WO2019168685A1 - Improvement of endothelial cell function - Google Patents

Abstract

Provided herein are compositions and methods for improving endothelial cell function, e.g., in a subject in need thereof. In some aspects, the compositions and methods involve enhancing or preserving endothelial cell viability. In some aspects, the compositions and methods involve improving leakiness of the endothelial cell barrier, or mitigating or improving vascular leak. In some aspects, the compositions and methods improve or enhance endothelial cell migration. In some aspects, the compositions and methods mitigate or dampen improper endothelial cell activation.

Description

IMPROVEMENT OF ENDOTHELIAL CELL FUNCTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 62/635685, filed February 27, 2018, the entire contents of which are hereby expressly incorporated by reference in its entirety.

BACKGROUND

[0002] Under normal physiological conditions, the endothelium forms a semi- permeable layer between the circulating blood and the elements of the wall of all blood vessels, either venous or arterial. Although the endothelium consists of a single layer of cells, its activity is very diversified. For example, endothelial cells synthesize and release factors that modulate angiogenesis, inflammatory responses, hemostasis, vascular tone, the synthesis and degradation of the extracellular matrix and vascular permeability, in response to many substances (e.g., circulating hormones, cytokines, drugs), as well as physical or chemical stimuli (shearing force, pressure change, pH), (Feletou and Vanhoutte, Am J Physiol Heart Circ Physiol 2006, 291 : 985-1002). The endothelial layer plays an important barrier function, preventing blood constituents such as blood borne substances, cells and serum from entering the underlying tissue. The barrier function is tightly regulated through a number of homo- and heterotopic interactions between molecules on neighboring endothelial cells as well as similar interaction with molecules on circulating blood cells. The breakdown of this barrier function leads to severe physiological consequences and injury to the underlying tissue. It is involved in the pathogenesis of inflammatory diseases, edema formation and angiogenesis, for instance.

[0003] Owing to the diverse activities of endothelial cells, proper endothelial cell function is important in maintaining homeostatis. Compromised or improper endothelial function is associated with several disease conditions. Accordingly, there is a need for compositions and methods for improving endothelial cell function, and/or normalizing endothelial cell function, and/or maintaining proper endothelial cell function. SUMMARY

[0004] The embodiments described herein relate to methods of improving endothelial cell function, normalizing endothelial cell function, and/or maintaining proper endothelial cell function. In some embodiments, the methods and compositions disclosed herein enhance or preserve endothelial cell viability. In some embodiments, the methods and compositions disclosed herein relate to improving leakiness of the endothelial cell barrier, or improving vascular leakiness. In some embodiments, the compositions and methods disclosed herein mitigate, inhibit, or dampen improper endothelial cell activation. In some embodiments, the compositions and methods disclosed herein enhance endothelial cell migration.

[0005] The methods can include the step of identifying a subject, e.g., a subject in need of improved endothelial function, and administering a composition comprising, consisting essentially of, or consisting of, regenerative cells to the subject. In some embodiments, the regenerative cells comprise a heterogeneous population of cells. For example, in some embodiments, the heterogeneous population of regenerative cells comprises stem and progenitor cells (e.g., endothelial progenitor cells, or endothelial precursor cells, or the like).

[0006] In some embodiments, the regenerative cells are derived from one or a combination of the following tissues: bone marrow, placenta, adipose tissue, skin, eschar tissue, endometrial tissue, adult muscle, corneal stroma, dental pulp, Wharton’s jelly, amniotic fluid, or umbilical cord.

[0007] In some embodiments, the regenerative cells have not been cultured prior to the administering step, such as e.g., placed in contact with a cellular growth media or serum for greater than or equal to 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, or 15 hours. In some embodiments, the regenerative cells are adherent cells (e.g., plastic adherent). In some embodiments, the regenerative cells are cultured for at least 5 passages in tissue culture.

[0008] In some embodiments, the regenerative cells are cryopreserved.

[0009] In some embodiments, the composition comprising regenerative cells includes an additive. For example, in some embodiments, the composition includes additives such as other cells, tissue, or tissue fragments. For example, in some embodiments, the composition can include undigested adipose tissue.

