2.5 U, 3 U, 4.5 U, 5 U, or more, of the MSC secretome is administered.
In some embodiments, a dosage of about 0.5 U or 1.5 U of the MSC secretome is administered.
In some embodiments, the MSC secretome composition is administered to the subject about 1, 2, 3, 4, 5, or up to 6 times daily.
In some embodiments, the MSC secretome composition is administered to the subject about 4 times daily.
In some embodiments, the MSC secretome composition comprising 1 U/mL of the MSC secretome is administered to the subject 4 times daily.
In some embodiments, the MSC secretome composition comprising 3 U/mL of the MSC secretome is administered to the subject 4 times daily.
In some embodiments, the treatment lasts for about 1, 2, 3, 4, 5, 6, 7, or 8 weeks, 1 month, 2 months, 3 months, 4 months, or longer.
In some embodiments, the treatment lasts for about 8 weeks.
In some embodiments, the LSCD is unilateral or bilateral. In some embodiments, the LSCD is partial or total. In some embodiments, the LSCD is at Stage 1, 2, or 3.
In some embodiments, the subject has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more lesions in the eye(s) affected by the LSCD.
In some embodiments, the subject exhibits normal corneal epithelium within the central 5 mm zone of the cornea or the visual axis in the eye(s) affected by the LSCD.
In some embodiments, the central 5 mm zone of the cornea or the visual axis is affected by the LSCD.
In some embodiments, the entire corneal surface is affected by the LSCD.
In some embodiments, the LSCD is detectable by one or more ocular examinations comprising BCVA (Best-Corrected Distance Visual Acuity), slit lamp examination, corneal photo without fluorescein, corneal photo with fluorescein, lesion size measurements, corneal sensitivity, corneal topography, or dilated ophthalmoscopy.
In some embodiments, prior to receiving the presently disclosed treatment, the subject has received and/or is refractory to one or more treatments comprising preservative free artificial tears, therapeutic contact lenses (such as bandage contact lens, the PROSE device, or scleral lens), topical Vitamin A ointment, short-term pulse topical corticosteroids (such as methylprednisolone 1%, loteprednol etabonate 0.5% or 0.2%, or prednisolone acetate 1%, and cyclosporine 0.05%), Prokera, Oxervate® (cenegermin-bkbj), limbal stem cell/limbal cell transplantation, contralateral conjunctival limbal autograft, oral mucosal epithelial transplantation, amniotic membrane transplantation, or keratoprosthesis.
In some embodiments, the LSCD is characterized by a reduction of about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%, of the limbal stem cell in the eye(s) affected as compared to an unaffected eye.
In some embodiments, the MSC secretome composition induces about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, wound healing in one or more of the lesions in the affected eye(s).
In some embodiments, the MSC secretome composition induces about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, reduction in the size of one or more of the lesions in the affected eye(s).
In some embodiments, the MSC secretome composition induces about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, increase in the population of limbal stem cells in the affected eye(s).
In some embodiments, the MSC secretome composition provided herein induces about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or more, increase in the population of limbal stem cells in the affected eye(s).
In some embodiments, the present invention provides a unit dosage formulation comprising the MSC secretome composition in accordance with the embodiments disclosed herein for treating limbal stem cell deficiency (LSCD).
In some embodiments, the formulation is prepared and packaged into a product container.
In some embodiments, the unit dosage formulation is for use in a treatment according to a method disclosed herein.
In some embodiments, the unit dosage formulation is formulated for topical administration.
In some embodiments, the formulation is prepared and packaged for administration for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or more, per day.
In some embodiments, the formulation is prepared and packaged in a dosage of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 drops, or more.
In some embodiments, the formulation is prepared and packaged in a dosage of about 0.1 U/mL, 0.5 U/mL, 1 U/mL, 2 U/mL, 3 U/mL, 4 U/mL, 5 U/mL, 6 U/mL, 7 U/mL, 8 U/mL, 9 U/mL, 10 U/mL, or more, of the MSC secretome.
In some embodiments, the formulation is prepared and packaged in a dosage of 1 U/mL, or 3 U/mL, of the MSC secretome.
In some embodiments, the formulation is sufficient to provide a dosage of 0.1 U, 0.5 U, 1 U, 1.5 U, 2 U, 2.5 U, 3 U, 4.5 U, 5 U, or more, of the MSC secretome.
In some embodiments, the formulation is sufficient to provide a dosage of 0.5 U or 1.5 U of the MSC secretome.
In some embodiments, the formulation is prepared and packaged for administration to the subject about 1, 2, 3, 4, 5, or up to 6 times daily.
In some embodiments, the formulation is prepared and packaged for administration to the subject about 4 times daily.
In some embodiments, the formulation is prepared and packaged for administration to the subject for about 1, 2, 3, 4, 5, 6, 7, or 8 weeks, 1 month, 2 months, 3 months, 4 months, or longer.
In some embodiments, the formulation is prepared and packaged for administration to the subject for about 8 weeks.
In some embodiments, the formulation is prepared and packaged for administration in a dosage of 1 U/mL, wherein 1 drop of the formulation is administered 4 times per day, optionally wherein the formulation is administered for about 8 weeks.
In some embodiments, the formulation is prepared and packaged for administration in a dosage of 3 U/mL of the MSC secretome, wherein 1 drop of the formulation is administered 4 times per day, optionally wherein the formulation is administered for about 8 weeks.
In some embodiments, the present invention provides a kit comprising the MSC secretome composition.
In some embodiments, the present invention provides use of the MSC secretome composition, or the unit dosage formulation, or the kit, for treating limbal stem cell deficiency (LSCD) in a subject in need thereof, according to any one of the preceding claims.
In some embodiments, the present invention provides for a method of making a anti-angiogenic mesenchymal stem cell (MSC) secretome composition comprising:
In some embodiments, culturing can be performed using a bioreactor system for culturing cells. In some embodiments, culturing can be performed using a bioreactor system for culturing stem cells. In some embodiments, culturing can be performed using a bioreactor system for culturing mesenchymal stem cells. In some embodiments, culturing can be performed using a media mixing technology. In some embodiments, culturing can be performed using a PBS Vertical Wheel™ Mixing Technology.
In some embodiments, in step (iv) processing the conditioned media in step (v) into the secretome composition comprises:
In some embodiments, step (c) comprises buffer exchanging with a buffer system selected from the group consisting of di/mono sodium phosphate, sodium citrate/citric acid, boric acid/sodium citrate, boric acid/sodium tetraborate, and citric acid/disodium phosphate.
In some embodiments, the filtering step (a) comprises the use of a 0.45 μm filter, a 0.22 μm filter, 0.8 μm filter, and 0.65 μm filter, a low protein binding PVDF membranes, and/or PES (polyethersulfone).
In some embodiments, the concentration step (b) comprises using hollow fiber filters, tangential flow filtration systems, or centrifugation based size exclusion techniques.
In some embodiments, centrifugation based size exclusion techniques employs a 3-10 kDa MW cutoff.
In some embodiments, the present invention provides a method of treatment of an ocular disease comprising administering to a patient in need thereof therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein or a composition made according to the methods described herein to a patient in need thereof.
In some embodiments, the composition is administered to a target area.
The present invention also provides a method for treating visual dysfunction following traumatic injury to ocular structures in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein or a composition made according to the methods described herein.
The present invention also provides a method for inducing and/or promoting ocular wound healing in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein or a composition made according to the methods described herein.
The present invention also provides a method for reducing and/or inhibiting neovascularization, reducing and/or inhibiting scarring, promoting and/or preserving vision, and/or increasing wound closure rate (e.g., decreasing would closure time) in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein or a composition made according to the methods described herein.
The present invention also provides a method for reducing and/or inhibiting neovascularization and reducing scarring in order to promote vision preservation in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein or a composition made according to the methods described herein.
In some embodiments, the mesenchymal stem cell secretome composition is formulated for topical administration.
In some embodiments, the mesenchymal stem cell secretome composition is formulated for subconjunctival injection.
The present invention also provides a method for characterizing a MSC secretome, wherein the method comprises:
The present invention also provides a method determining biopotency and stability of a MSC secretome comprising, wherein the method comprises:
The present invention also provides a method for determining MSC secretome lot consistency between a plurality of MSC secretome lots, wherein the method comprises:
In some embodiments, the results in (ii) from a physical component characterization identify an anti-angiogenic MSC secretome as described herein.
In some embodiments, the results in (ii) from safety analyses provide for a MSC secretome that exhibits blood compatibility, and low and/or no pyrogens and/or endotoxins.
In some embodiments, the results in (ii) from a stability assay provides for a MSC secretome that exhibits stability at 4° C., 20° C., and/or 25° C. (or room temperature) for at least 7 days.
In some embodiments, the results in (ii) from a proliferation assay provides for a MSC secretome that induces proliferation.
In some embodiments, the results in (ii) from a migration assay provides for a MSC secretome that induces migration.
In some embodiments, the results in (ii) from a neovascularization assay provides for a MSC secretome that inhibits or does not promote neovascularization.
In some embodiments, the results in (ii) from a differentiation/scarring assay provides for a MSC secretome that inhibits differentiation and/or scarring.
In some embodiments, the results in (ii) from an inflammation assay provides for a MSC secretome that inhibits inflammation.
In some embodiments, the method further comprises:
The present invention also provides a panel of tests and/or assays for characterizing a MSC secretome, wherein the panel comprises at least two characterization assays, wherein characterization assays are selected from the group consisting of physical component characterizations, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, neovascularization assays, differentiation/scarring assays, and/or inflammation assays.
The present invention also provides a panel of tests and/or assays for determining consistency between MSC secretome lots, wherein the panel comprises one or more characterization assays, wherein characterization assays are selected from the group consisting of physical component characterizations, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, neovascularization assays, differentiation/scarring assays, and/or inflammation assays.
In some embodiments, the physical component characterization identifies a MSC secretome as described herein.
In some embodiments, the results in (ii) from safety analyses provide for a MSC secretome that exhibits blood compatibility, and low and/or no pyrogens and/or endotoxins.
In some embodiments, the stability assay identifies for a MSC secretome that exhibits stability at 4° C., 20° C., and/or 25° C. (or room temperature) for at least 7 days.
In some embodiments, the proliferation assay identifies for a MSC secretome that induces proliferation.
In some embodiments, the migration assay identifies a MSC secretome that induces migration.
In some embodiments, the neovascularization assay identifies for a MSC secretome that inhibits or does not promote neovascularization.
In some embodiments, the differentiation/scarring assay identifies a MSC secretome that inhibits differentiation and/or scarring.
In some embodiments, the inflammation assay identifies a MSC secretome that inhibits inflammation.
In some embodiments, the physical component characterizations, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, neovascularization assays, differentiation/scarring assays, inflammation assays, and/or epithelial barrier integrity assays are all performed.
In some embodiments, the panel of tests and/or assays as described herein identify a MSC secretome as described herein.
In some embodiments, the panel of tests and/or assays as described herein includes at least one migration assay. In some embodiments, the migration assay is an in vitro wound closure assay. In some embodiments, the in vitro wound closure assay is selected from the group consisting of a “scratch assay” (also referred to as a “scratch wound assay”), a circular scratch wound method, a circular scratch wound assay, and a circular wound closure assay. In some embodiments, the MSC secretome is an anti-angiogenic MSC secretome and/or an anti-scarring MSC secretome.
In some embodiments, the MSC secretome is an anti-angiogenic MSC secretome and/or or an anti-scarring MSC secretome.
In some embodiments, the MSC secretome is an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.
The present invention also provides a stable mesenchymal stem cell (MSC) secretome formulation comprising:
The present invention also provides a stable mesenchymal stem cell (MSC) secretome formulation comprising:
The present invention also provides a stable mesenchymal stem cell (MSC) secretome formulation comprising:
The present invention also provides a stable mesenchymal stem cell (MSC) secretome formulation comprising:
The present invention also provides a mesenchymal stem cell (MSC) secretome composition comprising:
In some embodiments, the MSC secretome composition further comprises high levels of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1.
In some embodiments, the MSC secretome composition comprises 1 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1.
In some embodiments, the MSC secretome composition further comprises mid-range levels of at least one factor selected from the group consisting of Angiopoietin-1, Angiopoietin-2, Amphiregulin, Endostatin, Endothelin-1, Thrombospondin-2, Thrombospondin-1, Angiogenin, DPPIV, IGFBP-3, and uPA.
In some embodiments, the MSC secretome composition 400 pg/mL-3000 pg/mL of at least one factor selected from the group consisting of Angiopoietin-1, Angiopoietin-2, Amphiregulin, Endostatin, Endothelin-1, Thrombospondin-2, Thrombospondin-1, Angiogenin, DPPIV, IGFBP-3, and uPA.
In some embodiments, the MSC secretome composition 400 pg/mL, 500 pg/mL, 600 pg/mL, 700 pg/mL, 800 pg/mL, 900 pg/mL, 1000 pg/mL, 1100 pg/mL, 1200 pg/mL, 1300 pg/mL, 1400 pg/mL, 1500 pg/mL, 1600 pg/mL, 1700 pg/mL, 1800 pg/mL, 1900 pg/mL, 2000 pg/mL, 2100 pg/mL, 2200 pg/mL, 2300 pg/mL, 2400 pg/mL, 2500 pg/mL, 2600 pg/mL, 2700 pg/mL, 2800 pg/mL, 2900 pg/mL, or 3000 pg/mL of at least one factor selected from the group consisting of Angiopoietin-1, Angiopoietin-2, Amphiregulin, Endostatin, Endothelin-1, Thrombospondin-2, Thrombospondin-1, Angiogenin, DPPIV, IGFBP-3, and uPA.
In some embodiments, the MSC secretome composition further comprises at least one factor selected from the group consisting of Apolipoprotein A1, Complement Factor D, Complement factor H, Complement factor I, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, Complement receptor type 1 (CR1), C-reactive protein, Cystatin C, DKK-1, Emmprin, Osteopontin, Vitamin D Binding Protein, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.
In some embodiments, the MSC secretome composition comprises ratios of anti-angiogenic to pro-angiogenic wherein the ratio is >2, >3, >4, or >5.
In some embodiments of the MSC secretome composition the anti-angiogenic factors includes one or more factors selected from the group consisting of PEDF, lower levels of VEGF, and Serpin E1 and the pro-angiogenic factors includes one or more factors selected from the group consisting of VEGF, Angiogenin, IGFBP-3, uPA, Angio-1, Angio-2, Endothelin-1.
In some embodiments, the MSC secretome composition further comprises low levels for VEGF.
In some embodiments, the MSC secretome composition comprises 1 pg/mL-400 pg/mL of VEGF. In some embodiments, the MSC secretome composition comprises 1 pg/mL, 5 pg/mL, 10 pg/mL, 20 pg/mL, 30 pg/mL, 40 pg/mL, 50 pg/mL, 60 pg/mL, 70 pg/mL, 80 pg/mL, 90 pg/mL, 100 pg/mL, 110 pg/mL, 120 pg/mL, 130 pg/mL, 140 pg/mL, 150 pg/mL, 160 pg/mL, 170 pg/mL, 180 pg/mL, 190 pg/mL, 200 pg/mL, 210 pg/mL, 220 pg/mL, 230 pg/mL, 240 pg/mL, 250 pg/mL, 260 pg/mL, 270 pg/mL, 280 pg/mL, 290 pg/mL, 300 pg/mL, 310 pg/mL, 320 pg/mL, 330 pg/mL, 340 pg/mL, 350 pg/mL, 360 pg/mL, 370 pg/mL, 380 pg/mL, 390 pg/mL, or 400 pg/mL of VEGF.
In some embodiments of the MSC secretome composition the level of VEGF is 5-10 fold lower than the level of Serpin E1. In some embodiments of the MSC secretome composition the level of VEGF is 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold lower than the level of Serpin E1.
In some embodiments, the MSC secretome composition comprises one or more anti-angiogenic factors, and wherein the ratio of the sum of the concentration of the one or more anti-angiogenic factors relative to the concentration of VEGF is >2, >3, >4, or >5.
In some embodiments, the MSC secretome composition does not comprise and/or comprises very low levels of bFGF, PLGF, and PDGF.
In some embodiments, the MSC secretome composition comprises less than 1000 pg/mL of bFGF, PLGF, and PDGF.
In some embodiments, the MSC secretome composition has a pH of about 4.7 to about 7.5.
In some embodiments, the MSC secretome composition is formulated in a buffer system selected from the group consisting of di/mono sodium phosphate, sodium citrate/citric acid, boric acid/sodium citrate, boric acid/sodium tetraborate, and citric acid/disodium phosphate.
In some embodiments, the MSC secretome composition further comprises a tonicity modifying agent.
In some embodiments of the MSC secretome composition the tonicity modifying agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.
In some embodiments, the MSC secretome composition further comprises mono/di-sodium phosphate, mannitol, and trehalose, and wherein the composition has a pH of about pH 7.4.
In some embodiments, the MSC secretome composition further comprises divalent cations.
In some embodiments, the MSC secretome composition the divalent cations are selected from the group consisting of Mg2+, Ca2+, and Zn2+.
In some embodiments, the MSC secretome composition further comprises di-sodium phosphate/citric acid, mannitol, and trehalose, wherein the composition has a pH of about pH 6.4.
In some embodiments, the MSC secretome composition further comprises an adhesive agent.
In some embodiments of the MSC secretome composition the adhesive agent is selected from the group consisting of hypromellose, Poloxamer 407, Poloxamer 188, Poloxomer 237, Poloxomer 338, Hypromellose, polycarbophil, polyvinylpyrrolidone (PVP), PVA (polyvinyl alcohol), polyimide, sodium hyaluronate, gellan gum, poly(lactic acid-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), hydroxypropyl methylcellulose (HPMC), hydroxy methyl cellulose, hydroxy ethyl cellulose, sodium carboxy methyl cellulose, fibrin glue, polyethyelene glycol, and GelCORE.
In some embodiments, the MSC secretome composition does not comprise one or more components selected from the group consisting of: xenobiotic components; Phenol red; peptides and biomolecules <3 kDa; antibiotics; protein aggregates >200 nm; cells; non-exosome/non-Extracellular Vesicles cell debris; hormones; and L-glutamine.
In some embodiments, the MSC secretome composition comprises: HGF; Pentraxin-3 (TSG-14); VEGF; TIMP-1; Serpin E1; and <5 ng/mL IL-8.
In some embodiments, the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.
The present invention also provides a stable mesenchymal stem cell (MSC) secretome formulation comprising:
The present invention also provides a stable mesenchymal stem cell (MSC) secretome formulation comprising:
The present invention further provides a method of treatment for an ocular condition in a subject in need thereof comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition further comprises high levels of at least one factor selected from the group consisting of Serpin E1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1.
In some embodiments, the MSC secretome composition comprises 1 ng/ml-100 ng/ml of at least one factor selected from the group consisting of Serpin E1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1.
In some embodiments, the MSC secretome composition further comprises mid-range levels of at least one factor selected from the group consisting of Angiopoietin-1, Angiopoietin-2, Amphiregulin, Endostatin, Endothelin-1, Thrombospondin-2, Thrombospondin-1, Angiogenin, DPPIV, IGFBP-3, and uPA.
In some embodiments, the MSC secretome composition comprises 400 pg/mL-3000 pg/mL of at least one factor selected from the group consisting of Angiopoietin-1, Angiopoietin-2, Amphiregulin, Endostatin, Endothelin-1, Thrombospondin-2, Thrombospondin-1, Angiogenin, DPPIV, IGFBP-3, and uPA.
In some embodiments, the MSC secretome composition further comprises at least one factor selected from the group consisting of Apolipoprotein A1, Complement Factor D, Complement factor H, Complement factor I, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, Complement receptor type 1 (CR1), C-reactive protein, Cystatin C, DKK-1, Emmprin, Osteopontin, Vitamin D Binding Protein, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.
In some embodiments, the MSC secretome composition comprises ratios of anti-angiogenic to pro-angiogenic wherein the ratio is >2, >3, >4, or >5.
In some embodiments, the anti-angiogenic factors include one or more factors selected from the group consisting of PEDF, lower levels of VEGF, and Serpin E1 and pro-angiogenic: VEGF, Angiogenin, IGFBP-3, uPA, Angio-1, Angio-2, Endothelin-1.
In some embodiments, the MSC secretome composition further comprises low levels for VEGF.
In some embodiments, the MSC secretome comprises 1 pg/mL-400 pg/mL of VEGF.
In some embodiments, the level of VEGF is 5-10 fold lower than the level of Serpin E1. In some embodiments of the MSC secretome composition the level of VEGF is 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold lower than the level of Serpin E1.