[0010] In some embodiments, the regenerative cells are derived from adipose tissue, and the method includes the further step of processing adipose tissue to separate regenerative cells comprising stem and progenitor cells from mature adipocytes and connective tissue. By way of example, in some embodiments, the processing can include mechanically and/or enzymatically disaggregating adipose tissue to release the regenerative cells from mature adipocytes and connective tissue present in the adipose tissue. In embodiments, wherein the method includes enzymatic disaggregation of adipose tissue, the disaggregation can be enzymatically disaggregating the adipose tissue with collagenase, a neutral protease, or both.

[0011] In some embodiments, the compositions are administered to a subject having a condition or disease selected from the group consisting of: angina pectoris, myocardial infarction, coronary artery disease, hypertension, cerbrovascular accidents, transient ischemic attacks, chronic obstructive pulmonary disease, chronic hypoxic lung disease, pulmonary hypertension, renal hypertension, chronic renal disease, microvascular complications of diabetes, vasoocclusive complications of sickle cell anemia, erectile dysfunction, systemic sclerosis, peripheral vascular disease, hypercholesterolemia, hypertension, atherosclerosis, septic shock, stroke, heart disease, insulin resistance, tumor growth, metastasis, venous thrombosis and adult respiratory distress syndrome.

[0012] Embodiments disclosed herein also relate to compositions comprising, consisting of, or consisting essentially of regenerative cells for use in improving endothelial cell function, normalizing endothelial cell function, and/or maintaining proper endothelial cell function. As such, disclosed herein are compositions comprising, consisting of, or consisting essentially of regenerative cells for use in treating or ameliorating a condition selected from the group consisting of: angina pectoris, myocardial infarction, coronary artery disease, hypertension, cerebrovascular accidents, transient ischemic attacks, chronic obstructive pulmonary disease, chronic hypoxic lung disease, pulmonary hypertension, renal hypertension, chronic renal disease, microvascular complications of diabetes, vasoocclusive complications of sickle cell anemia, erectile dysfunction, systemic sclerosis, peripheral vascular disease, hypercholesterolemia, hypertension, atherosclerosis, septic shock, stroke, heart disease, insulin resistance, tumor growth, metastasis, venous thrombosis and adult respiratory distress syndrome, or for use in the manufacture of a medicament for treating or ameliorating any one or more of the aforementioned conditions or diseases.

[0013] Also disclosed herein are methods of improving endothelial cell function, normalizing endothelial cell function, and/or maintaining proper endothelial cell function, in a subject in need thereof, that include the step of identifying a subject in need of improved endothelial function (or a subject that has or is at risk of developing a condition selected from the group consisting of: angina pectoris, myocardial infarction, coronary artery disease, hypertension, cerebrovascular accidents, transient ischemic attacks, chronic obstructive pulmonary disease, chronic hypoxic lung disease, pulmonary hypertension, renal hypertension, chronic renal disease, microvascular complications of diabetes, vasoocclusive complications of sickle cell anemia, erectile dysfunction, systemic sclerosis, peripheral vascular disease, hypercholesterolemia, hypertension, atherosclerosis, septic shock, stroke, heart disease, insulin resistance, tumor growth, metastasis, venous thrombosis and adult respiratory distress syndrome,) and administering to the subject secretions from regenerative cells, and/or conditioned media from regenerative cells cultured in vitro.

[0014] Embodiments disclosed herein also relate to the compositions comprising, consisting of, or consisting essentially of secretions from regenerative cells, and/or conditioned media from regenerative cells cultured in vitro , for improving endothelial cell function, normalizing endothelial cell function, and/or maintaining proper endothelial cell function. As such, disclosed herein are compositions comprising, consisting of, or consisting essentially of regenerative cells for treating or ameliorating a condition selected from the group consisting of: angina pectoris, myocardial infarction, coronary artery disease, hypertension, cerebrovascular accidents, transient ischemic attacks, chronic obstructive pulmonary disease, chronic hypoxic lung disease, pulmonary hypertension, renal hypertension, chronic renal disease, microvascular complications of diabetes, vasoocclusive complications of sickle cell anemia, erectile dysfunction, systemic sclerosis, peripheral vascular disease, hypercholesterolemia, hypertension, atherosclerosis, septic shock, stroke, heart disease, insulin resistance, tumor growth, metastasis, venous thrombosis and adult respiratory distress syndrome, or for use in the manufacture of a medicament for treating or ameliorating any one or more of the aforementioned conditions or diseases. [0015] The following provides some of the preferred alternatives of the present invention.

[0016] 1. A method of improving endothelial cell function, comprising: identifying a subject in need of improvement of endothelial cell function; and administering an amount of a composition comprising regenerative cells sufficient to improve endothelial cell function to said subject.

[0017] 2. The method of alternative 1, wherein improving endothelial cell function comprises enhancing or preserving endothelial cell viability.

[0018] 3. The method of alternative 1 or 2, wherein improving endothelial cell function comprises improving leakiness of the endothelial cell barrier.

[0019] 4. The method of any one of alternatives 1-3, wherein improving endothelial cell function comprises mitigating or dampening improper endothelial cell activation

[0020] 5. The method of any one of alternatives 1-4, wherein improving endothelial cell function comprises enhancing endothelial cell migration.

[0021] 6. The method of any one of the preceding alternatives, wherein the regenerative cells comprise a heterogeneous population of cells.

[0022] 7. The method of alternative 6, wherein the heterogeneous population of regenerative cells comprises stem and progenitor cells.

[0023] 8. The method of any one of the preceding alternatives, wherein the regenerative cells are derived from a tissue selected from the group consisting of: bone marrow, placenta, adipose tissue, skin, eschar tissue, endometrial tissue, adult muscle, corneal stroma, dental pulp, Wharton’s jelly, amniotic fluid, and umbilical cord.

[0024] 9. The method of alternative 8, wherein the regenerative cells are adipose- derived regenerative cells.

[0025] 10. The method of any one of alternatives 1-9, wherein the regenerative cells have not been cultured prior to the administering step.

[0026] 11. The method of any one of alternatives 1-10, wherein the regenerative cells are adherent cells. [0027] 12. The method of any one of alternatives 1-9 or 11, wherein the regenerative cells are cultured for at least 5 passages in tissue culture.

[0028] 13. The method of any one of alternatives 1-12, wherein the regenerative cells are cryopreserved.

[0029] 14. The method of any one of alternatives 1-13, wherein the composition comprises an additive.

[0030] 15. The method of alternative 14, wherein the additive is selected from the group consisting of cells, tissue, and tissue fragments.

[0031] 16. The method of alternative 14 or 15, wherein the additive comprises undigested adipose tissue.

[0032] 17. The method of alternative 10, further comprising the steps of: processing adipose tissue to separate regenerative cells comprising stem and progenitor cells from mature adipocytes and connective tissue.

[0033] 18. The method of alternative 17, wherein the processing comprises mechanically or enzymatically disaggregating adipose tissue to release the regenerative cells from mature adipocytes and connective tissue present in the adipose tissue.

[0034] 19. The method of alternative 18, wherein the processing comprises enzymatically disaggregating the adipose tissue with collagenase, a neutral protease, or both.

[0035] 20. The method of any one of alternatives 1-19, wherein the subject has a condition selected from the group consisting of: angina pectoris, myocardial infarction, coronary artery disease, hypertension, cerbrovascular accidents, transient ischemic attacks, chronic obstructive pulmonary disease, chronic hypoxic lung disease, pulmonary hypertension, renal hypertension, chronic renal disease, microvascular complications of diabetes, vasoocclusive complications of sickle cell anemia, erectile dysfunction, systemic sclerosis, peripheral vascular disease, hypercholesterolemia, hypertension, atherosclerosis, septic shock, stroke, heart disease, insulin resistance, tumor growth, metastasis, venous thrombosis and adult respiratory distress syndrome. [0036] 21. A method of improving endothelial cell function, comprising: identifying a subject in need of improvement of endothelial cell function; and administering a composition comprising secretions from regenerative cells cultured in vitro , in an amount sufficient to improve endothelial cell function to said subject.

[0037] 22. The method of alternative 21, wherein the regenerative cells are derived from a tissue selected from the group consisting of: bone marrow, placenta, adipose tissue, skin, eschar tissue, endometrial tissue, adult muscle, corneal stroma, dental pulp, Wharton’s jelly, amniotic fluid, and umbilical cord.

[0038] 23. The method of alternative 21 or 22, wherein the regenerative cells are adipose-derived regenerative cells.

[0039] 24. The method of any one of alternative 21-23, wherein the regenerative cells are grown in EGM2 media.