In some embodiments, the MSC secretome composition comprises one or more anti-angiogenic factors, and wherein the ratio of the sum of the concentration of the one or more anti-angiogenic factors relative to the concentration of VEGF is >2, >3, >4, or >5.
In some embodiments, the MSC secretome composition does not comprise or comprises very low levels of bFGF, PLGF, and PDGF.
In some embodiments, the MSC secretome composition comprises less than 1000 pg/mL of bFGF, PLGF, and PDGF.
In some embodiments, the MSC secretome composition has a pH of about 4.7 to about 7.5.
In some embodiments, the MSC secretome composition is formulated in a buffer system selected from the group consisting of di/mono sodium phosphate, sodium citrate/citric acid, boric acid/sodium citrate, boric acid/sodium tetraborate, and citric acid/disodium phosphate.
In some embodiments, the MSC secretome composition further comprises a tonicity modifying agent.
In some embodiments, the tonicity modifying agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.
In some embodiments, the MSC secretome composition further comprises mono/di-sodium phosphate, mannitol, and trehalose, and wherein the composition has a pH of about pH 7.4.
In some embodiments, the MSC secretome composition further comprises divalent cations.
In some embodiments, the divalent cations are selected from the group consisting of Mg2+, Ca2+, and Zn2+.
In some embodiments, the MSC secretome composition further comprises di-sodium phosphate/citric acid, mannitol, and trehalose, and wherein the composition has a pH of about pH 6.4.
In some embodiments, the MSC secretome composition further comprises an adhesive agent.
In some embodiments, the adhesive agent is selected from the group consisting of hypromellose, Poloxamer 407, Poloxamer 188, Poloxomer 237, Poloxomer 338, Hypromellose, polycarbophil, polyvinylpyrrolidone (PVP), PVA (polyvinyl alcohol), polyimide, sodium hyaluronate, gellan gum, poly(lactic acid-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), hydroxypropyl methylcellulose (HPMC), hydroxy methyl cellulose, hydroxy ethyl cellulose, sodium carboxy methyl cellulose, fibrin glue, polyethyelene glycol, and GelCORE.
In some embodiments, the MSC secretome composition does not comprise one or more components selected from the group consisting of: xenobiotic components; Phenol red; peptides and biomolecules <3 kDa; antibiotics; protein aggregates >200 nm; cells; non-exosome/non-Extracellular Vesicles cell debris; hormones; and L-glutamine.
In some embodiments, the MSC secretome composition comprises: HGF; Pentraxin-3 (TSG-14); VEGF; TIMP-1; Serpin E1; and <5 ng/ml IL-8.
In some embodiments, the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition comprise an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.
The present invention also provides a method of treatment for an ocular condition in a subject in need thereof comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition is a stable mesenchymal stem cell (MSC) secretome formulation comprising:
The present invention also provides a method of treatment for an ocular condition in a subject in need thereof comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition is a stable mesenchymal stem cell (MSC) secretome formulation comprising:
The present invention also provides a stable mesenchymal stem cell (MSC) secretome formulation comprising:
The present invention also provides a stable mesenchymal stem cell (MSC) secretome formulation comprising:
The present invention also provides a stable mesenchymal stem cell (MSC) secretome formulation comprising:
The present invention also provides a stable mesenchymal stem cell (MSC) secretome formulation comprising:
The present invention also provides a stable mesenchymal stem cell (MSC) secretome formulation comprising:
The present invention also provides a stable mesenchymal stem cell (MSC) secretome formulation comprising:
In some embodiments of the stable mesenchymal stem cell (MSC) secretome formulation the formulation does not comprise hypromellose.
The present invention also provides a method of treatment for an ocular condition in a subject in need thereof comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition is a stable mesenchymal stem cell (MSC) secretome formulation comprising:
The present invention also provides a method of treatment for an ocular condition in a subject in need thereof comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition is a stable mesenchymal stem cell (MSC) secretome formulation comprising:
In some embodiments of method of treatment for an ocular condition, the MSC secretome composition and/or formulation used for the method of treatment does not comprise hypromellose.
In some embodiments of the methods described herein, the MSC secretome composition and/or formulation does not comprise hypromellose.
In some embodiments of the MSC secretome composition and/or formulation, the composition and/or formulation does not comprise hypromellose.
Limbal stem cell deficiency (LSCD) is a disease in which stem cells are partially or completely depleted, leaving the corneal epithelium of the eye unable to repopulate and maintain a stable ocular environment. LSCD can result from any condition or injury that decreases or eliminates the number of stem cells present in the eye. LSCD can be caused by chemical or mechanical injury, chronic contact lens use, infectious disease, autoimmune disorders, or other traumatizing events impacting the eye. LSCD can be partial (stem cells still present, but with diminished regenerative capacity) or complete (no stem cells remaining).
Presentation of LSCD overlaps with other conditions that can affect the eye, making differential diagnosis important. Patients with LSCD may present with ocular pain, irritation, light sensitivity, or vision disturbances. Diagnosis of LSCD consists of a clinical slit lamp examination and examination of the ocular epithelial surface for characteristic waterfall or whirling patterns. A hazy epithelium, scarring, and irregular ocular reflexes are all surface signs of LSCD. Diagnostic testing for LSCD is completed by in vivo confocal microscopy or corneal impression cytology for detection of goblet cells.
Treatment for LSCD depends on whether it is partial, complete, unilateral, or bilateral. For partial LSCD, there are multiple treatment modalities including lubrication and use of topical steroids. Scraping of the hazy, damaged epithelium may be performed to encourage generation of normal epithelium. For complete unilateral LSCD, contralateral conjunctival limbal autografts can be taken and applied to the eye with LSCD. For bilateral LSCD, keratolimbal allografts can be utilized in conjunction with immunosuppressive therapy to prevent rejection. If grafts are the chosen treatment, surface restoration is completed at the same time as the grafting procedure or shortly thereafter with intensive ocular lubrication. If grafting is not an option, keratoprosthesis may be introduced (American Academy of Ophthalmology, 2014; Kate, 2022).
Due to many different conditions and underlying causes that can contribute to the development of LSCD, treatments that can address multiple concerns associated with the condition are needed. It is clear patients would benefit from less invasive treatments targeted toward supporting regeneration of the stem cell population. The present disclosure provides herein a mesenchymal stem cell secretome that contains paracrine factors associated with limbal stem cell growth and maintenance. The product is being developed as a topical eye drop for treatment of LSCD.
The present invention provides mesenchymal stem cell secretome compositions for use in such treatments of LSCD, as well as methods for making such compositions. Such compositions, uses, and associated methods are described in further detail below.
Terms used in the claims and specification are defined as set forth below unless otherwise specified. In the case of direct conflict with a term used in a parent provisional patent application, the term used in the instant specification shall control.
“Limbal stem or progenitor cells” or “LSCs” include stem cells obtained from, e.g., the limbus, a region between cornea and conjunctiva of an eye. LSCs can proliferate and differentiate to give rise to corneal epithelial cells (CECs). In particular, LSCs are thought to be enriched in the LSC niche within the limbus. LSCs can be isolated from the limbus region comprising corneal limbus of an eye, margin between cornea and conjunctiva, border of cornea and sclera, corneoscleral limbus, a crypt region of the basal layer of limbal epithelium, a region comprising interpalisade rete ridge, or a region comprising Palisades of Vogt.
The limbus is not the only niche for LSCs or corneal stem cells, and studies have shown that LSCs were observed to reside in the ocular tissue outside the limbus, such as ocular surface including the cornea. See, Majo F, Rochat A, Nicolas M, Jaoudé G A, Barrandon Y. Nature. 2008 Nov. 13; 456(7219):250-4.
As used herein, “limbal stem cell deficiency” (LSCD), or sometimes referred to as “limbal epithelial cell deficiency” (LECD), includes but is not limited to severe or total (or complete), unilateral or partial LSCD or LECD, such as occurs in chemical or thermal injury, Steven-Johnson syndrome, contact lens-induced keratopathy, ocular cicatricial pemphigoid, congenital diseases of aniridia or ectodermal dysplasia, and multiple endocrine deficiency-associated keratitis.
As used herein “isolated” refers to material removed from its original environment and is thus altered “by the hand of man” from its natural state.
As used herein, “enriched” means to selectively concentrate or to increase the amount of one or more materials by elimination of the unwanted materials or selection and separation of desirable materials from a mixture (e.g., separate cells with specific cell markers from a heterogeneous cell population in which not all cells in the population express the marker).
As used herein, the term “substantially purified” means a population of cells substantially homogeneous for a particular marker or combination of markers. By substantially homogeneous is meant at least 90%, and preferably 95% homogeneous for a particular marker or combination of markers. As used herein, the term “multipotent stem cells” are true stem cells but can only differentiate into a limited number of types. For example, the bone marrow contains multipotent stem cells that give rise to all the cells of the blood but may not be able to differentiate into other cell types.
By the term “animal-free” when referring to certain compositions, growth conditions, culture media, etc. described herein, is meant that no non-human animal-derived materials, such as bovine serum, proteins, lipids, carbohydrates, nucleic acids, vitamins, etc., are used in the preparation, growth, culturing, expansion, storage or formulation of the certain composition or process. By “no non-human animal-derived materials” is meant that the materials have never been in or in contact with a non-human animal body or substance so they are not xeno-contaminated. Generally, clinical grade materials, such as recombinantly produced human proteins, are used in the preparation, growth, culturing, expansion, storage and/or formulation of such compositions and/or processes.
By the term “expanded”, in reference to cell compositions, means that the cell population constitutes a significantly higher concentration of cells than is obtained using previous methods. For example, the level of cells per gram of amniotic tissue in expanded compositions of AMP cells is at least 50-fold and up to 150-fold higher than the number of cells in the primary culture after 5 passages, as compared to about a 20-fold increase in such cells using previous methods. In another example, the level of cells per gram of amniotic tissue in expanded compositions of AMP cells is at least 30-fold and up to 100-fold higher than the number of cells in the primary culture after 3 passages. Accordingly, an “expanded” population has at least a 2-fold, and up to a 10-fold, improvement in cell numbers per gram of amniotic tissue over previous methods. The term “expanded” is meant to cover only those situations in which a person has intervened to elevate the number of the cells.
As used herein, “conditioned medium” is a medium in which a specific cell or population of cells has been cultured, and then removed. When cells are cultured in a medium, they may secrete cellular factors that can provide support to or affect the behavior of other cells. Such factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, chemokines, receptors, inhibitors and granules. The medium containing the cellular factors is the conditioned medium. Examples of methods of preparing conditioned media have been described in U.S. Pat. No. 6,372,494 which is incorporated by reference in its entirety herein. As used herein, conditioned medium also refers to components, such as proteins, that are recovered and/or purified from conditioned medium or from for example, MSC cells.
As used herein, the term “mesenchymal stem cell composition” or “MSC composition” means conditioned medium that has been derived from MSCs and in some instances has undergone further processing. In some embodiments, “MSC secretome” can refer to the crude conditioned media derived from the MSC. In some embodiments, “MSC secretome” can refer to the composition obtained from the crude conditioned media after it has been subjected to further processing as described herein.
As used herein, the term “suspension” means a liquid containing dispersed components, e.g., cytokines. The dispersed components may be fully solubilized, partially solubilized, suspended or otherwise dispersed in the liquid. Suitable liquids include, but are not limited to, water, osmotic solutions such as salt and/or sugar solutions, cell culture media, and other aqueous or non-aqueous solutions.
“Amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes.
An “amino acid substitution” refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (an amino acid sequence of a starting polypeptide) with a second, different “replacement” amino acid residue. An “amino acid insertion” refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, the present larger “peptide insertions,” can be made, e.g. insertion of about three to about five or even up to about ten, fifteen, or twenty amino acid residues. The inserted residue(s) may be naturally occurring or non-naturally occurring as disclosed above. An “amino acid deletion” refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.
“Polypeptide,” “peptide”, and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., Biol. Chem. 260:2605-2608, 1985; and Cassol et al, 1992; Rossolini et al, Mol. Cell. Probes 8:91-98, 1994). For arginine and leucine, modifications at the second base can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene. Polynucleotides used herein can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
As used herein, the term “secretome composition” refers to a composition comprising one or more substances which are secreted from a cell. In certain embodiments, a secretome composition may include one or more cytokines, one or more exosomes, and/or one or more microvesicles. A secretome composition may be purified or unpurified. In some embodiments, a secretome composition may further comprise one or more substances that are not secreted from a cell (e.g., culture media, additives, nutrients, etc.). In some a secretome composition does not comprise and or comprises only trace amounts of one or more substances that are not secreted from a cell (e.g., culture media, additives, nutrients, etc.).
The terms “treatment,” “treat,” or “treating,” and the like, as used herein covers any treatment of a human or nonhuman mammal (e.g., rodent, cat, dog, horse, cattle, sheep, and primates etc.), and includes preventing the disease or condition from occurring in a subject who may be predisposed to the disease or condition but has not yet been diagnosed as having it. It also includes inhibiting (arresting development of), relieving or ameliorating (causing regression of), or curing (permanently stopping development or progression) the disease, condition and/or any related symptoms. The terms “treatment,” “treat,” or “treating,” as used herein covers any treatment of a disease or condition of a mammal, particularly a human, and includes: (a) preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition, e.g., arresting its development; (c) relieving and or ameliorating the disease or condition, e.g., causing regression of the disease or condition; or (d) curing the disease or condition, e.g., stopping its development or progression. The population of subjects treated by the methods of the invention includes subjects suffering from the undesirable condition or disease, as well as subjects at risk for development of the condition or disease. In some embodiments, “treatment” (also “treat” or “treating”) refers to any administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder, and/or condition, and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively and/or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.
As used herein, a “wound” is any disruption, from whatever cause, of normal anatomy (internal and/or external anatomy) including but not limited to traumatic injuries such as mechanical (e.g. contusion, penetrating), thermal, chemical, electrical, radiation, concussive and incisional injuries; elective injuries such as operative surgery and resultant incisional hernias, fistulas, etc.; acute wounds, chronic wounds, infected wounds, and sterile wounds, as well as wounds associated with disease states (e.g. ocular contusion). A wound is dynamic and the process of healing is a continuum requiring a series of integrated and interrelated cellular processes that begin at the time of wounding and proceed beyond initial wound closure through arrival at a stable wound closure. These cellular processes are mediated or modulated by humoral substances including but not limited to cytokines, lymphokines, growth factors, and hormones. In accordance with the subject invention, “wound healing” refers to improving, by some form of intervention, the natural cellular processes and humoral substances of tissue repair such that healing is faster, and/or the resulting healed area has less scaring and/or the wounded area possesses tissue strength that is closer to that of uninjured tissue and/or the wounded tissue attains some degree of functional recovery.
As used herein, the terms “a” or “an” means one or more or at least one.
As used herein, a “therapeutically effective” or “effective” dosage or amount of a composition is an amount sufficient to have a positive effect on a given medical condition. If not immediate, the therapeutically effective or effective dosage or amount may, over period of time, provide a noticeable or measurable effect on a patient's health and well-being.
As used herein a “pharmaceutical composition” refers to an effective amount of the compositions described herein in combination with a delivery component. The pharmaceutical composition may optionally contain other components such as pharmaceutically suitable carriers and excipients, which may facilitate administration of a composition and/or its individual components to a subject.
The term “pharmaceutically acceptable carrier” refers to a carrier or a diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered compounds.
The term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.
As used herein, the terms “mix”, “mixing”, and the like describe a mechanical process or a mechanical treatment of the components. For example, mixing can be in the sense of carrying out repeated cycles of pressing and folding or comparable processing steps which lead to an intense compression and mixing of the provided hydrophobic matrices.
Adult stem cells can be harvested from a variety of adult tissues, including bone marrow, fat, and dental pulp tissue. While all adult stem cells are cable of self-renewal and are considered multipotent, their therapeutic functions vary depending on their origin. As a result, each type of adult stem cell has unique characteristics that make them suitable for certain diseases. Mesenchymal stem cells (MSCs) are typically derived from the mesoderm and are multipotent, nonhematopoietic (non-blood) stem cells isolated from (derived from) capable of differentiating into a variety of tissues, including osteoblasts (e.g., bone cells), chondrocytes (e.g., cartilage cells), myocytes (e.g., muscle cells) and adipocytes (e.g., fat cells which give rise to marrow adipose tissue). As used herein, “isolated” refers to cells removed from their original environment. Stem cells produce factors, such as growth factors, that regulate or are important for regulating multiple biological processes. A growth factor is an agent, such as a naturally occurring substance capable of stimulating cellular growth and/or proliferation and/or cellular differentiation. Typically, growth factors are proteins or steroid hormones. While the terms “growth factor” and “factor” and the like are used interchangeably herein, the term “biological factor” is not limited to growth factors.
Human mesenchymal stem cells (MSCs) can be characterized by the surface marker profile of CD45−/CD31−/CD73+/CD90+/CD105+/CD44+ (or any suitable subset thereof). (See Bourin et al., Cytotherapy 15(6):641-648 (2013)). Further, appropriate stem cells display the CD34+ positive at the time of isolation, but lose this marker during culturing. Therefore, the full marker profile for one stem cell type that may be used according to the present application includes CD45−/CD31−/CD73+/CD90+/CD105+. In another embodiment utilizing mouse stem cells, the stem cells are characterized by the Sca-1 marker, instead of CD34, to define what appears to be a homologue to the human cells described above, with the remaining markers remaining the same.
The phrase “conditioned medium” or “CM” refers to media which includes biological factors secreted by MSCs. This can also be referred to herein as the “secretome”, “MSC-CM”, “MSC secretome” and/or “MSC derived secretome”. Also provided are processed “conditioned medium” which included biological factors secreted by MSCs and which has been further processed by, for example, filtration, purification, and/or concentration procedures. The “conditioned medium” is obtained by culturing stem cells in media, as described herein in detail, and separating the resulting media, which contains stem cells and their secreted stem cell products (secretome) into conditioned medium that contains biological factors and fewer stem cells than were present prior to separation. The conditioned medium may be used in the methods described herein and is substantially free of stem cells (may contain a small percentage of stem cells) or free of stem cells. Biological factors that may be in the conditioned medium include, but are not limited to, proteins (e.g., cytokines, chemokines, growth factors, enzymes), nucleic acids (e.g., miRNA), lipids (e.g., phospholipids), polysaccharides, and/or combinations thereof. Any combination(s) of these biological factors may be either bound within or on the surface of extracellular vesicles (e.g., exosomes) or separate from extracellular vesicles.
According to the present description, compositions comprising conditioned medium comprising mesenchymal stem cell (MSC) secretome and/or mesenchymal stem cell (MSC) secretome (including processed MSC secretome) are provided herein.
In some embodiments, the MSC secretome is generally low for angiogenic factors. In some embodiments, the MSC secretome does not promote angiogenesis. In some embodiments, the MSC secretome exhibits anti-angiogenic properties. In some embodiments, the MSC secretome provides for reduced angiogenesis as compared to other secretome. In some embodiments, the MSC secretome provides for a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in angiogenesis. In some embodiments, the MSC secretome provides for a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in angiogenesis as compared to another secretome. In some embodiments, the MSC secretome provides for a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in angiogenesis as compared to the conditioned media prior to processing into the MSC secretome. In some embodiments, the MSC secretome has low angiogenesis induction. In some embodiments, the MSC secretome has reduced angiogenic response. In some embodiments, the MSC secretome has reduced angiogenic capacity. In some embodiments, the MSC secretome impairs and/or reduces the normal formation of blood vessels in presence of media supportive of angiogenesis. In some embodiments, the MSC secretome has reduced angiogenic capacity when the MSC secretome is compared to untreated control. In some embodiments, the MSC secretome has reduced angiogenic capacity as compared to a sample treated with serum containing media. In some embodiments, the MSC secretome attenuates an angiogenic response. In some embodiments, the MSC secretome reduces the angiogenic response induce by serum containing media. In some embodiments, a reduction in angiogenic response is induced by the MSC secretome when secretome plus serum containing media (reduced or no angiogenic response) is compared to serum containing media (angiogenic response). In some embodiments, an angiogenic response is indicated by tube formation in a cell based assay. In some embodiments, an angiogenic response is indicated by tube formation in an endothelial cell tube formation assay. In some embodiments, an angiogenic response is indicated by blood vessel formation in a CAM (Chick Chorioallantoic membrane) assay. In some embodiments, an angiogenic response is indicated by blood vessel formation in any blood vessel formation assay known in the art.