[0040] 25. The method of alternative 23, wherein the adipose-derived regenerative cells are grown in EGM2 media

[0041] 26. The method of any one of alternatives 21-25, wherein the subject has a condition selected from the group consisting of: angina pectoris, myocardial infarction, coronary artery disease, hypertension, cerbrovascular accidents, transient ischemic attacks, chronic obstructive pulmonary disease, chronic hypoxic lung disease, pulmonary hypertension, renal hypertension, chronic renal disease, microvascular complications of diabetes, vasoocclusive complications of sickle cell anemia, erectile dysfunction, systemic sclerosis, peripheral vascular disease, hypercholesterolemia, hypertension, atherosclerosis, septic shock, stroke, heart disease, insulin resistance, tumor growth, metastasis, venous thrombosis and adult respiratory distress syndrome.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1A is a graph showing the relative fluorescence units (RFU) of HUVECs cultured in the media indicated, as described in Example 1.

[0043] Figure IB (i)-(vii) are photographs taken of HUVEC cells treated and stained as described in Example 1, observed at 40X magnification. [0044] Figure 2 is a graph showing the RFUs of HUVECs cultured with ADRCs as indicated, and as described in Example 1.

[0045] Figure 3 is a graph showing the number of HUVECs/field that migrated in the transwell co-culutre assay as described in Example 2, below.

DETAILED DESCRIPTION

[0046] The embodiments disclosed herein relate to compositions and methods for improvement and/or maintenance of endothelial cell function.

Definitions

[0047] As used herein, the term“about,” when referring to a stated numeric value, indicates a value within plus or minus 10% of the stated numeric value.

[0048] As used herein, the term“derived” means isolated from or otherwise purified or separated from. For example, adipose-derived stem and other regenerative cells are isolated from adipose tissue. Similarly, the term“derived” does not encompass cells that are extensively cultured ( e.g ., placed in culture conditions in which the majority of dividing cells undergo 3, 4, 5 or less, cell doublings), from cells isolated directly from a tissue, e.g., adipose tissue, or cells cultured or expanded from primary isolates. Accordingly,“adipose derived cells,” including adipose-derived stem and other regenerative cells and combinations thereof, refers to cells obtained from adipose tissue, wherein the cells are not extensively cultured, e.g., are in their“native” form as separated from the adipose tissue matrix.

[0049] As used herein, a cell is“positive” for a particular marker when that marker is detectable. For example, an adipose derived regenerative cell is positive for, e.g., CD73 because CD73 is detectable on an adipose derived stem or regenerative cell in an amount that is detectably greater than background (in comparison to, e.g., an isotype control or an experimental negative control for any given assay). A cell is also positive for a marker when that marker can be used to distinguish the cell from at least one other cell type, or can be used to select or isolate the cell when present or expressed by the cell.

[0050] As used herein, "regenerative cells" refers to any heterogeneous or homogeneous population of cells obtained using the systems and methods of embodiments disclosed herein, which cause or contribute to complete or partial regeneration, restoration, or substitution of structure or function of an organ, tissue, or physiologic unit or system to thereby provide a therapeutic, structural or cosmetic benefit. Examples of regenerative cells include: adult stem cells, endothelial cells, endothelial precursor cells, endothelial progenitor cells, macrophages, fibroblasts, pericytes, smooth muscle cells, preadipocytes, differentiated or de-differentiated adipocytes, keratinocytes, unipotent and multipotent progenitor and precursor cells (and their progeny), or lymphocytes.

[0051] Accordingly, adipose-derived regenerative cells (“ADRCs”) as used herein refers to any heterogeneous or homogeneous cell population that contains one or more types of adipose-derived regenerative cells including adipose-derived stem cells, endothelial cells (including blood and lymphatic endothelial cells), endothelial precursor cells, endothelial progenitor cells, macrophages, fibroblasts, pericytes, smooth muscle cells, preadipocytes, kertainocytes, unipotent and multipotent progenitor and precursor cells (and their progeny), or lymphocytes. Adipose-derived stem cells comprise at least 0.1% of the cellular component of adipose-derived regenerative cells.