In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises:
In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises:
A mesenchymal stem cell (MSC) secretome composition comprising:
In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor which includes but is not limited to Apolipoprotein A1, Complement Factor D, Complement factor H, Complement factor I, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, Complement receptor type 1 (CR1), C-reactive protein, Cystatin C, DKK-1, Emmprin, Osteopontin, Vitamin D Binding Protein, MIF, RANTES, uPAR, IL-17a, GDF-15, and/or IFNγ. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor selected from the group consisting of Apolipoprotein A1, Complement Factor D, Complement factor H, Complement factor I, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, Complement receptor type 1 (CR1), C-reactive protein, Cystatin C, DKK-1, Emmprin, Osteopontin, Vitamin D Binding Protein, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.
In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor which includes but is not limited to a serpin family member, including serine protease inhibitors: Serpin F1, Serpin E1, Serpin A1, Serpin G1, Serpin H1, Serpin B6, Serpin E2, Serpin A3, Serpin C1, Serpin F2, Serpin I1), Serpin B1, Serpin B7, Serpin D1, Serpin B3, Serpin B8, Serpin B2, Serpin B12, Serpin A7, Serpin A4, and/or Serpin A6. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor which includes but is not limited to Serpin F1 (also referred to as PEDF), Serpin E1, and Serpin A1. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises Serpin F1 (also referred to as PEDF). In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises Serpin E1. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises Serpin A1.
In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor which includes but is not limited to proteins involved in anti-oxidation. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor which includes but is not limited to Catalase, Protein disulfide-isomerase, Protein disulfide-isomerase A3, Protein disulfide-isomerase A4, Protein disulfide-isomerase A6, Peroxiredoxin-6, Peroxiredoxin-1, Peroxiredoxin-2, and/or Peroxiredoxin-4. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises Catalase. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises Protein disulfide-isomerase. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises Protein disulfide-isomerase A3. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises Protein disulfide-isomerase A4. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises Protein disulfide-isomerase A6. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises Peroxiredoxin-6. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises Peroxiredoxin-1. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises Peroxiredoxin-2. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises Peroxiredoxin-4.
In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor which includes but is not limited to a matrix metalloproteinases. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor which includes but is not limited to MMP2, MMP1, and/or MMP14. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises MMP2. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises MMP1. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises MMP14.
In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor which includes but is not limited to a protein selected from the group consisting of soluble scavenger receptor cysteine-rich domain-containing protein SSC5D, tumor necrosis factor-inducible gene 6 protein (aka TSG-6), serum albumin, and latent transforming growth factor binding protein (LTGFBP-1), including various isoforms, LTGFBP-2, LTGFBP-3, and LTGFBP-4. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises soluble scavenger receptor cysteine-rich domain-containing protein SSC5D. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises tumor necrosis factor-inducible gene 6 protein (aka TSG-6). In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises serum albumin. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises LTGFBP-1. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises LTGFBP-2. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises LTGFBP-3. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises LTGFBP-4.
In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises Pentraxin-3, TIMP-1, Serpin E1, TSP-1, and HGF.
In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 2-16 ng/ml, or 9.8+/−0.5 ng/ml Pentraxin-3.
In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 10-200 ng/ml, or 90+/−21.5 ng/ml TIMP-1.
In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 10-100 ng/ml, or 49.2+/−9.8 ng/ml Serpin E1.
In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 0.1-10 ng/ml, or 2.0+/−0.3 ng/ml HGF.
In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 100-800 pg/mL, or 304+/−44 pg/ml VEGF.
In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 0.1-100 pg/mL, or <1 ng/ml IL-8.
In some embodiments, the IDO (Indoleamine-2,3-dioxygenase) enzyme activity less than about 250 μM. In some embodiments, the IDO (Indoleamine-2,3-dioxygenase) enzyme activity is from 0 μM to about 250 μM. In some embodiments, the IDO (Indoleamine-2,3-dioxygenase) enzyme activity is from 50 μM to about 250 μM L-Kynurenine/million MSC. In some embodiments, the IDO (Indoleamine-2,3-dioxygenase) enzyme activity is from 50 μM to about 200 μM L-Kynurenine/million MSC. In some embodiments, the IDO (Indoleamine-2,3-dioxygenase) enzyme activity is from 100 μM to about 250 μM L-Kynurenine/million MSC. In some embodiments, the IDO (Indoleamine-2,3-dioxygenase) enzyme activity is from 100 μM to about 200 μM L-Kynurenine/million MSC. In some embodiments, the IDO (Indoleamine-2,3-dioxygenase) enzyme activity is about 0 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 110 μM, about 120 μM, about 130 μM, about 140 μM, about 150 μM, about 160 μM, about 170 μM, about 180 μM, about 190 μM, about 200 μM, about 210 μM, about 220 μM, about 230 μM, about 240 μM, or about 250 μM L-Kynurenine/million MSC.
In some embodiments, the MSC secretome further comprises “threshold” ppm levels for at least one additional factor which includes but is not limited to sFLT-1, PEDF (Serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, Kallikrein 3, MCP-1, bFGF, Angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and/or HO-1. In some embodiments, the MSC secretome further comprises “threshold” ppm levels for at least one additional factor selected from the group consisting of sFLT-1, PEDF (Serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, Kallikrein 3, MCP-1, bFGF, Angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and HO-1. In some embodiments, the MSC secretome further comprises one additional factor in a concertation range of 200 pg/mL to 5000 pg/mL, wherein the one additional factor includes but is not limited to sFLT-1, PEDF (Serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, Kallikrein 3, MCP-1, bFGF, Angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and/or HO-1. In some embodiments, the MSC secretome further comprises 1000-3000 pg/mL of sFLT-1. In some embodiments, the MSC secretome further comprises 400-800 pg/mL of TSG-6.
In some embodiments, the MSC secretome further comprises 2000-8000 pg/mL of PEDF. In some embodiments, the MSC secretome further comprises 2000-7000 pg/mL of PEDF. In some embodiments, the MSC secretome further comprises 2000-6000 pg/mL of PEDF. In some embodiments, the MSC secretome further comprises 2000-5000 pg/mL of PEDF. In some embodiments, the MSC secretome further comprises 2000-4000 pg/mL of PEDF. In some embodiments, the MSC secretome further comprises 2000-3000 pg/mL of PEDF. In some embodiments, the MSC secretome further comprises 150-300 ng/mL PEDF. In some embodiments, the MSC secretome further comprises 200-300 ng/ml of PEDF. In some embodiments, the MSC secretome further comprises 200-275 ng/mL of PEDF. In some embodiments, the MSC secretome further comprises 225-275 ng/ml of PEDF. In some embodiments, the MSC secretome further comprises 150-300 ng/mL of PEDF. In some embodiments, the MSC secretome further comprises 273±27 ng/ml of PEDF.
In some embodiments, the MSC secretome further comprises “higher” levels of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome further comprises “higher” levels of Serpin E1. In some embodiments, the MSC secretome further comprises “higher” levels of Serpin A1. In some embodiments, the MSC secretome further comprises “higher” levels of TIMP-1. In some embodiments, the MSC secretome further comprises “higher” levels of Thrombospondin-1. In some embodiments, the MSC secretome further comprises “higher” levels of Pentraxin-3 (TSG-14). In some embodiments, the MSC secretome further comprises “higher” levels of Platelet Factor 4. In some embodiments, the MSC secretome further comprises “higher” levels of Serpin F1. In some embodiments, the MSC secretome comprises 1 ng/ml to 20 ng/ml at least one factor selected from the group consisting of Serpin E1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome comprises 1 ng/mL to 8 ng/mL at least one factor selected from the group consisting of Serpin E1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome comprises 2 ng/mL to 8 ng/mL at least one factor selected from the group consisting of Serpin E1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome comprises 3 ng/mL to 8 ng/ml at least one factor selected from the group consisting of Serpin E1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome comprises 4 ng/mL to 8 ng/ml at least one factor selected from the group consisting of Serpin E1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome comprises 5 ng/mL to 8 ng/mL at least one factor selected from the group consisting of Serpin E1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome comprises 6 ng/ml to 8 ng/mL at least one factor selected from the group consisting of Serpin E1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome comprises 2 ng/mL to 7 ng/mL at least one factor selected from the group consisting of Serpin E1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1.
In some embodiments, the MSC secretome composition further comprises “mid-range” levels of at least one factor including but not limited to Angiopoietin-1, Angiopoietin-2, Amphiregulin, Endostatin, Endothelin-1, Thrombospondin-2, and Thrombospondin-1, Angiogenin, DPPIV (Dipeptidyl peptidase-4), IGFBP-3, and/or uPA. In some embodiments, the MSC secretome composition further comprises “mid-range” levels of at least one factor selected from the group consisting of Angiopoietin-1, Angiopoietin-2, Amphiregulin, Endostatin, Endothelin-1, Thrombospondin-2, and Thrombospondin-1, Angiogenin, DPPIV, IGFBP-3, and uPA. In some embodiments, the MSC secretome composition further comprises “mid-range” levels of at least one factor selected from the group consisting of Angiogenin, DPPIV, IGFBP-3, and uPA. In some embodiments, the MSC secretome composition further comprises about 200 pg/mL to about 800 pg/mL of at least one factor selected from the group consisting of Angiogenin, DPPIV, IGFBP-3, and uPA. In some embodiments, the MSC secretome composition further comprises about 200 pg/mL to about 700 pg/mL, about 300 pg/mL to about 800 pg/mL, about 200 pg/mL to about 500 pg/mL, or about 300 pg/mL to about 500 pg/mL of at least one factor selected from the group consisting of Angiopoietin-1, Angiopoietin-2, Amphiregulin, Endostatin, Endothelin-1, Thrombospondin-2, and Thrombospondin-1, Angiogenin, DPPIV, IGFBP-3, and uPA. In some embodiments, the MSC secretome composition further comprises about 200 pg/mL, about 300 pg/mL, about 400 pg/mL, about 500 pg/mL, about 600 pg/mL, about 700 pg/mL, or about 800 pg/mL of at least one factor selected from the group consisting of Angiopoietin-1, Angiopoietin-2, Amphiregulin, Endostatin, Endothelin-1, Thrombospondin-2, and Thrombospondin-1, Angiogenin, DPPIV, IGFBP-3, and uPA. In some embodiments, the MSC secretome composition comprises about 200 pg/mL to about 800 pg/mL, about 300 pg/mL to 800 pg/mL, about 200 pg/mL to about 500 pg/mL, or about 300 pg/mL to about 500 pg/mL of Angiogenin. In some embodiments, the MSC secretome composition further comprises about 200 pg/mL to about 800 pg/mL, about 300 pg/mL to about 800 pg/mL, about 200 pg/mL to about 500 pg/mL, or about 300 pg/mL to about 500 pg/mL of DPPIV. In some embodiments, the MSC secretome composition comprises about 200 pg/mL to about 800 pg/mL, about 300 pg/mL to about 800 pg/mL, about 200 pg/mL to 500 pg/mL, or about 300 pg/mL to about 500 pg/mL of IGFBP-3. In some embodiments, the MSC secretome composition comprises 200 pg/mL to about 800 pg/mL, about 300 pg/mL to 800 pg/mL, about 200 pg/mL to 500 pg/mL, or about 300 pg/mL to about 500 pg/mL of uPA.
In some embodiments, the MSC secretome further comprises “low” levels of VEGF. In some embodiments, the MSC secretome further comprises about 1 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises about 10 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises about 20 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises about 30 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises about 40 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises about 50 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises about 60 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises about 70 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises about 80 pg/mL of VEGF.
In some embodiments, the MSC secretome further comprises about 90 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises about 100 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises about 125 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises about 150 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises about 175 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 1 pg/mL to about 400 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 10 pg/mL to about 400 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 50 pg/mL to about 350 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 50 pg/mL to about 300 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 10 pg/mL to about 300 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 100 pg/mL to about 300 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises less than about 200 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises less than about 200 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 0 pg/mL to about 200 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 0 pg/mL to about 200 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 10 pg/mL to about 200 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 20 pg/mL to about 200 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 30 pg/mL to about 200 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 40 pg/mL to about 200 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 50 pg/mL to about 200 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 60 pg/mL to about 200 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 70 pg/mL to about 200 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 80 pg/mL to about 200 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 90 pg/mL to about 200 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 100 pg/mL to about 200 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 10 pg/mL to about 150 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 20 pg/mL to about 150 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 30 pg/mL to about 150 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 40 pg/mL to about 150 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 50 pg/mL to about 150 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 60 pg/mL to about 150 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 70 pg/mL to about 150 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 80 pg/mL to about 150 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 90 pg/mL to about 150 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 100 pg/mL to about 150 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 10 pg/mL to about 100 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 20 pg/mL to about 100 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 30 pg/mL to about 100 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 40 pg/mL to about 100 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 50 pg/mL to about 100 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 60 pg/mL to about 100 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 70 pg/mL to about 100 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 80 pg/mL to about 100 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 90 pg/mL to about 100 pg/mL of VEGF. In some embodiments, the MSC secretome further comprises 100 pg/mL to about 100 pg/mL of VEGF.
In some embodiments of the MSC secretome composition the level of VEGF is 5-10 fold lower than the level of Serpin E1. In some embodiments of the MSC secretome composition the level of VEGF is 6-10 fold lower than the level of Serpin E1. In some embodiments of the MSC secretome composition the level of VEGF is 7-10 fold lower than the level of Serpin E1. In some embodiments of the MSC secretome composition the level of VEGF is 8-10 fold lower than the level of Serpin E1. In some embodiments of the MSC secretome composition the level of VEGF is 9-10 fold lower than the level of Serpin E1. In some embodiments of the MSC secretome composition the level of VEGF is 5-fold lower than the level of Serpin E1. In some embodiments of the MSC secretome composition the level of VEGF is 6-fold lower than the level of Serpin E1. In some embodiments of the MSC secretome composition the level of VEGF is 7-fold lower than the level of Serpin E1. In some embodiments of the MSC secretome composition the level of VEGF is 8-fold lower than the level of Serpin E1. In some embodiments of the MSC secretome composition the level of VEGF is 9-fold lower than the level of Serpin E1. In some embodiments of the MSC secretome composition the level of VEGF is 10-fold lower than the level of Serpin E1.
In some embodiments, the MSC secretome composition does not comprise and/or comprises very low levels of bFGF, PLGF, and PDGF. In some embodiments, the MSC secretome composition comprises less than about 200 pg/mL, less than about 150 pg/mL, less than about 100 pg/mL, less than about 75 pg/mL, less than about 50 pg/mL, or less than about 25 pg/mL bFGF, PLGF, and/or PDGF. In some embodiments, the MSC secretome composition comprises less than about 200 pg/mL, less than about 150 pg/mL, less than about 100 pg/mL, less than about 75 pg/mL, less than about 50 pg/mL, or less than about 25 pg/mL bFGF, PLGF, and PDGF. In some embodiments, the MSC secretome composition does not comprise bFGF, PLGF, and/or PDGF. In some embodiments, the MSC secretome composition does not comprise bFGF, PLGF, and PDGF. In some embodiments, the MSC secretome composition comprises less than about 200 pg/mL, less than about 150 pg/mL, less than about 100 pg/mL, less than about 75 pg/mL, less than about 50 pg/mL, or less than about 25 pg/mL of bFGF. In some embodiments, the MSC secretome composition does not comprise bFGF. In some embodiments, the MSC secretome composition comprises less than about 200 pg/mL, less than about 150 pg/mL, less than about 100 pg/mL, less than about 75 pg/mL, less than about 50 pg/mL, or less than about 25 pg/mL of PLGF. In some embodiments, the MSC secretome composition does not comprise PLGF. In some embodiments, the MSC secretome composition comprises less than about 200 pg/mL, less than about 150 pg/mL, less than about 100 pg/mL, less than about 75 pg/mL, less than about 50 pg/mL, or less than about 25 pg/mL of PDGF. In some embodiments, the MSC secretome composition does not comprise PDGF. In some embodiments, the MSC secretome composition does not comprise bFGF. In some embodiments, the MSC secretome composition does not comprise PLGF. In some embodiments, the MSC secretome composition does not comprise PDGF. In some embodiments, the MSC secretome composition comprises very low levels of bFGF, PLGF, and PDGF. In some embodiments, the MSC secretome composition comprises very low levels of bFGF. In some embodiments, the MSC secretome composition comprises very low levels of PLGF. In some embodiments, the MSC secretome composition comprises very low levels of PDGF.
In some embodiments, the MSC secretome composition comprises Apolipoprotein A1, Complement Factor D, Complement factor H, Complement factor I, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, Complement receptor type 1 (CR1), C-reactive protein, Cystatin C, DKK-1, Emmprin, Osteopontin, vitamin D Binding Protein, MIF, RANTES, uPAR, IL-17a, GDF-15, and/or IFNγ.
In some embodiments, the MSC secretome further comprises “higher” levels of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 1 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 1 ng/mL-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 1 ng/mL-200 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 1 ng/ml-100 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng/mL-300 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng/ml-200 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng/ml-100 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 20 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 20 ng/ml-300 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 20 ng/mL-200 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 20 ng/ml-100 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 30 ng/ml-400 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 30 ng/mL-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 30 ng/ml-200 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 30 ng/ml-100 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 4 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 40 ng/mL-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 40 ng/ml-200 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 40 ng/ml-100 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 50 ng/mL-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 50 ng/mL-300 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 50 ng/ml-200 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 50 ng/ml-100 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 60 ng/mL-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 60 ng/mL-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 60 ng/ml-200 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 60 ng/mL-100 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 70 ng/mL-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 70 ng/mL-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 70 ng/ml-200 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 70 ng/ml-100 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 80 ng/mL-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 80 ng/ml-300 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 80 ng/ml-200 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 80 ng/ml-100 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 90 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 90 ng/ml-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 90 ng/mL-200 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 90 ng/mL-100 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 100 ng/mL-400 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 100 ng/ml-300 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 100 ng/ml-200 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 110 ng/mL-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 110 ng/ml-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 110 ng/ml-200 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 120 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 120 ng/mL-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 120 ng/ml-200 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 130 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 130 ng/mL-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 130 ng/mL-200 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 140 ng/ml-400 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 140 ng/ml-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 140 ng/ml-200 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 150 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 150 ng/ml-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 150 ng/mL-200 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 160 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 160 ng/ml-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 160 ng/ml-200 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 170 ng/mL-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 170 ng/ml-300 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 170 ng/ml-200 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 180 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 180 ng/mL-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 180 ng/ml-200 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 190 ng/ml-400 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 190 ng/ml-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 190 ng/ml-200 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 200 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 200 ng/mL-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1 In some embodiments, the MSC secretome composition comprises 210 ng/mL-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 210 ng/mL-300 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 220 ng/mL-400 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 220 ng/ml-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 230 ng/mL-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 230 ng/mL-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 240 ng/mL-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 240 ng/ml-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 250 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 250 ng/ml-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 260 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 260 ng/ml-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 270 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 270 ng/ml-300 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 280 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 280 ng/mL-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 290 ng/mL-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 290 ng/ml-300 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 310 ng/ml-400 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 320 ng/mL-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 330 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 340 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 350 ng/mL-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 360 ng/ml-400 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 370 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 380 ng/ml-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 390 ng/mL-400 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng/ml-90 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng/mL-80 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 20 ng/mL-100 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 30 ng/ml-100 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 40 ng/ml-100 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 50 ng/ml-100 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng/ml-70 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng/ml-60 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng/ml-50 ng/mL of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1.
In some embodiments, the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition is formulated at a pH of about pH 4.5 to about pH 8. In some embodiments, the MSC secretome composition is formulated at a pH of about pH 4.7 to about pH 7.8. In some embodiments, the MSC secretome composition is formulated at a pH of about pH 5.0 to about pH 7.5. In some embodiments, the MSC secretome composition is formulated at a pH of about pH 5.5 to about pH 7.5. In some embodiments, the MSC secretome composition is formulated at a pH of about pH 6 to about pH 7.5.
In some embodiments, the MSC secretome composition is formulated at a pH of about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.4, about pH 8.0. In some embodiments, the MSC secretome composition is formulated at a pH of about pH 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.
In some embodiments, the MSC secretome composition does not comprise certain components. In some embodiments, the MSC secretome composition does not comprise certain components found in cellular media. In some embodiments, the MSC secretome composition does not comprise one or more components selected from the group consisting of xenobiotic components (for example, animal serum); Phenol red; peptides and biomolecules <3 kDa; antibiotics; protein aggregates (for example, protein aggregates >200 nm); cells; cell debris (cell debris do not include exosomes/Extracellular Vesicles (EVs); for example, non-exosome, non-EV cell debris); hormones (for example, hormones include, but are not limited to insulin and/or hydrocortisone); and/or L-glutamine. In some embodiments, the MSC secretome composition does not comprise xenobiotic components. In some embodiments, the MSC secretome composition does not comprise Phenol red. In some embodiments, the MSC secretome composition does not comprise peptides and biomolecules <3 kDa. In some embodiments, the MSC secretome composition does not comprise antibiotics. In some embodiments, the MSC secretome composition does not comprise protein aggregates (for example, protein aggregates >200 nm). In some embodiments, the MSC secretome composition does not comprise cells. In some embodiments, the MSC secretome composition does not comprise cell debris (cell debris do not include exosomes/EVs; for example, non-exosome, non-EV cell debris). In some embodiments, the MSC secretome composition does not comprise hormones (for example, hormones include, but are not limited to insulin and/or hydrocortisone. In some embodiments, the MSC secretome composition does not comprise L-glutamine.
In some embodiments, the MSC secretome further comprises mannitol, lactose, sorbitol, xylitol, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, dextrose, and/or combinations thereof. In some embodiments, the MSC secretome further comprises phosphate. In some embodiments, the phosphate source is sodium phosphate or potassium phosphate. In some embodiments, the phosphate source is sodium phosphate. In some embodiments, the phosphate source is potassium phosphate. In some embodiments, the MSC secretome further comprises mono/di-sodium phosphate, mannitol, and trehalose, wherein the composition has a pH of about pH 7.4.
In some embodiments, the MSC secretome composition can comprise one or more additional agents including but not limited to glycine, glycerol, sodium chloride, potassium chloride, and/or dextrose. In some embodiments, the MSC secretome composition can comprise one or more additional agents selected from the group consisting of glycine, glycerol, sodium chloride, potassium chloride, and dextrose. In some embodiments, the MSC secretome composition can comprise one or more additional agents selected from the group consisting of glycine and glycerol, and dextrose. In some embodiments, the MSC secretome composition can comprise one or more additional agents selected from the group consisting of sodium chloride and potassium chloride.
In some embodiments, the MSC secretome composition is formulated in a buffer system. In some embodiments, the MSC secretome composition is formulated in a buffer system including but not limited to di/mono sodium phosphate, sodium citrate/citric acid, boric acid/sodium citrate, boric acid/sodium tetraborate, and/or citric acid/disodium phosphate. In some embodiments, the MSC secretome composition is formulated in a buffer system selected from the group consisting of di/mono sodium phosphate, sodium citrate/citric acid, boric acid/sodium citrate, boric acid/sodium tetraborate, and/or citric acid/disodium phosphate. In some embodiments, the MSC secretome composition is formulated in a di/mono sodium phosphate buffer system. In some embodiments, the MSC secretome composition is formulated in sodium citrate/citric acid buffer system. In some embodiments, the MSC secretome composition is formulated in a boric acid/sodium citrate buffer system. In some embodiments, the MSC secretome composition is formulated in a boric acid/sodium tetraborate buffer system. In some embodiments, the MSC secretome composition is formulated in a citric acid/disodium phosphate buffer system.
In some embodiments, the phosphate source is sodium phosphate or potassium phosphate. In some embodiments, the phosphate source is sodium phosphate. In some embodiments, the phosphate source is potassium phosphate. In some embodiments, the MSC secretome composition comprises di-sodium phosphate/citric acid, mannitol, and trehalose, wherein the composition has a pH of about pH 6.4.
In some embodiments, the MSC secretome composition further comprises a tonicity adjusting or tonicity modifying agent. In some embodiments, tonicity adjusting or tonicity modifying agent includes but is not limited to NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and/or glycerin. In some embodiments, tonicity adjusting or tonicity modifying agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and/or glycerin.
In some embodiments, the MSC secretome composition further comprises an adhesive agent. In some embodiments, the MSC secretome composition further comprises an adhesive agent including but not limited to hypromellose, Poloxamer 407, Poloxamer 188, Poloxomer 237, Poloxomer 338, Hypromellose, HEC, polycarbophil, polyvinylpyrrolidone (PVP), PVA (polyvinyl alcohol, polyimide, sodium hyaluronate, gellan gum, poly(lactic acid-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), hydroxypropyl methylcellulose (HPMC), hydroxy methyl cellulose, hydroxy ethyl cellulose, sodium carboxy methyl cellulose, fibrin glue, polyethyelene glycol, and GelCORE. In some embodiments, the adhesive agent is hypromellose. In some embodiments, the adhesive agent is fibrin glue. In some embodiments, the adhesive agent is a polyethyelene glycol. In some embodiments, the adhesive agent is GelCORE (see, Sani, et al., Science Advances, Vol. 5, no. 3 (2019)).
In some embodiments, the MSC secretome composition comprises (a) processed conditioned medium comprising the MSC secretome produced by any one of the methods described herein; and (b) a polymer. In some embodiments, the MSC secretome composition comprises conditioned medium comprising the MSC secretome which is produced as described herein and a polymer. In some embodiments, the MSC secretome composition comprises processed conditioned medium comprising the MSC secretome which is produced as described herein and a polymer. In some embodiments the polymer can be a biodegradable polymer from which the MSC secretome and/or processed MSC secretome components can be released. In some embodiments, the polymer enables sustained (slow) release of the MSC secretome components.
In some embodiments, the MSC secretome compositions provided herein are in the form of a therapeutic bandage (e.g., a polymer impregnated with MSC secretome composition). The therapeutic bandage may be configured as needed, depending on the application. In some embodiments, the bandage is in the form or a patch or is configured as mesh.
In some embodiments, the MSC secretome compositions exhibit bio-penetrance, for example, ocular penetration, corneal penetration, and/or corneal permeation. In some embodiments, the MSC secretome composition exhibits the ability to be absorbed by the eye. In some embodiments, the MSC secretome composition exhibits inherent bio-penetrance. In some embodiments, the MSC secretome composition exhibits excipient-enabled bio-penetrance. In some embodiments, the MSC secretome composition exhibits bio-penetrance due to upregulation of the smaller factors. In some embodiments, the MSC secretome composition exhibits bio-penetrance due to the presence of a biopreservative. In some embodiments, the MSC secretome composition exhibits bio-penetrance due to the presence of the biopreservative benzalkonium chloride.
In some embodiments, the MSC secretome compositions exhibit long half-life and/or have increased stability as compared to other treatments. In some embodiments, the MSC secretome compositions as provided herein allow for an upregulation of proteins that are allow for increased stability of the MSC secretome. In some embodiments, the MSC secretome compositions as provided herein allow for upregulating chaperone proteins to improve stability of other proteins in the MSC secretome.
In some embodiments, the MSC secretome compositions exhibit ultrapotency when administered to a subject in need thereof. In some embodiments, the MSC secretome compositions allow for therapeutic efficacy with one drop or one administration per day.
Further descriptions of the MSC secretome composition and related use are provided in International Application No. PCT/US20/27093 and US Patent Publication No. US 2021/0369617, which are incorporated herein by reference in their entireties.
According to the present invention, conditioned medium (and, thus, mesenchymal stem cell secreted factors) can be obtained from mesenchymal stem cells obtained from the patient or individual to be treated (the patient in need thereof) or from another (donor) individual, such as a young and/or healthy donor and/or from mesenchymal stem cells obtained commercially. For example, MSC obtained from the individual to be treated (autologous stem cells) or from a donor (allogeneic stem cells), can be used to produce the conditioned medium described herein, which can then be further processed into a MSC secretome composition as described herein. In some embodiments, MSCs can also be obtained from commercial suppliers. In some embodiments, commercially obtained MSCs can used in MSC secretome production.
According to the present invention, the method of making a anti-angiogenic mesenchymal stem cell (MSC) secretome composition comprising:
In some embodiments, culturing can be performed using a bioreactor system for culturing cells. In some embodiments, culturing can be performed using a bioreactor system for culturing stem cells. In some embodiments, culturing can be performed using a bioreactor system for culturing mesenchymal stem cells. In some embodiments, culturing can be performed using a media mixing technology. In some embodiments, culturing can be performed using a PBS Vertical Wheel™ Mixing Technology.
In some embodiments, in step (iv) processing the conditioned media in step (v) into the secretome composition comprises:
In some embodiments, step (c) comprises buffer exchanging with a buffer system selected from the group consisting of di/mono sodium phosphate, sodium citrate/citric acid, boric acid/sodium citrate, boric acid/sodium tetraborate, and citric acid/disodium phosphate.
In some embodiments, the filtering step (a) comprises the use of a 0.45 μm filter, a 0.22 μm filter, 0.8 μm filter, and 0.65 micron, a low protein binding PVDF membranes, and/or PES (polyethersulfone). In some embodiments, the filtering step (a) comprises the use of a 0.45 μm filter. In some embodiments, the filtering step (a) comprises the use of a 0.22 μm filter. In some embodiments, the filtering step (a) comprises the use of 0.8 μm filter. In some embodiments, the filtering step (a) comprises the use of 0.65 micron. In some embodiments, the filtering step (a) comprises the use of low protein binding PVDF membranes. In some embodiments, the filtering step (a) comprises the use of PES (polyethersulfone).
In some embodiments, the concentration step (b) comprises using hollow fiber filters, tangential flow filtration systems, or centrifugation based size exclusion techniques. In some embodiments, the concentration step (b) comprises using a hollow fiber filters technique. In some embodiments, the concentration step (b) comprises using a tangential flow filtration system. In some embodiments, the concentration step (b) comprises using a centrifugation based size exclusion technique.
In some embodiments, the centrifugation based size exclusion techniques employs a 3-10 kDa MW cutoff. In some embodiments, the centrifugation based size exclusion techniques employs at least a 3 kDa MW cutoff, at least a 4 kDa MW cutoff, at least a 5 kDa MW cutoff, at least a 6 kDa MW cutoff, at least a 7 kDa MW cutoff, at least a 8 kDa MW cutoff, at least a 9 kDa MW cutoff, at least a 10 kDa MW cutoff, at least a 11 kDa MW cutoff, at least a 12 kDa MW cutoff, at least a 13 kDa MW cutoff, at least a 14 kDa MW cutoff, at least a 15 kDa MW cutoff, at least a 16 kDa MW cutoff, at least a 17 kDa MW cutoff, at least a 18 kDa MW cutoff, at least a 19 kDa MW cutoff, at least a 20 kDa MW cutoff, at least a 21 kDa MW cutoff, at least a 22 kDa MW cutoff, at least a 23 kDa MW cutoff, at least a 24 kDa MW cutoff, at least a 25 kDa MW cutoff, at least a 26 kDa MW cutoff, at least a 27 kDa MW cutoff, at least a 28 kDa MW cutoff, at least a 29 kDa MW cutoff, and/or at least a 30 kDa MW cutoff.
In some embodiments, the method produces an MSC secretome composition and/or formulation as described herein above. In some embodiments, the first and/or second culture medium are MSC Media and/or MSC-XF.
MSCs, or cells differentiated from MSCs, can be made to produce a conditioned media comprising the desired secretome, e.g., which comprises desired cytokines and/or desired therapeutic properties as described herein. For example, the secretome can be produced from MSCs of a super donor cell line. The secretome can also be produced from MSCs obtained commercially. In come embodiments, allogeneic MSCs (and/or cells derived therefrom) and/or allogeneic MSC-derived secretome compositions can be prepared and stored for large groups of individuals. Allogeneic MSCs (and/or cells derived therefrom) and/or MSC-derived secretome compositions can be made in advance so that they are ready when people need them. In certain embodiments, MSCs (and/or cells derived therefrom) and/or MSC-derived secretome compositions can be processed to manufacture a more concentrated solution or composition (e.g., a mesenchymal stem cell derived secretome composition or MSC secretome composition as described herein).
In some embodiments, the initial culture medium and the first culture medium are different. In some embodiments, the initial culture medium and the first culture medium are the same. Non-limiting examples of cell culture medium or media useful in culturing MSCs to produce conditioned media comprising the MSC secretome according to the present invention include hMSC Media Booster XFM, hMSC High Performance Basal Media, Minimum Essential Medium Eagle (MEME), ADC-1, LPM (Bovine Serum Albumin-free), F10 (HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and without Fitton-Jackson Modification), StemPro, MSCGro, MesenCult, NutriStem, Basal Medium Eagle (BME—with the addition of Earle's salt base), Dulbecco's Modified Eagle Medium (DMEM—with or without serum), Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium, Medium M199 (M199E—with Earle's sale base), Medium M199 (M199H—with Hank's salt base), Minimum Essential Medium Alpha (MEM-alpha), Minimum Essential Medium Eagle (MEM-E—with Earle's salt base), Minimum Essential Medium Eagle (MEM-H—with Hank's salt base) and Minimum Essential Medium Eagle (MEM-NAA with non-essential amino acids), among numerous others, including medium 199, CMRL 1415, CMRL 1969, CMRL 1066, NCTC 135, MB 75261, MAB 8713, DM 145, Williams' G, Neuman & Tytell, Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, MCDB 153. A preferred medium for use in the present invention is MEM-alpha. These and other useful media are available from GIBCO, Grand Island, N.Y., USA and Biological Industries, Bet HaEmek, Israel, among others. A number of these media are summarized in Methods in Enzymology, Volume LVIII, “Cell Culture”, pp. 62 72, edited by William B. Jakoby and Ira H. Pastan, published by Academic Press, Inc.
In some embodiments, the cell culture medium for mesenchymal stem cells can be a serum-free medium. In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with serum. In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented human platelet lysate. In some embodiments, the serum can include fetal bovine serum (FBS). In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with serum such as fetal serum of bovine or other species. In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with other components to facilitate cell growth and/or promote cell health, such as mercaptoethanol and/or antibiotics. In some embodiments, the cell culture medium for mesenchymal stem cells is not supplemented with antibiotics.
In some embodiments, the oxygen percentage is varied to facilitate cell growth and/or promote cell health. In some embodiments, the oxygen is at 5%, 10%, 15%, 20%, or 25% volume to facilitate cell growth and/or promote cell health. In some embodiments, the mesenchymal stem cells are grown under partial oxygen pressure to facilitate cell growth and/or promote cell health. In some embodiments, the mesenchymal stem cells are grown under a low oxygen partial pressure environment to facilitate cell growth and/or promote cell health.
In one aspect, the present invention is directed to conditioned medium (CM) comprising biological factors secreted by mesenchymal stem cells, which can be referred to as conditioned media comprising the MSC secretome. The conditioned medium can be obtained by culturing mesenchymal stem cells in media, as described herein, and separating the resulting media, which contains mesenchymal stem cells and their secreted mesenchymal stem cell products (referred to as biological factors and/or the secretome) into the components parts of the conditioned medium contain the secretome and mesenchymal stem cells grown in the conditioned media. The conditioned medium once separated comprises the mesenchymal stem cell secretome and can be further processed and/or used according to the methods described herein and is substantially free of mesenchymal stem cells (may contain a small percentage of stem cells and/or trace amounts of stem cells) or free of mesenchymal stem cells. The MSC secretome comprises a variety of biological factors including hormones, cytokines, extracellular matrix, proteins, vesicles, antibodies, chemokines, receptors, inhibitor, and granules. As described herein, the conditioned medium or media (CM or conditioned media comprising the MSC secretome) comprising the MSC secretome can be further processed, producing concentrated, conditioned medium (pCM or concentrated MSC secretome).
In some embodiments, the conditioned media comprising the MSC secretome or concentrated MSC secretome is produced by culturing mesenchymal stem cells in culture medium, replacing culture medium in which the mesenchymal stem cells have been cultured. In some embodiments, the resultant conditioned media comprising the MSC secretome is harvested (collected), then processed to produce concentrated MSC secretome. In certain embodiments, processing of the harvested conditioned media comprising the MSC secretome includes removal of some, most, or essentially all of the medium, or removal of some, most, or essentially all of selected components of the conditioned medium.
In some embodiments, the harvested conditioned media comprising the MSC secretome is filtered to produce concentrated MSC secretome. In some embodiments, the harvested conditioned media comprising the MSC secretome is ultra-filtered to produce concentrated MSC secretome.
In one aspect, provided herein are methods of producing processed conditioned medium, comprising (a) culturing stem cells in a cell culture medium, thereby generating conditioned medium that comprises factors secreted by the mesenchymal stem cells (e.g., conditioned media comprising the mesenchymal stem cell secretome); (b) harvesting the conditioned medium thereby producing harvested conditioned medium (e.g., harvested mesenchymal stem cell secretome); and (c) filtering harvested conditioned medium (e.g., harvested mesenchymal stem cell secretome) to produce processed conditioned medium (mesenchymal stem cell secretome). In some embodiments, the stem cells of (a) are cultured (have been cultured) in growth medium prior to being cultured in growth factor-free medium. Thus, in some embodiments, the methods comprise: (a) culturing mesenchymal stem cells in a first growth medium; (b) replacing the first growth medium with a second growth medium and culturing the stem cells in the second growth medium, thereby generating conditioned media comprising the mesenchymal stem cell secretome; (c) harvesting the conditioned media comprising the mesenchymal stem cell secretome, thereby producing harvested conditioned medium comprising the mesenchymal stem cell secretome; and (d) filtering harvested conditioned medium to produce processed conditioned medium comprising the mesenchymal stem cell secretome.
In some embodiments, the MSC secretome of the present invention is further processed. In some embodiments, the MSC secretome of the present invention is further processed using techniques known in the art, including but not limited to extraction, freeze-thawing, homogenization, permeabilization, centrifugation, density gradient centrifugation, CsCl gradient centrifugation, iodixanol gradient centrifugation, ultracentrifugation, fractionation, precipitation, SDS-PAGE, native PAGE, size exclusion chromatography, liquid chromatography, gas chromatography, hydrophobic interaction chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, affinity chromatography, heparin sulfate affinity chromatography, sialic acid affinity chromatography, immunoaffinity chromatography, metal binding chromatography, nickel column chromatography, epitope tag purification, or lyophilization, or any combination thereof.
In some embodiments, the MSC secretome of the present invention is enriched by one or more of the following methods: affinity-based enrichment, size based enrichment, cation or anion based enrichment, and fraction to enrich for favorable attributes.
In some embodiments, the stem cells are mesenchymal stem cells. Mesenchymal stem cells (MSCs) are multipotent (capable of differentiating into multiple, but not all, cell lineages) nonhematopoietic (non-blood) stem cells isolated from (derived from) a variety of adult tissues, including bone marrow and adipose tissue. In certain embodiments, the mesenchymal stem cells are isolated from bone marrow. “Isolated” refers to cells removed from their original environment. MSCs may differentiate into cells of mesodermal lineage, for example, adipocytes, osteoblasts, and chondrocytes. MSCs have a small cell body with few cell processes that are long and thin. The cell body contains a large, round nucleus with a prominent nucleolus, which is surrounded by finely dispersed chromatin particles, giving the nucleus a clear appearance. The remainder of the cell body contains a small amount of Golgi apparatus, rough endoplasmic reticulum, mitochondria, and polyribosomes. The cells, which are long and thin, are widely dispersed and the adjacent extracellular matrix is populated by a few reticular fibrils but is devoid of the other types of collagen fibrils [Brighton, et al. 1991 The Journal of Bone and Joint Surgery 73(6):832-47]. MSCs described herein may express the following molecular marker (protein molecule characteristic of plasma membrane of a cell or cell type) profiles: bone morphogenic protein receptor “1” (BMPR+); CD34+Scal+Lin″; CD44+; c-kit+; Sca-1+; Thy-1+; NOTCH3; JAG1; ITGA11. MSCs may also express other cell type-specific markers (see, the World Wide Web at stemcells.nih.gov; Kaltz, et al. 2010 Exp Cell Res October 1; 316(16):2609-17, incorporated herein by reference]. MSCs described herein may be identified based on colony-forming unit assays to detect the multipotent differentiation potential of the MSCs (to what cell types the MSCs give rise). However, cells that are somewhat differentiated (progenitor cells) can also be used.