[0052] Similarly,“bone marrow-derived regenerative cells” (“BMRCs”) refers to any heterogeneous or homogeneous cell population that contains one or more types of bone marrow-derived regenerative cells including bone marrow-derived stem cells, endothelial cells (including blood and lymphatic endothelial cells), endothelial precursor cells, endothelial progenitor cells, macrophages, fibroblasts, pericytes, smooth muscle cells, preadipocytes, keratinocytes, unipotent and multipotent progenitor and precursor cells (and their progeny), or lymphocytes.

[0053] In some contexts, the term “progenitor cell” refers to a cell that is unipotent, bipotent, or multipotent with the ability to differentiate into one or more cell types, which perform one or more specific functions and which have limited or no ability to self- renew. Some of the progenitor cells disclosed herein may be pluripotent.

[0054] As used herein the phrase "adherent cells" refers to a homogeneous or heterogeneous population of cells which are anchorage dependent, i.e., require attachment to a surface in order to grow in vitro.

[0055] In some contexts, the term“adipose tissue-derived cells” refers to cells extracted from adipose tissue that has been processed to separate the active cellular component ( e.g ., the cellular component that does not include adipocytes and/or red blood cells) from the mature adipocytes and connective tissue. Separation may be partial or full. That is, the“adipose tissue-derived cells” may or may not contain some adipocytes and connective tissue and may or may not contain some cells that are present in aggregates or partially disaggregated form (for example, a fragment of blood or lymphatic vessel comprising two or more cells that are connected by extracellular matrix). This fraction is referred to herein as“adipose tissue-derived cells,”“adipose derived cells,”“adipose derived regenerative cells” or“ADC.” Typically, ADC refers to the pellet of cells obtained by washing and separating the cells from the adipose tissue e.g., after digestion of adipose tissue with an enzyme, such as collagenase. The pellet is typically obtained by concentrating a suspension of cells released from the connective tissue and adipose tissue matrix. By way of example, the pellet can be obtained by centrifuging a suspension of adipose-derived cells so that the cells aggregate at the bottom of a centrifuge container, e.g., the stromal vascular fraction. In some embodiments, the adipose-derived cell populations described herein include, among other cell types, leukocytes. In some embodiments, the adipose-derived cell populations described herein include, among other regenerative cell types, endothelial cells.

[0056] In some contexts, the term“adipose tissue” refers to a tissue containing multiple cell types including adipocytes and vascular cells. Adipose tissue includes multiple regenerative cell types, including adult stem cells (ASCs), endothelial progenitor and precursor cells, or pericytes and the like. Accordingly, adipose tissue refers to fat, including the connective tissue that stores the fat.

[0057] In some contexts, the term“unit of adipose tissue” refers to a discrete or measurable amount of adipose tissue. A unit of adipose tissue may be measured by determining the weight and/or volume of the unit. In reference to the disclosure herein, a unit of adipose tissue may refer to the entire amount of adipose tissue removed from a subject, or an amount that is less than the entire amount of adipose tissue removed from a subject. Thus, a unit of adipose tissue may be combined with another unit of adipose tissue to form a unit of adipose tissue that has a weight or volume that is the sum of the individual units.

[0058] In some contexts, the term“portion” refers to an amount of a material that is less than a whole. A minor portion refers to an amount that is less than 50%, and a major portion refers to an amount greater than 50%. Thus, a unit of adipose tissue that is less than the entire amount of adipose tissue removed from a subject is a portion of the removed adipose tissue.

[0059] The compositions and embodiments disclosed herein are useful as a therapy for subjects in need of an improvement in endothelial cell function and/or maintenance of proper endothelial cell function. Accordingly, the term“subject” can refer to any mammal including, but not limited to mice, rats, rabbits, guinea pigs, pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, or humans. In some embodiments, the subject is a human. The term "subject" can be used interchangeably with the terms "individual" and "patient" herein.

[0060] As explained in further detail below, in some embodiments, the subject has a condition associated with improper endothelial cell function, or a condition or disorder associated with, or caused in whole or in part by, endothelial dysfunction. Accordingly, in some embodiments, the subject can have one or more conditions selected from the group consisting of: angina pectoris, myocardial infarction, coronary artery disease, hypertension, cerbrovascular accidents, transient ischemic attacks, chronic obstructive pulmonary disease, chronic hypoxic lung disease, pulmonary hypertension, renal hypertension, chronic renal disease, microvascular complications of diabetes, vasoocclusive complications of sickle cell anemia, erectile dysfunction, systemic sclerosis, peripheral vascular disease, hypercholesterolemia, hypertension, atherosclerosis, septic shock, stroke, heart disease, insulin resistance, tumor growth, metastasis, venous thrombosis and adult respiratory distress syndrome.