Further descriptions of the methods of producing and manufacturing the MSC secretome composition disclosed herein are provided in International Application No. PCT/US20/27093 and US Patent Publication No. US 2021/0369617, which are incorporated herein by reference in their entireties.
i. MSC Secretome—Processing
The conditioned medium comprising the MSC secretome described herein can in some embodiments be collected and filtered and/or purified to remove cell particulate and/or other detrimental components. For example, as described above under step (v) harvesting the second culture media from step (iv) as conditioned media. The filtration membranes used herein may be selected from any of those known in the art having a suitable membrane and configuration, such that they are capable of retaining the desired MSC secretome components while allowing the cell particulate and/or other detrimental components pass through. Thus, one may employ any suitable membrane which permits the retention of cells under the fluid dynamic conditions selected whilst allowing the detrimental components to pass through for removal. In some embodiments, an upper limit of pore size of about 5 microns and a lower limit of about 0.1 microns would be suitable. In some embodiments, filtration can be performed using a micropore filter. In some embodiments, filtration can be performed using a 0.5 μm to a 0.2 μm filter. In some embodiments, filtration can be performed using a 0.5 μm, 0.45 μm, 0.4 μm, 0.35 μm, 0.3 μm, 0.25 μm, 0.22 μm and/or a 0.2 μm filter. In some embodiments, filtration can be performed using a 0.45 μm filter. In some embodiments, filtration can be performed using a 0.22 μm filter. In some embodiments, filtration/purification can be performed using a low protein binding polyvinylidene difluoride (PVDF) membranes. In some embodiments, filtration/purification can be performed using polyethersulfone (PES).
In some embodiments, the filtering is by ultra-filtration. In some embodiments, the conditioned medium is filtered using a filter size of 3 kD (to achieve purification, desalting, and concentration in the processed conditioned medium of molecules larger than the filter size). In some embodiments, a filter size of less than 3 kD is used to filter the conditioned medium, while in other embodiments a filter size of greater than 3 kD is used, depending on the application for which the processed conditioned medium is used. In other embodiments, ultra-filtration of harvested conditioned medium is carried out using a filter of a different pore size (e.g., 2 kD, <2 kD or >2 kD) selected to determine the size of components of the resulting processed conditioned medium comprising the MSC secretome.
In some embodiments, the detrimental components in the growth supporting media are removed by medium exchange, preferably via “cross-flow filtration”. Cross-flow filtration refers to a mode of filtration where a suspension of MSC secretome cells flows substantially parallel to a filter which is permeable to a component of the suspension other than cells. The cross-flow filtration process is characterized by a set of fluid dynamic parameters including Re=Reynolds number, γw=wall shear rate, ΔP=pressure drop and TMP=transmembrane pressure. Re, γw and ΔP will depend on the geometry of the filtration system, flow conditions and fluid properties. Such cross-flow processes can, in some embodiments, include hollow fiber filtration systems as well. See, for example, U.S. Pat. No. 5,053,334, incorporated herein by reference in its entirety.
In some embodiments, the MSC secretome can be further subject to concentrated in the absence of filtration and/or after filtration. In some embodiments, the MSC secretome can be concentrated using hollow fiber tangential flow technology, or
In some embodiments, the MSC secretome can be concentrated using centrifugation based size exclusion technique, for example, amicons and/or centricons can be employed during the centration step. In some embodiments, the size cutoff is a 3-10 kDa MW cutoff. In some embodiments, the molecular weight cutoff for use during centrifugation based size exclusion technique concentration methods is at least about 3 kDa, at least about 4 kDa, at least about 5 kDa, at least about 6 kDa, at least about 7 kDa, at least about 8 kDa, at least about 9 kDa, or at least about 10 kDa.
In some embodiments, the MSC secretome is concentrated about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, or about 100-fold. In some embodiments, the MSC secretome is concentrated about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, or about 100-fold as compared to the conditioned media prior to concentration
In some embodiments, the MSC secretome is further buffer exchanged after the concentration step into the final formulation buffer. In some embodiments, the MSC secretome is further buffer exchanged after the concentration step into the final formulation buffer without an adhesive agent. In some embodiments, buffer exchange comprises altering the buffer components of the MSC secretome. In some embodiments, the MSC secretome is not diluted during the buffer exchange step. In some embodiments, the MSC secretome is diluted less than 1%, less than 5%, less than 10%, less than 15%, less than 20%, or less than 25% during the buffer exchange step.
In some embodiments, the MSC secretome is buffer exchanged after the concentration step such that all traces of culture media components are removed. In some embodiments, the MSC secretome is buffer exchanged after the concentration step such that less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% or about 0% of the culture media components remain.
ii. MSC Secretome—Formulating
The present invention further provides an MSC secretome composition comprising the MSC secretome described herein for treating LSCD in a subject.
In some embodiments, the invention provides an MSC secretome composition comprising a therapeutically effective amount of the MSC secretome described herein.
In some embodiments, the invention is an MSC secretome composition comprising a therapeutically effective amount of the MSC secretome described herein, and a pharmaceutically acceptable carrier. In some embodiments, the invention is an MSC secretome composition comprising a therapeutically effective amount of the MSC secretome, and a pharmaceutically acceptable carrier.
As used herein a “an MSC secretome composition” refers to a preparation of the MSC secretome compounds described herein, optionally with other chemical components such as physiologically suitable carriers and excipients.
In some embodiments, the invention is an MSC secretome composition is in a unit dosage formulation. An MSC secretome composition containing the MSC secretome described herein may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art. In some embodiments, the term “unit dosage formulations” as used herein refers to formulations containing an effective dose, or an appropriate fraction thereof, of the active ingredient.
MSC secretome compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the MSC secretome into preparations which, can be used pharmaceutically. The carriers must be acceptable in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
In some embodiments, the MSC secretome is prepared in a formulation comprising about 0.5 μg-20 μg per 1 mL of MSC secretome. In some embodiments, the MSC secretome is prepared in a formulation comprising 0.004% to 0.0375% per mL of MSC secretome.
In some embodiments, the MSC secretome is prepared in a formulation comprising about 2 μg-8 μg per 1 mL of MSC secretome. In some embodiments, the MSC secretome is prepared in a formulation comprising 0.008% to 0.015% per mL of MSC secretome.
In some embodiments, the MSC secretome is prepared in a formulation comprising 2 mg-3 mg per mL of monobasic sodium phosphate. In some embodiments, the MSC secretome is prepared in a formulation comprising 4% to 5% per mL of monobasic sodium phosphate.
In some embodiments, the MSC secretome is prepared in a formulation comprising 11 mg-12 mg per mL of dibasic sodium phosphate. In some embodiments, the MSC secretome is prepared in a formulation comprising 21.5% to 23% per mL of dibasic sodium phosphate.
In some embodiments, the MSC secretome is prepared in a formulation comprising 11.5 mg-13 mg per mL of mannitol. In some embodiments, the MSC secretome is prepared in a formulation comprising 23% to 25% per mL of mannitol.
In some embodiments, the MSC secretome is prepared in a formulation comprising 23 mg-25 mg per mL of trehalose dihydrate. In some embodiments, the MSC secretome is prepared in a formulation comprising 46% to 48% per mL of trehalose dihydrate.
In some embodiments, the MSC secretome is prepared in a formulation that does not comprise hypromellose. In some embodiments, the MSC secretome is prepared in a formulation that optionally comprises hypromellose. In some embodiments, the MSC secretome is prepared in a formulation comprising 0.5 mg-2 mg per mL of hypromellose. In some embodiments, the MSC secretome is prepared in a formulation comprising 1% to 3% per mL of hypromellose.
In some embodiments, the MSC secretome is prepared in a formulation comprising hydrochloric acid and/or sodium hydroxide. In some embodiments, the MSC secretome is prepared in a formulation comprising hydrochloric acid. In some embodiments, the MSC secretome is prepared in a formulation comprising sodium hydroxide. In some embodiments, the hydrochloric acid and/or sodium hydroxide is employed to obtain the desired pH.
In some embodiments, the MSC secretome is prepared in a formulation comprising the components as provided in Table 1 below:
In some embodiments, the MSC secretome formulation provided in Table 2 has a pH of about 5.5.
In some embodiments, the MSC secretome formulation provided in Table 3 has a pH of about 6.2.
In some embodiments, the MSC secretome formulation provided in Table 4 has a pH of about 7.2. In some embodiments, the MSC secretome is formulated with Water for Injection in accordance with USP standards.
In some embodiments of the invention, the MSC secretome is processed to achieve certain ingredient ratios/concentrations as well as properties for the MSC secretome.
In some embodiments, the MSC secretome composition comprises ratios of anti-angiogenic to pro-angiogenic wherein the ratio is >1. In some embodiments, the MSC secretome composition comprises ratios of anti-angiogenic to pro-angiogenic wherein the ratio is >2, >3, >4, or >5. In some embodiments, the MSC secretome composition comprises an increased concentration of pro-angiogenic factors (relative to the concentration of pro-angiogenic factors in conditioned medium from which the MSC secretome composition is produced). In some embodiments, the MSC secretome composition comprises a sum of several anti-angiogenic factors that exceeds the level of VEGF. In some embodiments, the MSC secretome composition comprises a sum of several anti-angiogenic factors such that the ratio of the more than 1 anti-angiogenic factor to VEGF is >2, >3, >4, or >5. In some embodiments, the MSC secretome composition comprises one or more anti-angiogenic factors, and wherein the ratio of the sum of the concentration of the one or more anti-angiogenic factors relative to the concentration of VEGF is >2, >3, >4, or >5. In some embodiments, pro-angiogenic factors include but are not limited to Serpin E1 to VEGF-A. In some embodiments, the pro-angiogenic factor is Serpin E1. In some embodiments, the pro-angiogenic factor is VEGF-A.
In some embodiments of the invention, the MSC secretome is processed to achieve certain potency performance criteria. In some embodiments, the buffer exchange step promotes obtaining a potent MSC secretome.
Extracellular Vesicles are membrane bound particles that carry cargo of soluble and insoluble substances mentioned above. The term “Extracellular Vesicles” refers a group of secreted or shedded vesicles of various species. These are generally divided into the following subtypes: 1) microvesicles or Shed microvesicles which typically exhibit a size range of 50-1500 nm; 2) exosomes which typically exhibit a size range of 30-120 nm; and 3) vesicles which typically exhibit a size range of less than 500 nm (i.e., <500 nm). (See, for example, WO2019016799, incorporated by reference herein in its entirety.) In some embodiments, the MSC secretome can be analyzed for particle count and/or to quantitate the extracellular vesicles (EVs) present in the secretome.
In some embodiments, EVs are present in a concentration of about 2.5×10{circumflex over ( )}5/uL, 2.6×10{circumflex over ( )}5/uL, 2.7×10{circumflex over ( )}5/uL, 2.8×10{circumflex over ( )}5/uL, 2.9×10{circumflex over ( )}5/uL, 3.0×10{circumflex over ( )}5/uL, 3.1×10{circumflex over ( )}5/uL, 3.2×10{circumflex over ( )}5/uL, 3.3×10{circumflex over ( )}5/uL, 3.4×10{circumflex over ( )}5/uL, 3.5×10{circumflex over ( )}5/uL, 3.6×10{circumflex over ( )}5/uL, 3.7×10{circumflex over ( )}5/uL, 3.8×10{circumflex over ( )}5/uL, 3.9×10{circumflex over ( )}5/uL, 4.0×10{circumflex over ( )}5/uL, 4.1×10{circumflex over ( )}5/uL, 4.2×10{circumflex over ( )}5/uL, 4.3×10{circumflex over ( )}5/uL, 4.4×10{circumflex over ( )}5/uL, 4.5×10{circumflex over ( )}5/uL, 4.6×10{circumflex over ( )}5/uL, 4.7×10{circumflex over ( )}5/uL, 4.8×10{circumflex over ( )}5/uL, 4.9×10{circumflex over ( )}5/uL, or about 5.0×10{circumflex over ( )}5/uL. In some embodiments, EVs are present in a concentration of about 3.8×10{circumflex over ( )}5/uL+/−0.8×10{circumflex over ( )}5.
In some embodiments, EVs are present in a concentration of about 2.5×10{circumflex over ( )}5/uL, 2.6×10{circumflex over ( )}5/uL, 2.7×10{circumflex over ( )}5/uL, 2.8×10{circumflex over ( )}5/uL, 2.9×10{circumflex over ( )}5/uL, 3.0×10{circumflex over ( )}5/uL, 3.1×10{circumflex over ( )}5/uL, 3.2×10{circumflex over ( )}5/uL, 3.3×10{circumflex over ( )}5/uL, 3.4×10{circumflex over ( )}5/uL, 3.5×10{circumflex over ( )}5/uL, 3.6×10{circumflex over ( )}5/uL, 3.7×10{circumflex over ( )}5/uL, 3.8×10{circumflex over ( )}5/uL, 3.9×10{circumflex over ( )}5/uL, 4.0×10{circumflex over ( )}5/uL, 4.1×10{circumflex over ( )}5/uL, 4.2×10{circumflex over ( )}5/uL, 4.3×10{circumflex over ( )}5/uL, 4.4×10{circumflex over ( )}5/uL, 4.5×10{circumflex over ( )}5/uL, 4.6×10{circumflex over ( )}5/uL, 4.7×10{circumflex over ( )}5/uL, 4.8×10{circumflex over ( )}5/uL, 4.9×10{circumflex over ( )}5/uL, or about 5.0×10{circumflex over ( )}5/uL and average 110-120 nm in diameter. In some embodiments, EVs are present in a concentration of about 2.5×10{circumflex over ( )}5/uL, 2.6×10{circumflex over ( )}5/uL, 2.7×10{circumflex over ( )}5/uL, 2.8×10{circumflex over ( )}5/uL, 2.9×10{circumflex over ( )}5/uL, 3.0×10{circumflex over ( )}5/uL, 3.1×10{circumflex over ( )}5/uL, 3.2×10{circumflex over ( )}5/uL, 3.3×10{circumflex over ( )}5/uL, 3.4×10{circumflex over ( )}5/uL, 3.5×10{circumflex over ( )}5/uL, 3.6×10{circumflex over ( )}5/uL, 3.7×10{circumflex over ( )}5/uL, 3.8×10{circumflex over ( )}5/uL, 3.9×10{circumflex over ( )}5/uL, 4.0×10{circumflex over ( )}5/uL, 4.1×10{circumflex over ( )}5/uL, 4.2×10{circumflex over ( )}5/uL, 4.3×10{circumflex over ( )}5/uL, 4.4×10{circumflex over ( )}5/uL, 4.5×10{circumflex over ( )}5/uL, 4.6×10{circumflex over ( )}5/uL, 4.7×10{circumflex over ( )}5/uL, 4.8×10{circumflex over ( )}5/uL, 4.9×10{circumflex over ( )}5/uL, or about 5.0×10{circumflex over ( )}5/uL and average 112-116 nm in diameter. In some embodiments, EVs are present in a concentration of about 2.5×10{circumflex over ( )}5/uL, 2.6×10{circumflex over ( )}5/uL, 2.7×10{circumflex over ( )}5/uL, 2.8×10{circumflex over ( )}5/uL, 2.9×10{circumflex over ( )}5/uL, 3.0×10{circumflex over ( )}5/uL, 3.1×10{circumflex over ( )}5/uL, 3.2×10{circumflex over ( )}5/uL, 3.3×10{circumflex over ( )}5/uL, 3.4×10{circumflex over ( )}5/uL, 3.5×10{circumflex over ( )}5/uL, 3.6×10{circumflex over ( )}5/uL, 3.7×10{circumflex over ( )}5/uL, 3.8×10{circumflex over ( )}5/uL, 3.9×10{circumflex over ( )}5/uL, 4.0×10{circumflex over ( )}5/uL, 4.1×10{circumflex over ( )}5/uL, 4.2×10{circumflex over ( )}5/uL, 4.3×10{circumflex over ( )}5/uL, 4.4×10{circumflex over ( )}5/uL, 4.5×10{circumflex over ( )}5/uL, 4.6×10{circumflex over ( )}5/uL, 4.7×10{circumflex over ( )}5/uL, 4.8×10{circumflex over ( )}5/uL, 4.9×10{circumflex over ( )}5/uL, or about 5.0×10{circumflex over ( )}5/uL and average 114 nm in diameter. In some embodiments, EVs are present in a concentration of about 3.8×10{circumflex over ( )}5/uL+/−0.8×10{circumflex over ( )}5 and average 114 nm in diameter.
i. MSC Secretome—Therapeutic Properties
The MSC secretome of the present disclosure exhibits a variety of therapeutic properties, including for example, anti-angiogenic properties (blood vessels and/or lymphatic vessels), anti-fibrotic properties, anti-inflammatory properties, properties promoting cell migration and proliferation, mitogenic promoting properties, anti-oxidative stress/damage properties,
In some embodiments, anti-angiogenic (blood vessels and/or lymphatic vessels) properties can be determined by the presence and/or level of one or more factors in the MSC secretome. In some embodiments, the anti-angiogenic factors include but are not limited to one or more of PEDF, sFLT-1, lower levels of VEGF, and/or Serpin E1. In some embodiments, the anti-angiogenic factors include but are not limited to one or more of PEDF, lower levels of VEGF, and/or Serpin E1. In some embodiments, the anti-angiogenic factor is PEDF. In some embodiments, the anti-angiogenic factor is sFLT-1. In some embodiments, the anti-angiogenic factor corresponds to lower levels of VEGF. In some embodiments, the anti-angiogenic factor is Serpin E1.
In some embodiments, pro-angiogenic (blood vessels and/or lymphatic vessels) properties can be determined by the presence and/or level of one or more factors in the MSC secretome. In some embodiments, the pro-angiogenic factors includes one or more factors selected from the group consisting of VEGF, Angiogenin, IGFBP-3, uPA, Angio-1, Angio-2, Endothelin-1. In some embodiments, the pro-angiogenic factor is VEGF. In some embodiments, the pro-angiogenic factor is Angiogenin. In some embodiments, the pro-angiogenic factors is IGFBP-3. In some embodiments, the pro-angiogenic factor is uPA. In some embodiments, the pro-angiogenic factor is Angio-1. In some embodiments, the pro-angiogenic factor is Angio-2. In some embodiments, the pro-angiogenic factor is Endothelin-1.
In some embodiments, the MSC secretome exhibits anti-fibrotic properties. In some embodiments, such anti-fibrotic properties can be assayed for using standard assays. In some embodiments, the present of various factors and/or activities with regard to the MSC secretome are indicative of anti-fibrotic properties. In some embodiments, factors which are indicative of anti-fibrotic properties include but are not limited to FGF7 and/or FGF10. In some embodiments, the factor indicative of anti-fibrotic properties is FGF7. In some embodiments, the factor indicative of anti-fibrotic properties is FGF10. In some embodiments, the factor indicative of anti-fibrotic properties is HGF. In some embodiments, activities indicative of anti-fibrotic properties include, but are not limited to, activation of SMAD, inhibition of TGFβ pathway, inhibition of myofibroblast differentiation, and/or inhibition of excess ECM deposition. In some embodiments, activities indicative of anti-fibrotic properties include activation of SMAD. In some embodiments, activities indicative of anti-fibrotic properties include inhibition of TGFβ pathway. In some embodiments, activities indicative of anti-fibrotic properties include inhibition of myofibroblast differentiation. In some embodiments, activities indicative of anti-fibrotic properties include inhibition of excess ECM deposition.
In some embodiments, the MSC secretome exhibits anti-inflammatory properties. In some embodiments, the MSC secretome inhibits inflammation. In some embodiments, the MSC secretome inhibits inflammation by 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (e.g., complete reduction in inflammation). In some embodiments, the MSC secretome prevents degranulation of mast cells.
In some embodiments, the MSC secretome promotes cell migration and proliferation, including for example, mitogenic and motogenic activities. In some embodiments, the MSC secretome promotes mitogenic activities. In some embodiments, the MSC secretome promotes motogenic activities. In some embodiments, the MSC secretome comprises FGF7, which provides for the cell migration and proliferation activities of the MSC secretome.
In some embodiments, the MSC secretome comprises FGF7, which provides for the cell migration and proliferation activities of the MSC secretome.
In some embodiments, the MSC secretome comprises HGF, which provides for the cell migration and proliferation activities of the MSC secretome.
In some embodiments, the MSC secretome comprises anti-apoptotic agents, which provides for the cell migration and proliferation activities of the MSC secretome. In some embodiments, the MSC secretome comprises anti-apoptotic agents include but are not limited to FGF-2, HGF and IGF-1, and which provide for the cell migration and proliferation activities of the MSC secretome. In some embodiments, the MSC secretome comprises anti-apoptotic agents selected from the group the consisting of FGF-2, HGF and IGF-1, and which provide for the cell migration and proliferation activities of the MSC secretome.
In some embodiments, the MSC secretome comprises NGF, which provides for the cell migration and proliferation activities of the MSC secretome.
In some embodiments, the MSC secretome provides for anti-oxidative stress and or reduction in cellular damage. In some embodiments, the MSC secretome comprises anti-oxidative stress and reduction in cellular damage factors. In some embodiments, the anti-oxidative stress and reduction in cellular damage factors include but are not limited to SOD-1, SOD-2, SOD-3, HO-1. In some embodiments, the anti-oxidative stress and reduction in cellular damage factor is selected from the group consisting of SOD-1, SOD-2, SOD-3, HO-1.
ii. MSC Secretome—Biophysical/Biochemical Properties
In some embodiments, the present invention provides methods for characterization of the MSC secretome. In some embodiments, the MSC secretome characterization will include: 1) a comprehensive and/or quantitative mapping of the molecular entities in the MSC secretome; 2) measuring the contributions of select factors to biological activity; and 3) measuring biophysical parameters. In some embodiments, in order to determine the properties of the MSC secretome, various potency assays can be performed on the MSC secretome as described herein. In some embodiments, the MSC secretome can be subjected to a comprehensive and/or quantitative mapping of the molecular entities in the MSC secretome; 2) measuring the contributions of select factors to biological activity; and 3) measuring biophysical parameters. In some embodiments, characterization assays include but are not limited to biophysical assays, biochemical assays, and bioassays. In some embodiments, characterization assays can include but are not limited to physical component characterizations, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation/scarring assays, inflammation assays, immune assays, gliosis assays, tissue explant survival and function assays, organoid development or survival/function, epithelial barrier integrity assays, retinal degeneration assays, and/or assays of inherited retinal disease including human and animal retinal explants, neural protection/neurotrophic assays.
In some embodiments, the characterization of the MSC secretome comprises a method employing a combination of bioanalytical techniques. In some embodiments, the characterization of the MSC secretome comprises determining the physical components of the MSC secretome. In some embodiments, characterization of the MSC secretome includes employing protein arrays, enzyme-linked immunosorbent assays (ELISAs), mass spectrometry, and immunoblotting. In some embodiments, the MSC secretome characterization can be used to identify the molecules in the MSC secretome. In some embodiments, protein arrays can be employed to identify factors in the MSC secretome. In some embodiments, mass spectrometry can be employed to determine the presence of one or more factors in the MSC secretome. In some embodiments, quantitative techniques can be employed to measure the levels of one or more factors. In some embodiments, quantitative techniques such as ELISA can be employed to measure the levels of each factor.
In some embodiments, the secretome comprises protein factors and extracellular vesicles (EVs). In some embodiments, the MSC secretome comprises trophic factors. In some embodiments, the protein factors of the MSC secretome comprise Pentraxin-3, TIMP-1, Serpin E1, TSP-1, HGF. In some embodiments, the MSC secretome comprises EVs. In some embodiments, the MSC secretome is analyzed for simple lipid content in order to quantitatively measure total lipid. In some embodiments, the EV fraction if the MSC secretome can be evaluated for EV markers. In some embodiments, the EV fraction if the MSC secretome can be evaluated for EV markers, including but not limited to AUX, TSG101, CD63, CD9, and CD8.
In some embodiments, the secretome comprises extracellular vesicles (EVs) in a size range of 30-200 nm and 1×108 to 5×109 EVs per mL.
In some embodiments, depletion studies can be performed to distill the individual contributions of critical factors. In some embodiments, using an antibody-based pulldown method, defined factors can be removed from the MSC secretome. In some embodiments, depletion can be verified by western blot and then evaluated by one or more bioassays, as described herein below. In some embodiments, depletion studies can be performed to evaluate the contributions of the protein fraction and the EV fraction. In some embodiments, TIMP-1 and/or Serpin E1 can be depleted. In some embodiments, TIMP-1 and/or Serpin E1 can be depleted.
In some embodiments, oxidative stress prevention assays can be performed on the MSC secretome. In some embodiments, the MSC secretome prevents corneal epithelium damage. In some embodiments, the MSC secretome reduces the presence of inflammation. In some embodiments, the MSC secretome reduces the presence of inflammation as determined by an increase in the present of anti-inflammation markers. In some embodiments, the MSC secretome reduces the presence of inflammation as determined by an increase in the present of anti-inflammation markers, such as, for example, IL-8. Detailed description of oxidative stress prevention/oxidative stress assays can be found in U.S. patent application Ser. No. 18/349,107, which is incorporated herein by reference in its entirety.
In some embodiments, the MSC secretome can be evaluated for blood compatibility and implementing tests for sterility as well as pyrogen and endotoxin levels. In some embodiments, the MSC secretome can be evaluated blood compatibility. In some embodiments, evaluating blood compatibility includes assays for hemolysis and hemagglutination. In some embodiments, the MSC secretome does not exhibit detrimental effects with systemic exposure. In some embodiments, the MSC secretome does not exhibit detrimental effects with systemic exposure, such as with severe ocular burns. In some embodiments, the MSC secretome does not exhibit hemagglutination activity. In some embodiments, the MSC secretome does not induce hemolysis. In some embodiments, the MSC secretome does not induce hemolytic activity.
In some embodiments, the MSC secretome can be sterile such that it can be administered as part of a pharmaceutical formulation. In some embodiments, the MSC secretome can be free or substantially free of endotoxins. In some embodiments, the MSC secretome can be free or substantially free of microorganisms.
In some embodiments, the biophysical characteristics of the MSC secretome can be evaluated and/or determined. In some embodiments, the fluorescence, static light scattering and dynamic light scatting to characterize protein stability metrics. In some embodiments, the following parameters can be measured to further characterize the secretome: thermal melting, thermal aggregation, Delta G, and/or viscosity. In some embodiments, a thermal melting assay is employed to determine MSC secretome stability. In some embodiments, a thermal aggregation assay is employed to determine MSC secretome stability. In some embodiments, delta G is employed as a measure for determining MSC secretome stability. In some embodiments, viscosity is measured as an MSC secretome characteristic. In some embodiments, viscosity is to determine MSC secretome stability.
In some embodiments, biophysical metrics can be employed to establish stability parameters for characterizing different MSC secretome formulations.
In some embodiments, the MSC secretome is stable at −20° C., 4° C., and room temperature (20° C.), for at least 7 days. In some embodiments, the MSC secretome is stable −20° C., 4° C., and room temperature (20° C.), for at least 14 days. In some embodiments, the MSC secretome is stable for at least 7 days, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month. In some embodiments, the MSC secretome is stable for at least 7 days, at least 14 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, or at least 3 months at about −20° C. In some embodiments, the MSC secretome is stable for at least 7 days, at least 14 days, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month at about 4° C. In some embodiments, the MSC secretome is stable for at least 7 days, at least 14 days, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month at about 20° C. (or room temperature).
In some embodiments, the MSC secretome is stable for at least 7 days at about −20° C. In some embodiments, the MSC secretome is stable for at least 7 days at about 4° C. In some embodiments, the MSC secretome is stable for at least 7 days at about 20° C. In some embodiments, the MSC secretome is stable for at least 7 days at about 25° C. (room temperature).
In some embodiments, the MSC secretome is stable for at least 14 days at about −20° C. In some embodiments, the MSC secretome is stable for at least 14 days at about 4° C. In some embodiments, the MSC secretome is stable for at least 14 days at about 20° C. (or room temperature). In some embodiments, the MSC secretome is stable for at least 14 days at about 25° C. (room temperature).
The corneal epithelium, more precisely, the apical surface of the epithelium has a major contribution to the overall barrier properties of the cornea and change to the corneal barrier serves as a sensitive factor for biocompatibility analysis. In some embodiments, the biophysical characteristics of the MSC secretome can be evaluated and/or determined such as by an epithelial barrier integrity assay. In some embodiments, the epithelial barrier integrity assay is a transepithelial electrical resistance (TEER). In some embodiments, the transepithelial electrical resistance (TEER) can be assessed to measure overall barrier properties. In some embodiments, 3D tissues can be transferred into 24-well plates containing 2 mL of TEER buffer and incubated for 10 min. In some embodiments, TEER can be measured using an epithelial volt-ohm meter EVOMÒ and the EndOhm-12 chamber (World Precision, Sarasota, FL). In some embodiments, at the end of the procedure, tissues can be used for tissue viability assessment using the following formula:
% Barrier integrity=100×[TEER(treated tissue)/TEER(placebo control)]
In some embodiments, TEER can be employed to evaluate the effect on barrier integrity after topical application of the MSC secretome. In some embodiments, TEER can be employed to evaluate the effect on barrier integrity after topical application of the MSC secretome following corneal epithelial damage caused by topical exposure to nitrogen mustard (NM) utilizing the EpiCorneal tissue model (MatTek Corp). In some embodiments, MSC secretome can be applied topically, for example at 6 μg/ml (diluted in Placebo solution), as described in Example 6. In some embodiments, EpiCorneal tissues were cultured in 5 ml medium at standard culture conditions for 24 h.
In some embodiments, bioassays can be employed to characterize the MSC secretome. In some embodiments, bioassays can be related to corneal wound healing: epithelial cell migration and proliferation, stromal cell differentiation (e.g., scarring); neovascularization, and inflammation. In some embodiments, bioassays can be employed to evaluate the ability of the MSC secretome to mediate corneal wound healing: epithelial cell migration and proliferation, stromal cell differentiation (scarring); neovascularization; and inflammation.
In some embodiments, the MSC secretome can be evaluated for the ability of the MSC secretome to promote proliferation and migration. In some embodiments, the MSC secretome can be evaluated for the ability of the MSC secretome to promote proliferation. In some embodiments, the MSC secretome can be evaluated for the ability of the MSC secretome to promote migration. In some embodiments, the MSC secretome promotes proliferation and/or migration. In some embodiments, the MSC secretome promotes proliferation. In some embodiments, the MSC secretome promotes migration. In some embodiments, the MSC secretome can be evaluated use a transwell migration assay to determine proliferation promoting ability.
In some embodiments, a migration assay can be employed to evaluate for the ability of the MSC secretome to promote migration. In some embodiments, a migration assay can be employed to evaluate for the ability of the MSC secretome to promote migration, wherein the migration assay is an in vitro wound closure assay. In some embodiments, the migration assay can include a “scratch assay” (also referred to as a “scratch wound assay”). In some embodiments, the MSC secretome promotes migration and this promotion of migration is determined and/or examined utilizing a “scratch assay”. Generally, a scratch assay method is based on when artificial gap, also referred to as a “scratch”, occurs on a confluent cell monolayer. The “scratch” can be monitored for the cells on the edge of the newly created gap migrating toward the opening to close/cover the “scratch”. See, for example, Liang, C., Park, A. & Guan, J. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2, 329-333 (2007).)
In some embodiments, the migration assay can include a transwell migration assay employing corneal epithelial cells (or other cell surrogate once validation)—(e.g., wound closure) can be performed on the MSC secretome. In some embodiments, a transwell migration assay employing corneal epithelial as a test for wound closure potency of the MSC secretome. In some embodiments, the MSC secretome promotes wound closure as determined using a transwell migration assay.
In some embodiments, in vitro wound closure assays include but are not limited to a “scratch assay” (also referred to as a “scratch wound assay”) or a circular scratch wound method or circular scratch wound assay or circular wound closure assay.
In some embodiments, human corneal epithelial cell proliferation assays can be performed on the MSC secretome. In some embodiments, human corneal epithelial cell proliferation assays are indicative of a test for wound closure properties of the MSC secretome. In some embodiments, the MSC secretome promotes wound closure as determined using a human corneal epithelial cell proliferation assay.
In some embodiments, a circular scratch wound method or circular scratch wound assay or circular wound closure assay can be employed. In some embodiments, the Oris™ Cell Migration Assay platform can be employed (see, also, as described herein in Example 6).
In some embodiments, an endothelial cell tube formation assay can be performed on the MSC secretome. In some embodiments, an endothelial cell tube formation assays can be indicative that the MSC secretome is not pro-angiogenic. In some embodiments, an endothelial cell tube formation assay provides a measure of the angiogenic potential of the MSC secretome. In some embodiments, the MSC secretome exhibits anti-angiogenic properties. In some embodiments, the MSC secretome is anti-angiogenic properties. In some embodiments, an endothelial cell tube formation assay provides the ratio of anti-angiogenesis signals and pro-angiogenesis signals. In some embodiments, an endothelial cell tube formation assay negative result will confirm the anti: pro ratio is high and will ensure the MSC secretome will not promote neovascularization. In some embodiments, an endothelial cell tube formation assay negative result will confirm the anti:pro ratio is high and will ensure the MSC secretome will not promote CNV (choroidal neovascularization) or neovascularization in general. In some embodiments, an inhibition of TGFb induced myofibroblast differentiation assay can be performed on the MSC secretome. In some embodiments, an inhibition of TGFb induced myofibroblast differentiation assay can be performed on the MSC secretome to show that the MSC secretome prevents scarring. In some embodiments, the MSC secretome prevents scarring. In some embodiments, the MSC secretome prevents scarring corneal opacity. In some embodiments, the MSC secretome has low angiogenesis induction. In some embodiments, the MSC secretome has reduced angiogenic response. In some embodiments, the MSC secretome has reduced angiogenic capacity. In some embodiments, the MSC secretome impairs and/or reduces the normal formation of blood vessels in presence of media supportive of angiogenesis. In some embodiments, the MSC secretome has reduced angiogenic capacity when the MSC secretome is compared to untreated control. In some embodiments, the MSC secretome has reduced angiogenic capacity as compared to a sample treated to serum containing media. In some embodiments, the MSC secretome attenuates an angiogenic response. In some embodiments, the MSC secretome reduces the angiogenic response induce by serum free media. In some embodiments, a reduction in angiogenic response is induced by the MSC secretome when secretome plus serum containing media (reduced or no angiogenic response) is compared to serum containing media (angiogenic response). In some embodiments, an angiogenic response is indicated by tube formation in a cell-based assay. In some embodiments, an angiogenic response is indicated by tube formation in an endothelial cell tube formation assay.
In some embodiments, the present invention provides a composition that induces ocular wound healing comprising a mesenchymal stem cell (MSC) secretome and a tonicity modifying agent, wherein the ability of the composition to promote ocular wound healing is indicated by a wound healing assay comprising:
In some embodiments, the corneal cells are corneal epithelial cells. In some embodiments, the corneal cells are corneal keratocytes (or fibroblasts).
In some embodiments, the layer of corneal cells is a confluent monolayer.
In some embodiments, the wound gap is introduced by mechanically disrupting the layer of corneal cells. In some embodiments, the wound gap is introduced by chemically disrupting the layer of corneal cells. In some embodiments, the wound gap comprises a linear gap. In some embodiments, the wound gap comprises a circular gap.
In some embodiments, the determining whether the wound gap closes in step (c) comprises detecting and quantitating daily the migration and/or proliferation of the corneal cells within the wound gap. In some embodiments, the migration and/or proliferation of the corneal cells is characterized as the number of corneal cells migrated and/or proliferated within the wound gap. In some embodiments, determining whether the wound gap heals in step (c) comprises measuring the size of the wound gap daily, wherein the size of the wound gap is expressed as a percentage of the initial size of the wound gap measured immediately after the wound gap is introduced.
In some embodiments, the size of the wound gap is characterized as the surface area of the wound gap. In some embodiments, the size of the wound gap is characterized as the width of the wound gap.
In some embodiments, the step (c) is performed within a period of 2-4 days after the completion of step (b). In some embodiments, the step (c) is performed within a period of 2 days after the completion of step (b). In some embodiments, the step (c) is performed within a period of 3 days after the completion of step (b).
In some embodiments, the wound healing assay further comprises, prior to administering the composition to the corneal cells, a step of concentrating the composition, and optionally a step of buffer exchanging for the composition. In some embodiments, the wound healing assay further comprises, prior to administering the composition to the corneal cells, a step of diluting the composition, and optionally a step of buffer exchanging for the composition.
In some embodiments, the composition comprises at least 45 μg/ml secretome proteins.
In some embodiments, the MSC secretome can be evaluated for the ability to prevent differentiation and prevent scarring. In some embodiments, the MSC secretome prevents and/or impairs scarring. In some embodiments, the MSC secretome prevents scarring. In some embodiments, the MSC secretome reduces scarring as compared to other standard treatments. In some embodiments, the MSC secretome prevents and/or impairs differentiation. In some embodiments, the MSC secretome prevents and/or impairs myofibroblast differentiation. In some embodiments, the MSC secretome reduces the loss of corneal transparency. In some embodiments, the MSC secretome reduces the loss of corneal transparency by preventing and/or impairing myofibroblast differentiation.
In some embodiments, the MSC secretome can be evaluated for the ability of the MSC secretome to modulate factors involved in differentiation. In some embodiments, the MSC secretome can be evaluated the ability of the MSC secretome to modulate factors involved in differentiation, including but not limited to TGFB2, Collagen I, Collagen III (normally upregulated during differentiation), TFGB3, MMP-2, and MMP-9 (normally downregulated during differentiation. In some embodiments, the MSC secretome modulates factors selected from the group consisting of TGFB2, Collagen I, Collagen III (normally upregulated during differentiation), TFGB3, MMP-2, and MMP-9 (normally downregulated during differentiation. In some embodiments, the MSC secretome induces a decrease in factors upregulated during normal differentiation. In some embodiments, the MSC secretome induces an increase in factors downregulated during normal differentiation. In some embodiments, the MSC secretome induces a decrease in expression of factors such as SMA. In some embodiments, the MSC secretome induces a decrease in expression of factors such as SMA which is indicative of MSC secretome potency.
In some embodiments, the MSC secretome can be evaluated for the ability to prevent neovascularization. In some embodiments, the MSC secretome prevents, impairs, inhibits, and/or reduces neovascularization. In some embodiments, the MSC secretome inhibits or does not promote neovascularization. In some embodiments, the MSC secretome can be evaluated for the ability to prevent angiogenesis. In some embodiments, the MSC secretome prevents, impairs, inhibits, and/or reduces angiogenesis. In some embodiments, the MSC secretome inhibits angiogenesis.
In some embodiments, the MSC secretome can be further evaluated using depletion assays. In some embodiments, the MSC secretome can be depleted of specified factors. In some embodiments, the MSC secretome can be depleted of specified factors, including for example, but not limited to TIMP-1 and/or Serpin E1. In some embodiments, the MSC secretome can be depleted of TIMP-1 and/or Serpin E1. In some embodiments, the MSC secretome can be depleted of TIMP-1. In some embodiments, the MSC secretome can be depleted of Serpin E1.
In some embodiments, the MSC secretome can be evaluated for the ability to prevent, impair, inhibit, and/or reduce inflammation. In some embodiments, the MSC secretome prevents, impairs, inhibits, and/or reduces inflammation. In some embodiments, the MSC secretome inhibits inflammation. In some embodiments, the MSC secretome is characterized in vitro and/or in vivo to determine the ability to prevent, impair, inhibit, and/or reduce inflammation. In some embodiments, the MSC secretome prevents, impairs, inhibits, and/or reduces inflammation in vitro and/or in vivo. In some embodiments, the MSC secretome prevents, impairs, inhibits, and/or reduces inflammation in vitro. In some embodiments, the MSC secretome prevents, impairs, inhibits, and/or reduces inflammation or in vivo. In some embodiments, a tissue model can be employed to characterizing preventing, impairing, inhibiting, and/or reducing inflammation in vitro. In some embodiments, a 3D tissue model can be employed to characterizing preventing, impairing, inhibiting, and/or reducing inflammation in vitro. In some embodiments, a nitrogen mustard (NM) gas burn model can be used to evaluate preventing, impairing, inhibiting, and/or reducing inflammation in vitro. In some embodiments, a nitrogen mustard (NM) gas burn model can be used to evaluate preventing, impairing, inhibiting, and/or reducing inflammation in vitro and as a surrogate for in vivo conditions. In some embodiments, the cytokine profile in response to treatment with and/or administration of the MSC secretome can be determined. In some embodiments, the levels of specific cytokines can be determined. In some embodiments, the level of IL-8 can be determined. In some embodiments, the level of IL-8 expression can be reduced in tissues treated with the MSC secretome. In some embodiments, the level of IL-8 expression is reduced in tissues treated with the MSC secretome and this is indicative of preventing, impairing, inhibiting, and/or reducing inflammation.
Further descriptions of the characterization assays for the MSC secretome composition are provided in International Application Nos. PCT/US20/27093, PCT/US23/73573, and PCT/US23/69840; and US Patent Publication No. US 2021/0369617, which are incorporated herein by reference in their entireties.