Improving Endothelial Function

[0061] Embodiments herein relate to improving endothelial function. Many methods of detecting compromised endothelial function are known in the art and are useful with the embodiments disclosed herein. By way of example, methods of detecting endothelial dysfunction are described in U.S. Patent No’s. 6649421, 6445945, 6908436, 6654628, US6376169, U.S. Patent Application Publication No’s US20060264755A1, US20030134332A1, US20080119741 Al, US20050228303A1, International Patent

Application Publication No. W02000074551A2, Japanese Patent Application No. JP2003144395A, and the like, the methods described in each of the aforementioned are herein expressly incorporated by reference in their entireties. In some embodiments, the methods disclosed herein include measuring an improvement in endothelial function. Improvement in endothelial function can be assessed by any of the methods described above. In some embodiments, a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or any percentage within a range defined by any two of the aforementioned percentages, improvement in endothelial function is observed, following administration of any one or more of the compositions disclosed herein, to a subject in need thereof.

Regenerative Cells

[0062] In the embodiments disclosed herein, regenerative cells are used for improving endothelial cell function e.g., improving or promoting endothelial cell viability, improving or promoting endothelial cell migration, reducing or mitigating endothelial barrier leakage or vascular leakage, reducing or mitigating edema, reducing or mitigating improper endothelial cell activation, and the like. As mentioned above, a population of“regenerative cells” disclosed herein can be a homogeneous or heterogeneous population of cells, which cause or contribute to a complete or partial regeneration, restoration, or substitution of structure or function of an organ, tissue, or physiologic unit or system to thereby provide a therapeutic, structural or cosmetic benefit. Examples of regenerative cells include, but are not limited to, adult stem cells, endothelial cells, endothelial precursor cells, endothelial progenitor cells, macrophages, fibroblasts, pericytes, smooth muscle cells, preadipocytes, differentiated or de-differentiated adipocytes, keratinocytes, unipotent and multipotent progenitor and precursor cells (and their progeny), or lymphocytes.

[0063] The regenerative cells disclosed herein can be isolated from various tissues, including, but not limited to, bone marrow, placenta, adipose tissue, skin, eschar tissue, endometrial tissue, adult muscle, corneal stroma, dental pulp, Wharton’s jelly, amniotic fluid, or umbilical cord. The regenerative cells disclosed herein can be isolated from the tissues above using a variety of approaches, which are well-known to those skilled in the art.

[0064] By way of example only, regenerative cells can be isolated from adipose tissue by a process, wherein tissue is excised or aspirated. Excised or aspirated tissue can be washed, and then enzymatically or mechanically disaggregated in order to release cells bound in the adipose tissue matrix. Once released, the cells can be suspended. By way of example only, regenerative cells useful in the embodiments disclosed herein can be isolated using the methods and/or devices described in U.S. Patent No’s. 7390484; 7585670, 7687059, 8309342, 8440440, US Patent Application Publication No’s. 2013/0164731, 2013/0012921, 2012/0164113, US2008/0014181. International Patent Application

Publication No. W02009/073724, or WO/2013030761, each of which is hereby expressly incorporated by reference in its entirety.

[0065] Exemplary, non-limiting methods for isolation of regenerative cells from bone marrow useful in the embodiments disclosed herein are described in U.S. Patent No’s 5879940, U.S. Patent Application Publication No’s 2013/0101561, 2013/0266541 European Patent Application Publication No. EP2488632A1, EP0241578A2, EP2624845A2, International Patent Application Publication No. WO2011047289A1, WO 1996038482A, each of which is hereby expressly incorporated by reference in its entirety.

[0066] Exemplary, non-limiting methods for isolation of regenerative cells from placental tissue useful in the embodiments disclosed herein are described in U.S. Patent No. 8580563, U.S. Patent Application Publication No. 20130040281, International Patent Application Publication No. W02003089619A, Klein, et al. (2011) Methods Mol Biol. 698:75-88, Vellasamy, et al. (2012) World J Stem Cells 4(6): 53-61; Timmins, et al. (2012) Biotechnol Bioeng. 109(7): 1817-26; Semenov, et al. (2010) Am J Obstet Gynecol 202: 193- e. l3, each of which is hereby expressly incorporated by reference in its entirety.