The present disclosure also provides methods of treatment using the MSC secretome of the present disclosure. In particular, the MSC secretome finds use in the treatment of ocular conditions. In particular, the MSC secretome finds use in the treatment of ocular conditions, including but not limited to ocular diseases. In some embodiments, the ocular disease is associated with the ocular surface. In some embodiments, the ocular disease is associated with damaged ocular tissue and/or damaged ocular tissue indications. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, including accelerating wound healing. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, including reducing scarring. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, including reducing inflammation. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, including reducing inflammation and thus promoting growth. In some embodiments, the MSC secretome finds use in treating ocular conditions such as reducing inflammation at the ocular surface. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, including reducing neovascularization. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, including reducing neovascularization in the cornea. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, including dry eye treatment (including, for example, treatment of severe dry eye, including where the epithelial cells are damaged). In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, such as restoring the integrity to damaged ocular tissue. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, such as accelerating the healing of damaged ocular tissue. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, such as treating a retinal condition. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, such as treating a macular disease. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, such as regenerating damaged ocular nerve tissue. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, such as regenerating damaged ocular nerve tissue associated with PCED. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, such as PCED. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, such as inflammatory damage to the eye surface. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, such as for example GvHD and/or Sjogren's syndrome. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, such as limbal stem cell deficiency (LSCD).
In some embodiments, the MSC secretome composition provided herein finds use in the treatment of limbal stem cell deficiency (LSCD).
In some embodiments, the LSCD arises due an injury or disease or disorder selected from the group consisting of chemical burns, thermal burns, radiation injury, aniridia, sclerocornea, multiple endocrine neoplasia, Stevens Johnson syndrome, ocular cicatricial pemphigoid, collagen vascular diseases, chronic non-auto-immune inflammatory disorders arising from contact lens use, dry eye disease, rosacea, staph marginal, keratitis (including bacterial, fungal & viral keratitis), pterygia or neoplasm, limbal stem cell deficiency arising after multiple eye surgeries or excision of pterygia or neoplasm or cryotherapy; and limbal stem cell deficiency arising as a result of medication toxicity from a medication such as a medication selected from the group consisting of preservatives (e.g., thimerosal, benzalkonium), topical anesthetics, pilocarpine, beta blockers, mitomycin, 5-fluorouracil, silver nitrate, and oral medications causing Stevens Johnson syndrome. In some embodiments, the LSCD arises due to ocular autoimmune degenerations, ultraviolet and ionizing radiation, contact lens wear, acute thermal or chemical burns, allergic/atopic conjunctivitis, drug toxicity, or ocular surgery involving the limbus.
In some embodiments, the present invention further comprises, prior to the disclosed methods of treatment for LSCD, characterizing the LSCD by one or more ocular examinations including but not limited to BCVA (Best-Corrected Distance Visual Acuity), slit lamp examination, corneal photo without fluorescein, corneal photo with fluorescein, lesion size measurements, corneal sensitivity, corneal topography, and dilated ophthalmoscopy.
In some embodiments, the LSCD is unilateral. In some embodiments, the LSCD is bilateral. In some embodiments, the LSCD is unilateral. In some embodiments, the LSCD is partial. In some embodiments, the LSCD is unilateral. In some embodiments, the LSCD is complete or total.
In some embodiments, the LSCD is present in one eye of the subject. In some embodiments, the LSCD is present in both eyes of the subject.
In some embodiments, the LSCD is at stage 1. In some embodiments, the LSCD is at stage 2. In some embodiments, the LSCD is at stage 3.
In some embodiments, the subject has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more lesions in the eye(s) affected by the LSCD.
In some embodiments, the subject exhibits normal corneal epithelium within the central 5 mm zone of the cornea or the visual axis in the eye(s) affected by the LSCD. In some embodiments, the central 5 mm zone of the cornea or the visual axis is affected by the LSCD. In some embodiments, the entire corneal surface is affected by the LSCD.
In some embodiments, one or more of the lesions exhibit about less than 50% of limbal involvement. In some embodiments, one or more of the lesions exhibit about 50-100% limbal involvement. In some embodiments, one or more of the lesions exhibit 100% of limbal involvement.
In some embodiments, the LSCD is characterized by a reduction of about 1-100% of the limbal stem cell in the eye(s) affected as compared to an unaffected eye. In some embodiments, the LSCD is characterized by a reduction of about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%, of the limbal stem cell in the eye(s) affected as compared to an unaffected eye. In some embodiments, the LSCD is characterized by a reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, of the limbal stem cell in the eye(s) affected. In some embodiments, the LSCD is characterized by a reduction of about 1-50% of the limbal stem cell in the eye(s) affected. In some embodiments, the LSCD is characterized by a reduction of about 50% of the limbal stem cell in the eye(s) affected. In some embodiments, the LSCD is characterized by a reduction of about 50-100% of the limbal stem cell in the eye(s) affected.
In some embodiments, prior to receiving the presently disclosed treatment, the subject has received and/or is refractory to one or more treatments comprising preservative free artificial tears, therapeutic contact lenses (such as bandage contact lens, the PROSE device, or scleral lens), topical Vitamin A ointment, short-term pulse topical corticosteroids (such as methylprednisolone 1%, loteprednol etabonate 0.5% or 0.2%, or prednisolone acetate 1%, and cyclosporine 0.05%), Prokera, Oxervate® (cenegermin-bkbj), or surgical procedures including but not limited to limbal stem cell/limbal cell transplantation, contralateral conjunctival limbal autograft, oral mucosal epithelial transplantation, amniotic membrane transplantation, or keratoprosthesis.
In some embodiments, the MSC secretome composition provided herein suppresses progression of the LSCD in the subject. In some embodiments, the MSC secretome composition provided herein reverses, halt, or slows down the progression of the LSCD in the subject.
In some embodiments, the MSC secretome composition provided herein induces wound healing in one or more of the lesions in the affected eye(s). In some embodiments, the MSC secretome composition provided herein induces 1-100% wound healing in one or more of the lesions in the affected eye(s). In some embodiments, the MSC secretome composition provided herein induces about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, wound healing in one or more of the lesions in the affected eye(s).
In some embodiments, the MSC secretome composition provided herein reduces the size of one or more of the lesions in the affected eye(s). In some embodiments, the MSC secretome composition provided herein induces a 1-100% reduction in the size of one or more of the lesions in the affected eye(s). In some embodiments, the MSC secretome composition provided herein induces about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, reduction in the size of one or more of the lesions in the affected eye(s).
In some embodiments, the MSC secretome composition provided herein induces increase in the population of limbal stem cells in the affected eye(s). In some embodiments, the MSC secretome composition provided herein induces an 1-100% increase in the population of limbal stem cells in the affected eye(s). In some embodiments, the MSC secretome composition provided herein induces about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, increase in the population of limbal stem cells in the affected eye(s).
In some embodiments, the MSC secretome composition provided herein induces increase in the population of limbal stem cells in the affected eye(s). In some embodiments, the MSC secretome composition provided herein induces an 1- to 10-fold increase in the population of limbal stem cells in the affected eye(s). In some embodiments, the MSC secretome composition provided herein induces about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or more, increase in the population of limbal stem cells in the affected eye(s).
In some embodiments, reduced in symptoms and/or decreased disease state by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% is indicative of therapeutic efficacy of the MSC secretome composition for treating LSCD.
In some embodiments, the MSC secretome finds use in accelerating wound healing. In some embodiments, the MSC secretome finds use in reducing scarring. In some embodiments, the MSC secretome finds use in reducing inflammation. In some embodiments, the MSC secretome finds use in reducing inflammation and thus promoting growth. In some embodiments, the MSC secretome finds use in reducing inflammation at the ocular surface. In some embodiments, the MSC secretome finds use in reducing neovascularization. In some embodiments, the MSC secretome finds use in reducing neovascularization in the cornea. In some embodiments, the MSC secretome finds use in the protection and repair of retinal epithelial cells and retinal ganglion cells. In some embodiments, the MSC secretome finds use in induction of trabecular meshwork regeneration and reduction of intraocular pressure.
In some embodiments, the mesenchymal stem cell secretome is administered for the treatment of an ocular disease. In some embodiments, treatment comprises administering to a patient in need thereof therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein to a patient in need thereof. In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof in order to promote or induce ocular wound healing. In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof in order to reduce and/or inhibit neovascularization, reduce and/or inhibit scarring, promote and/or preserve vision, and/or increasing wound closure rate (e.g., decreasing wound closure time). In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof in order to prevent, reduce, and/or inhibit neovascularization. In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof in order to prevent, reduce, and/or inhibit reducing scarring. In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof in order to promote and/or preserve vision. In some embodiments, the mesenchymal stem cell secretome is administered to promote and/or induce closing wound faster wound closure (e.g., reduce the amount of time required for wound closure). In some embodiments, the mesenchymal stem cell secretome prevents, reduces, and/or inhibits or does not promote neovascularization and reducing scarring in order to promote vision preservation. In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof in order to prevent, reduce, and/or inhibit neovascularization and reducing scarring in order to promote vision preservation. In some embodiments, the mesenchymal stem cell secretome prevents, reduces, and/or inhibits inflammation. In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof in order to prevent, reduce, and/or inhibit inflammation.
In some embodiments, the mesenchymal stem cell secretome is administered for the treatment of a visual dysfunction following traumatic injury to ocular structures. In some embodiments, treatment comprises administering to a patient in need thereof a therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein.
In some embodiments, the mesenchymal stem cell secretome is administered for the treatment of a traumatic injury of the optic nerve degeneration following concussive injury. In some embodiments, the concussive injury to the eye is selected from the group consisting of ocular contusion and blunt injury to the eye. In some embodiments, the mesenchymal stem cell secretome is administered for the treatment of a traumatic injury of the optic nerve. In some embodiments, treatment comprises administering to a patient in need thereof a therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein.
In some embodiments, the mesenchymal stem cell secretome is administered for ameliorating optic nerve degeneration following concussive injury to the eye. In some embodiments the method for ameliorating optic nerve degeneration comprises administering to the patient a therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein. In some embodiments, the concussive injury to the eye is selected from the group consisting of ocular contusion and blunt injury to the eye. In some embodiments, the concussive injury to the eye an ocular contusion. In some embodiments, the concussive injury to the eye a blunt injury to the eye.
Efficacy readouts can include a reduced in symptoms and/or decreased disease state, including for example, increased quality of life. In some embodiments, reduced in symptoms and/or decreased disease state by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% is indicative of therapeutic efficacy. In some embodiments, reduction in inflammation by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% is indicative of therapeutic efficacy. In some embodiments, a reduction in scarring by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% is indicative of therapeutic efficacy. In some embodiments, a reduction in neovascularization by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% is indicative of therapeutic efficacy.
In some embodiments, the disease or conditions an ocular disease or ocular condition. In some embodiments, the disease or condition is a visual dysfunction following traumatic injury to ocular structures. In some embodiments, the disease or condition is a concussive (e.g., blunt or non-blunt) injury to the eye. In some embodiments, the disease or condition is a burn, including a chemical burn to the eye.
In some embodiments, the mesenchymal stem cell secretome is administered to a particular targeted area. In some embodiments, the particular targeted area is the eye. In some embodiments, the mesenchymal stem cell secretome is administered to a particular targeted area and is formulated so as not to spread to other surrounding areas.
In some embodiments, the mesenchymal stem cell secretome is administered to a particular targeted area and is formulated so as not to spread to other surrounding areas.
In some embodiments, the mesenchymal stem cell secretome is administered to a particular targeted area and is formulated to stay in the targeted area for at least 1 minute, at least about 2 minutes, 3 at least about minutes, at least about 4 minutes, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 60 minutes, at least about 70 minutes, at least about 80 minutes, at least about 90 minutes, or at least about 2 hours.
In some embodiments, the mesenchymal stem cell secretome is administered to an affected area immediately after the wound or injury. In some embodiments, the mesenchymal stem cell secretome is administered to an affected area within 15 seconds, 30 seconds, 1 minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, or 96 hours.
In some embodiments, the mesenchymal stem cell secretome is administered topically. In some embodiments, the mesenchymal stem cell secretome is administered by subconjunctival injection. In some embodiments, the mesenchymal stem cell secretome is administered by intravitreal injection. In some embodiments, the MSC secretome compositions exhibit ultrapotency when administered to a subject in need thereof. In some embodiments, the mesenchymal stem cell secretome is administered topically once, two, three, four, five, and/or up to six times daily. In some embodiments, the MSC secretome compositions allow for therapeutic efficacy with one drop or one administration per day. In some embodiments, one drop is administered 1, 2, 3, 4, 5, or 6 times per day. In some embodiments, one drop is administered at 1 hour, 2 hour, 3 hour, or 4 hour intervals. In some embodiments, one drop is administered at least once per day for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In some embodiments, one drop is administered at least twice per day for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In some embodiments, one drop is administered at least 3 times per day for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In some embodiments, one drop is administered at least 4 times per day for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In some embodiments, one drop is administered at least 5 times per day for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In some embodiments, one drop is administered at least 6 times per day for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks.
In some embodiments, the MSC secretome composition is administered topically to a subject having LSCD. In some embodiments, the MSC secretome composition provided herein is topically administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, the MSC secretome composition is topically administered to the subject as needed during a day.
In some embodiments, about 0.1-10 U/mL of the MSC secretome composition is topically administered to the subject in need thereof. In some embodiments, the MSC secretome composition administered in accordance with the embodiments herein has a concentration of about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 U/mL, or more.
In some embodiments, about 0.1-10 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily.
In some embodiments, about 0.1 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 0.1 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 0.5 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 0.5 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 1 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 1 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 1.5 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 1.5 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 2 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 2 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 2.5 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 2.5 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 3 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 3 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 3.5 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 3.5 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 4 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 4 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 4.5 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 4.5 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 5 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 5 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 5.5 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 5.5 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 6 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 6 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 6.5 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 6.5 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 7 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 7 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 7.5 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 7.5 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 8 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 8 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 8.5 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 8.5 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 9 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 9 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 9.5 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 9.5 U/mL of the MSC secretome composition is administered to the subject as needed during a day. In some embodiments, about 10 U/mL of the MSC secretome composition is administered to the subject in need thereof about once, two, three, four, five, or up to six times daily. In some embodiments, about 10 U/mL of the MSC secretome composition is administered to the subject as needed during a day.
In some embodiments, about 1 U/mL of the MSC secretome composition is administered about four times a day to a subject having LSCD. In some embodiments, about 3 U/mL of the MSC secretome composition is administered about four times a day to a subject having LSCD. In some embodiments, about 1-3 drops of the MSC secretome composition having a concentration of about 1 U/mL or 3 U/mL is administered about four times a day to a subject having LSCD in accordance with the embodiments provided herein.
In some embodiments, about 0.3-1.5 mL of the MSC secretome composition having a concentration of about 1 U/mL or about 3 U/mL is administered to the subject about four times a day. In some embodiments, about 0.3-1.5 mL of the MSC secretome composition having a concentration of about 1 U/mL or about 3 U/mL is administered to the subject as needed during a day. In some embodiments, about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 mL of the MSC secretome composition having a concentration of about 1 U/mL or about 3 U/mL is administered to the subject about four times a day. In some embodiments, about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 mL of the MSC secretome composition having a concentration of about 1 U/mL or about 3 U/mL is administered to the subject as needed during a day.
In some embodiments, about 0.1-5 U of the MSC secretome composition is administered to the subject about once, two, three, four, five, or up to six times daily. In some embodiments, about 0.1 U of the MSC secretome composition is administered to the subject about once, two, three, four, five, or up to six times daily. In some embodiments, about 0.1 U of the MSC secretome composition is administered to the subject as needed daily. In some embodiments, about 0.5 U of the MSC secretome composition is administered to the subject about once, two, three, four, five, or up to six times daily. In some embodiments, about 0.5 U of the MSC secretome composition is administered to the subject as needed daily. In some embodiments, about 1 U of the MSC secretome composition is administered to the subject about once, two, three, four, five, or up to six times daily. In some embodiments, about 1 U of the MSC secretome composition is administered to the subject as needed daily. In some embodiments, about 1.5 U of the MSC secretome composition is administered to the subject about once, two, three, four, five, or up to six times daily. In some embodiments, about 1.5 U of the MSC secretome composition is administered to the subject as needed daily. In some embodiments, about 2 U of the MSC secretome composition is administered to the subject about once, two, three, four, five, or up to six times daily. In some embodiments, about 2 U of the MSC secretome composition is administered to the subject as needed daily. In some embodiments, about 2.5 U of the MSC secretome composition is administered to the subject about once, two, three, four, five, or up to six times daily. In some embodiments, about 2.5 U of the MSC secretome composition is administered to the subject as needed daily. In some embodiments, about 3 U of the MSC secretome composition is administered to the subject about once, two, three, four, five, or up to six times daily. In some embodiments, about 3 U of the MSC secretome composition is administered to the subject as needed daily. In some embodiments, about 3.5 U of the MSC secretome composition is administered to the subject about once, two, three, four, five, or up to six times daily. In some embodiments, about 3.5 U of the MSC secretome composition is administered to the subject as needed daily. In some embodiments, about 4 U of the MSC secretome composition is administered to the subject about once, two, three, four, five, or up to six times daily. In some embodiments, about 4 U of the MSC secretome composition is administered to the subject as needed daily. In some embodiments, about 4.5 U of the MSC secretome composition is administered to the subject about once, two, three, four, five, or up to six times daily. In some embodiments, about 4.5 U of the MSC secretome composition is administered to the subject as needed daily. In some embodiments, about 5 U of the MSC secretome composition is administered to the subject about once, two, three, four, five, or up to six times daily. In some embodiments, about 5 U of the MSC secretome composition is administered to the subject as needed daily.
In some embodiments, the MSC secretome composition is administered to the subject daily for about 1, 2, 3, 4, 5, 6, 7, or 8 weeks, 1 month, 2 months, 3 months, 4 months, or longer.
In some embodiments, 1-3 drops of the MSC secretome composition at 0.1-10 U/mL is administered to the subject about once, two, three, four, five, or up to six times daily, for about 1, 2, 3, 4, 5, 6, 7, or 8 weeks, 1 month, 2 months, 3 months, 4 months, or longer. In some embodiments, 1 drop of the MSC secretome composition at 1 U/mL is administered the subject 4 times daily for about 8 weeks. In some embodiments, 1 drop of the MSC secretome composition at 3 U/mL is administered the subject 4 times daily for about 8 weeks.
In some embodiments of the method of treatment for LSCD in a subject in need thereof comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition comprises:
In some embodiments, the MSC secretome composition for use in the methods of treatment further comprises high levels of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1.
In some embodiments, the MSC secretome composition for use in the methods of treatment comprises 1 ng/ml-100 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition for use in the methods of treatment comprises 1 ng/ml-200 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition for use in the methods of treatment comprises 1 ng/ml-300 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1. In some embodiments, the MSC secretome composition for use in the methods of treatment comprises 1 ng/mL-400 ng/ml of at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1.
In some embodiments, the MSC secretome composition for use in the methods of treatment further comprises mid-range levels of at least one factor selected from the group consisting of Angiopoietin-1, Angiopoietin-2, Amphiregulin, Endostatin, Endothelin-1, Thrombospondin-2, Thrombospondin-1, Angiogenin, DPPIV, IGFBP-3, and uPA.
In some embodiments, the MSC secretome composition for use in the methods of treatment comprises 400 pg/mL-3000 pg/mL of at least one factor selected from the group consisting of Angiopoietin-1, Angiopoietin-2, Amphiregulin, Endostatin, Endothelin-1, Thrombospondin-2, Thrombospondin-1, Angiogenin, DPPIV, IGFBP-3, and uPA.
In some embodiments, the MSC secretome composition for use in the methods of treatment further comprises at least one factor selected from the group consisting of Apolipoprotein A1, Complement Factor D, Complement factor H, Complement factor I, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, Complement receptor type 1 (CR1), C-reactive protein, Cystatin C, DKK-1, Emmprin, Osteopontin, Vitamin D Binding Protein, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.
In some embodiments, the MSC secretome composition for use in the methods of treatment comprises ratios of anti-angiogenic to pro-angiogenic wherein the ratio is >2, >3, >4, or >5. In some embodiments, the anti-angiogenic factors include one or more factors selected from the group consisting of PEDF, lower levels of VEGF, and Serpin E1 and pro-angiogenic: VEGF, Angiogenin, IGFBP-3, uPA, Angio-1, Angio-2, Endothelin-1.
In some embodiments, the MSC secretome composition for use in the methods of treatment further comprises low levels for VEGF. In some embodiments, the MSC secretome for use in the methods of treatment comprises 1 pg/mL-400 pg/mL of VEGF. In some embodiments, the level of VEGF is 5-10 fold lower than the level of Serpin E1. In some embodiments, the MSC secretome composition for use in the methods of treatment comprises one or more anti-angiogenic factors, and wherein the ratio of the sum of the concentration of the one or more anti-angiogenic factors relative to the concentration of VEGF is >2, >3, >4, or >5.
In some embodiments, the MSC secretome composition for use in the methods of treatment does not comprise or comprises very low levels of bFGF, PLGF, and PDGF.
In some embodiments, the MSC secretome composition for use in the methods of treatment comprises less than 1000 pg/mL of bFGF, PLGF, and PDGF.
In some embodiments, the MSC secretome composition for use in the methods of treatment has a pH of about 4.7 to about 7.5.
In some embodiments, the MSC secretome composition for use in the methods of treatment is formulated in a buffer system selected from the group consisting of di/mono sodium phosphate, sodium citrate/citric acid, boric acid/sodium citrate, boric acid/sodium tetraborate, and citric acid/disodium phosphate.
In some embodiments, the MSC secretome composition for use in the methods of treatment further comprises a tonicity modifying agent. In some embodiments, the tonicity modifying agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.
In some embodiments, the MSC secretome composition for use in the methods of treatment further comprises mono/di-sodium phosphate, mannitol, and trehalose, and wherein the composition has a pH of about pH 7.4.
In some embodiments, the MSC secretome composition for use in the methods of treatment further comprises divalent cations. In some embodiments, the divalent cations are selected from the group consisting of Mg2+, Ca2+, and Zn2+.
In some embodiments, the MSC secretome composition for use in the methods of treatment further comprises di-sodium phosphate/citric acid, mannitol, and trehalose, and wherein the composition has a pH of about pH 6.4.
In some embodiments, the MSC secretome composition for use in the methods of treatment further comprises an adhesive agent. In some embodiments, the adhesive agent is selected from the group consisting of hypromellose, Poloxamer 407, Poloxamer 188, Poloxomer 237, Poloxomer 338, Hypromellose, polycarbophil, polyvinylpyrrolidone (PVP), PVA (polyvinyl alcohol), polyimide, sodium hyaluronate, gellan gum, poly(lactic acid-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), hydroxypropyl methylcellulose (HPMC), hydroxy methyl cellulose, hydroxy ethyl cellulose, sodium carboxy methyl cellulose, fibrin glue, polyethyelene glycol, and GelCORE.
In some embodiments, the MSC secretome composition for use in the methods of treatment does not comprise one or more components selected from the group consisting of: xenobiotic components; Phenol red; peptides and biomolecules <3 kDa; antibiotics; protein aggregates >200 nm; cells; non-exosome/non-Extracellular Vesicles cell debris; hormones; and L-glutamine.
In some embodiments, the MSC secretome composition for use in the methods of treatment comprises: HGF; Pentraxin-3 (TSG-14); VEGF; TIMP-1; Serpin E1; and <5 ng/ml IL-8.
In some embodiments, 1 U/mL of the MSC secretome composition comprises:
In some embodiments, 1 U/mL of the MSC secretome composition comprises:
In some embodiments, 1 U/mL of the MSC secretome composition comprises:
In some embodiments, 1 U/mL of the MSC secretome composition comprises:
In some embodiments, the MSC secretome for use in the methods of treatment composition comprises:
In some embodiments, the MSC secretome composition for use in the methods of treatment comprise an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.
In some embodiments, the present disclosure provides a method of treatment for LSCD in a subject in need thereof comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition is a stable mesenchymal stem cell (MSC) secretome formulation comprising:
In some embodiments, the present disclosure provides a method of treatment for LSCD in a subject in need thereof comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition is a stable mesenchymal stem cell (MSC) secretome formulation comprising:
In some embodiments, the MSC secretome composition comprising:
In some embodiments, the present disclosure provides a method of treatment for LSCD in a subject in need thereof comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition is a stable mesenchymal stem cell (MSC) secretome formulation comprising:
In some embodiments, the present disclosure provides a method of treatment for LSCD in a subject in need thereof comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition is a stable mesenchymal stem cell (MSC) secretome formulation comprising:
In some embodiments, the present disclosure provides a method of treatment for LSCD in a subject in need thereof comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition is a stable mesenchymal stem cell (MSC) secretome formulation comprising:
In some embodiments, the present disclosure provides a method of treatment for LSCD in a subject in need thereof comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition is a stable mesenchymal stem cell (MSC) secretome formulation comprising:
A kit can include an MSC secretome in a container or the conditioned media for use in preparing an MSC secretome, also in a container, as disclosed herein, and instructions for use. Additionally, a kit can include components for mixing to prepare a solution for use in an LSCD treatment, and instructions for mixing and use.
The container can include at least one vial, well, test tube, flask, bottle, syringe, or other container means, into which an MSC secretome in a container or the conditioned media for use in preparing an MSC secretome, and in some instances, suitably aliquoted. Where an additional component is provided, the kit can contain additional containers into which this component may be placed. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Containers and/or kits can include labeling with instructions for use and/or warnings.
The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all FIGURES and all references, Genbank sequences, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.
The present invention can provide kits comprising a panel of tests and/or assays for characterizing a MSC secretome, wherein the panel comprises at least two characterization assays, wherein characterization assays are selected from the group consisting of physical component characterizations, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, neovascularization assays, differentiation/scarring assays, inflammation assays, and/or an epithelial barrier integrity assays. In some embodiments, the panel of tests and/or assays identifies a MSC secretome as described herein.
The present invention can provide kits comprising a panel of tests and/or assays for determining consistency between MSC secretome lots, wherein the panel comprises one or more characterization assays, wherein characterization assays are selected from the group consisting of physical component characterizations, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, neovascularization assays, differentiation/scarring assays, inflammation assays, and/or an epithelial barrier integrity assays. In some embodiments, the panel of tests and/or assays identifies a MSC secretome as described herein.
The objective of this study is to investigate the safety and efficacy of bone marrow-derived MSC secretome ophthalmic solution compared to vehicle in subjects who have a documented clinical diagnosis of LSCD.
This is a multi-center, randomized, controlled, double-masked Phase 1 study designed to evaluate the safety and efficacy of 2 doses of topically-administered MSC-S ophthalmic solution (1 U/mL and 3 U/mL) compared with vehicle in subjects who have a clinical diagnosis of LSCD of at least X days' duration at the time of screening. Eligible subjects will be randomly assigned in a 1:1:1 ratio to one of 3 treatment groups and administer study medication (3 U/mL MSC-S, 1 U/mL MSC-S, or vehicle) four times daily (QID) for 8 weeks into the Study Eye (defined below).
The study has 3 periods: Screening Period (7 to 10 days before Day 1), 8-week Treatment Period (Day 1 through Day 56), and a 6-month Follow-up Period (Day 57 through Day 238). There are 9 planned site visits (Screening, Day 1/Baseline, and Days 7, 14, 28, 42, 56, 70, and 238) and 2 non-site visits (telephone calls, Days 21 and 150).
At Visit 1 (the Screening visit), the Investigator will identify an LSCD as the Study Lesion, based on the criteria below. The Study Eye will be defined as the eye with the Study Lesion. If both eyes have a Study Lesion, the Study Eye will be selected as described in Inclusion Criterion #6.
At Visit 1 (Screening), individuals of any gender or race will be eligible for study participation if they:
Are 18 years of age or older.
Provide written informed consent and Health Insurance Portability and Accountability Act (HIPAA) authorization prior to any study-related procedures.
Have a clinical diagnosis of LSCD (secondary to contact lens wear, acute thermal or chemical burns, allergic/atopic conjunctivitis, drug toxicity, or ocular surgery involving the limbus) of at least X days' duration prior to Screening, that is refractory to one or more non-surgical treatments (e.g., preservative free artificial tears, therapeutic contact lenses).
NOTE: The primary cause of the LSCD must be documented on the subject's record.
Have a best-corrected visual acuity (BCVA) of 20/40 to 20/100 Snellen (0.3-0.7 Logarithm of the Minimum Angle of Resolution [Log MAR])
Be willing and able, during the 7- to 10-day washout between the Screening and Day 1/Baseline visits, to self-administer in the Study Eye: preservative-free moxifloxacin ophthalmic solution 0.5% (e.g., Vigamox®) up to 3 times per day (TID), and preservative-free artificial tears (Refresh Classic) up to 4 times per day (QID) as needed. NOTE: the subject must also be willing to continue self-administering these treatments throughout the Treatment Period, along with their randomized study drug. If the Investigator's standard of care (SoC) dosing regimen for moxifloxacin differs from the protocol (e.g., more than 3 times per day), the Sponsor and Medical Monitor will consider use of the Investigator's SoC during the study.
The LSCD is measured during slit-lamp examination at Screening and Day 1/Baseline, as follows:
NOTE: If there is more than one LSCD in the eye, the largest LSCD (based on the estimated area calculated using the 2 linear measurements [length multiplied by width]) will be designated as the Study Lesion.
If bilateral LSCD is present at Screening or Day 1/Baseline, the subject will be eligible, provided:
NOTE: The eye with the larger LSCD will be selected as the Study Eye. The LSCD in the Fellow Eye may be treated with the Investigator's SoC (such as lubrication, bandage soft contact lens, pressure patching, debridement of the edges of the defect, scleral lens, tarsorrhaphy, autologous serum eye drops), excluding treatment with Oxervate™.
Female subjects must be one of the following: a) postmenopausal (defined as no menses for 1 year); b) surgically sterilized or partner is surgically sterilized; c) practicing abstinence as a lifestyle choice (not as a form of contraception); or d) consistently and correctly using protocol-specified birth control for the duration of the study (e.g., a hormonal contraceptive, intrauterine device, diaphragm with spermicide, or condom with spermicide; see Appendix 3). Female subjects who are neither postmenopausal nor surgically sterile require a negative urine pregnancy test at the Screening and Day 1/Baseline visits.
Have the ability and willingness to comply with study procedures.
For subjects to be eligible at Visit 1 (Screening) and/or Visit 2 (Day 1/Baseline), they may not have the criteria listed below:
Any use of topical ophthalmic ointments, gels, or pharmaceutically active products in the Study Eye that are known to impact epithelial healing at any time after the Screening visit and for the duration of the Treatment Period. Exceptions include moxifloxacin and Refresh Classic (i.e., protocol-required washout treatment) and intraocular pressure (IOP)-lowering medications. Subjects using topical ophthalmic IOP-lowering medications must be stable on the medication and dose regimen for at least 30 days by Day 1/Baseline, in order to be eligible for entry.
Anticipated use of topical ocular treatment in the Study Eye at any time after the Screening visit and for the duration of the Treatment Period. No topical treatments can be administered in the Study Eye during the Treatment Period, except for those explicitly permitted or required by the study protocol.
Any active infectious ocular etiology related to LSCD in the Study Eye.
Any active ocular infection (bacterial, viral, fungal, or protozoal) or active ocular inflammation not related to LSCD in the Study Eye.
Severe corneal burns in the Study Eye.
The circumference affected by limbal blood vessel ischemia greater than 75% of the circumference in the Study Eye.
Severe blepharitis or severe meibomian gland disease in the Study Eye.
Severe eyelid abnormalities in the Study Eye, contributory to the persistence of the LSCD.
Evidence of calcific corneal plaque (Band Keratopathy) in the Study Eye.
Evidence of corneal ulceration involving the posterior third of the corneal stroma, corneal melting, Descemetocele or impending or actual perforation in the Study Eye.
An anticipated need for punctal occlusion in the Study Eye, from the Screening visit through the end of the Treatment Period.
NOTE: Subjects with punctal occlusion or punctal plugs inserted prior to the study are eligible for enrollment if the punctal occlusion will be maintained during the Treatment Period.
Previous use of Oxervate in the Study Eye within the past 30 days prior to Screening.
NOTE: Subjects who have a history of treatment with Oxervate in the Fellow Eye are eligible (whether treatment was deemed successful or not); however, Oxervate may not be used concomitantly during study participation.
History of any surgical procedure(s) for the treatment of the study LSCD (e.g., complete tarsorrhaphy, conjunctival flap, grafting) in the Study Eye. Exceptions: Subjects undergoing amniotic membrane transplantation are eligible if there is no evidence of amniotic membrane at the Screening visit.
History of any other ocular surgery (including laser or refractive surgical procedures) in the Study Eye within the 90 days prior to the Screening visit, except if the ocular surgery was thought by the Investigator to be the cause of the LSCD.
NOTE: Debridement of the damaged epithelium at the LSCD edge is not allowed if within 14 days of Screening visit through the end of the Treatment Period.
Known hypersensitivity to any of the components of the study drug, preservative-free moxifloxacin ophthalmic solution 0.5% (e.g., Vigamox), preservative-free artificial tears (Refresh Classic), or procedural medications (e.g., fluorescein).
Any anticipated use of therapeutic contact lenses or contact lenses for refractive correction in the Study Eye from the Screening visit through the end of the Treatment Period. Subjects using Prokera® are eligible if Prokera is removed at least one day prior to Screening visit.
Any use of Botox (botulinum toxin) injections to induce pharmacologic blepharoptosis in the 90 days prior to the Screening visit, or anticipated use at any time during the Treatment Period.
Any anticipated use of systemic doxycycline from the Screening visit through the end of the Treatment Period.
Any use of chemotherapeutic agents within 7 days prior to the Screening visit, or anticipated use at any time during the Treatment Period.
Any changes in the dose or regimen of any systemic medications that could influence epithelial healing (e.g., systemic steroids or biologic agents) have been made within 30 days prior to the Screening visit or changes are anticipated at any time during the Treatment Period.
Evidence of current drug or alcohol abuse or addiction, in the opinion of the Investigator.
Participated in or used another investigational agent in another clinical study, within 30 days prior to Screening (participation in a non-interventional observational study is permitted).
Female subjects who are pregnant, breastfeeding, or planning a pregnancy during their projected study participation.
Current diagnosis or history of any ocular or systemic disorder or condition that might hinder the efficacy of the study drug or its evaluation, could possibly interfere with the interpretation of study results, or could be judged by the Investigator to be incompatible with the study visit schedule or conduct. (Examples include progressive or degenerative corneal or retinal conditions such as active acute Stevens-Johnson, systemic infection, optic neuritis, uveitis, neoplastic diseases, poorly controlled diabetes, poorly controlled autoimmune disease, poorly controlled graft versus host disease.)
Individuals who were deemed eligible at Screening will be excluded from study entry at Day 1/Baseline if there is objective clinical evidence of improvement in the LSCD during the Washout period between the Screening and Day 1/Baseline visits.
At the Screening and Day 1/Baseline visits, the Investigator will measure the Study Lesion via slit-lamp examination and record the longest length and the widest perpendicular measurements. The measurements from both visits will be entered into the Electronic Data Capture (EDC)/Interactive Web-response System (IWRS). Subjects will be excluded at the Day 1/Baseline visit if both linear measurements have decreased by more than 10% since the Screening visit. The EDC/IWRS will determine eligibility for this criterion.
Study drug will be self-administered as 1 drop QID in the Study Eye for 8 weeks.
Duration of treatment: 8 weeks
All efficacy analyses will be conducted using the Full Analysis Set, which will include all subjects randomized to treatment; subjects will be analyzed based on their randomization assignment. Safety analyses will be conducted using the Safety Set, which will include all subjects who received at least 1 dose of study drug; subjects will be analyzed based on the actual treatment received.
Descriptive statistics and categorical displays will be used to summarize the efficacy and safety analyses, as appropriate. For ocular safety assessments conducted in both eyes, results for both eyes will be listed by subject; descriptive or categorical summaries will be tabulated for the Study Eye only.
An independent central reading center will be used to evaluate slit-lamp photographs with corneal fluorescein staining.
Further detail on statistical methods will be provided in a Statistical Analysis Plan (SAP).
aSlit-lamp photographs taken at Screening and Day 1/Baseline will not be submitted to the reading center until after the Day 1/Baseline visit is complete. At the Screening and Baseline visits, the longest length and widest perpendicular measurement of the Study Lesion will be entered into the EDC/IWRS. The subject will be randomized into the study only if both measurements do NOT decrease by more than 10% from the Screening measurement to the Day 1/Baseline measurement.
bIf the Investigator's SoC dosing regimen for moxifloxacin differs from the protocol (e.g., more than 3 times per day), the Sponsor and Medical Monitor will consider use of Investigator's SoC during the study. At his or her discretion, the Investigator may direct the subject to continue using one or both of the non-study-drug treatments (moxifloxacin and/or artificial tears) through Day 70, if it is believed to be medically indicated based on ocular assessments of the Study Eye at the Day 56 visit. In that case, the site will dispense moxifloxacin and/or artificial tears to the subject at Day 56, and the subject will return the unused portion on Day 70.
cThe first dose of study drug is to be self-administered by the subject at the study site on Day 1 under supervision of site staff for training purposes. Study diary training will occur at the same time. Training may be repeated at any visit as needed. The date and time of administration of all doses of study drug and non-study drugs (moxifloxacin and Refresh Classic) will be recorded by the subject in the study diary.
dIf a subject discontinues the Treatment Period prior to Day 56, the last study visit should include all ocular assessments of the Study Eye as would be assessed at the Day 56 visit.
eIf a subject discontinues the Follow-up Period prior to Day 238, the last study visit should include all ocular assessments of the Study Eye as would be assessed at the
fEach time study drug or non-study drugs are dispensed, a new diary should be dispensed to the subject. Subjects should be instructed to bring their diary to each study visit during the Treatment Period to be collected and reviewed to assess compliance.
Both chemical burns and large mechanical epithelial debridement injury in rodents represent etiologies associated with LSCD development, and animal models based on both types of corneal injury have been used. The chemical burn rat model results in severe corneal pathologies, which is representative of human LSCD including neovascularization. The mechanical debridement mouse model involves mechanical depletion of the corneal epithelium and associated limbal stem cell compartment. Bone marrow-derived MSC secretome (MSC-S) has been evaluated in both animal models.
Using the mechanical debridement model, 34 female C57BL/6 mice were anesthetized and a 3 mm diameter area was marked on the surface of the cornea with a trephine, followed by debridement (mechanical scraping) with an Alger Brush II to remove the epithelial layer. Fluorescein staining was immediately completed to assess baseline wound size.
Rats were assigned to treatment arms dosed either with vehicle control (2% HPMC, n=9, TID), MSC-S 0.6 μg/mL (n=8, TID), MSC-S 6 μg/mL (n=10, TID), MSC-S 60 μg/mL (n=8, TID), or MSC-S 6 μg/mL (n=9, six times per day).
Measurement of lesion size by fluorescein staining occurred at baseline and throughout the study duration, ending on day 15. Additional secondary assessments included opacity, neovascularization and neovascular vessel size, body weight, and digital imaging.
By day 3 of the study, decreased wound size was observed for all treatment arms with 0.6 and 6 μg/mL treatment groups achieving >90% closure. At Day 4, for both the 0.6 and 6 μg/mL MSC-S treatment groups, 57% of eyes achieved complete wound closure (defined as >99.5% closure) compared to the vehicle control arm, which only showed complete wound closure in 28% of eyes.
Using the mechanical debridement model of LSCD, 34 female C57BL/6 mice were anesthetized and a 3 mm area was marked on the surface of the cornea with a trephine, followed by debridement (mechanical scraping) with an Alger Brush II to remove the epithelial layer. Fluorescein staining was immediately completed to assess baseline wound size.
Mice were assigned to treatment arms and received two times a day (BID) bilateral topical administration of a vehicle (0.1% HPMC), MSC-S 2.4 U/mL, MSC-S 17.5 U/mL, MSC-S 134 U/mL, or Treatment “Dep”, which consisted of a sample of MSC-S that had been depleted of key proteins involved in wound healing.
Measurement of lesion size by fluorescein staining occurred at baseline and throughout the study duration, ending on day 8. Additional secondary assessments included opacity, neovascularization, body weight, and digital imaging.
By day 4 of the study, decreased wound size was observed for all treatment arms with 2.4 U/mL and 17.5 U/mL treatment groups achieving >90% closure. At day 5, the 2.4 U/mL and 17.5 U/mL treatment groups had 88% and 75% of eyes achieving complete wound closure (defined as >99.5% closure), respectively, compared to the vehicle control arm, which had only 33% of eyes achieving complete wound closure. For the group treated with MSC-S depleted of key proteins, only 50% of the eyes achieved complete closure by day 5.
The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the compositions, systems and methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
All headings and section designations are used for clarity and reference purposes only and are not to be considered limiting in any way. For example, those of skill in the art will appreciate the usefulness of combining various aspects from different headings and sections as appropriate according to the spirit and scope of the invention described herein.
All references cited herein are hereby incorporated by reference herein in their entireties and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled.
This application claims priority to U.S. Provisional Application No. 63/619,718, filed Jan. 10, 2024, which is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63619718 | Jan 2024 | US |