[0067] Exemplary, non-limiting methods for isolation of regenerative cells from skin useful in the embodiments disclosed herein are described in Toma, et al. (2001), Nat Cell Biol 3(9):778-84; Nowak, et al. (2009), Methods Mol Biol. 482:215-32; U.S Patent Application Publication No. 2007/0248574, each of which is hereby expressly incorporated by reference in its entirety.

[0068] Exemplary, non-limiting methods for isolation of regenerative cells from eschar tissue useful in the embodiments disclosed herein are described in Van der Veen, et al. (2012), Cell Transplant. 2l(5):933-42, hereby expressly incorporated by reference in its entirety.

[0069] Exemplary, non-limiting methods for isolation of regenerative cells from endometrial tissue useful in the embodiments disclosed herein are described in U.S. Patent Application Publication No. 2013/0156726, 2008/0241113, each of which is hereby expressly incorporated by reference in its entirety.

[0070] Exemplary, non-limiting methods for isolation of regenerative cells from muscle tissue useful in the embodiments disclosed herein are described in U.S. Patent No. 6337384, U.S. Patent Application Publication No. 2001/019966, 2011/0033428, 2005/0220775, each of which is hereby expressly incorporated by reference m its entirety.

[0071] Exemplary, non-limiting methods for isolation of regenerative cells from corneal tissue useful m the embodiments disclosed herein are described in U.S. Patent Application Publication No. 20050841 19, Sharifi, et al. (2010) Biocell. 34(l):53-5, each of which is hereby expressly incorporated by reference in its entirety.

[0072] Exemplary, non-limiting methods for isolation of regenerative cells from dental pulp useful in the embodiments disclosed herein are described in U.S. Patent Application Publication No. 2012/0251504, Gronthos, et al. (2011) Methods Mol Biol. 698: 107-21 ; Suchanek, et al. Acta Medica (Hradec Kralove). 2007;50(3): 195-201 ; Yildirm, Sibel,“Isolation Methods of Dental Pulp Stem Cells,” in Dental Pulp Stem Cells: Springer Briefs in Stem Cells, pp. 41-51, © 2013, Springer New York, New York, NY, each of which is hereby expressly incorporated by reference in its entirety.

[0073] Exemplary, non-limiting methods for isolation of regenerative cells from Wharton’s jelly useful in the embodiments disclosed herein are described in U.S. Patent Application Publication No’s. 2013/0183273, 2011/0151556, International Patent

Application Publication No. WO 04/072273 Al, Sheshareddy, et al. (2008) Methods Cell Biol. 86: 101-19, Mennan, et al. (2013) BioMed Research International, Article ID 916136, Corotchi, et al. (2013) Stem Cell Research & Therapy 4:81, each of which is hereby expressly incorporated by reference in its entirety.

[0074] Exemplary, non-limiting methods for isolation of regenerative cells from amniotic fluid useful in the embodiments described herein are described in U.S. Patent No. 8021876, International Patent Application Publication No. WO 2010/033969A1, WO 2012/014247A1, WO 2009/052132, U.S. Patent Application Publication No. 2013/0230924, 2005/0054093, each of which is hereby expressly incorporated by reference in its entirety.

[0075] Exemplary, non-limiting methods for isolation of regenerative cells from the umbilical cord useful in the embodiments described herein are described in U.S. Patent Application Publication No. 20130065302, Reddy, et al. (2007 ), Methods Mol Biol. 407: 149- 63, Hussain, et al. (2012) Cell Biol Int. 36(7):595-600, Pham, et al. (2014) Journal of Translational Medicine 2014, 12:56, Lee, et al. (2004) Blood 103(5): 1669-1675, each of which is hereby expressly incorporated by reference in its entirety. The regenerative cells in the methods and compositions described herein can be a heterogeneous population of cells that includes stem and other regenerative cells. In some embodiments, the regenerative cells in the methods and compositions described herein can include stem and endothelial precursor cells. In some embodiments, the regenerative cells can include stem and pericyte cells. In some embodiments, the regenerative cells can include stem cells and leukocytes. For example, in some embodiments, the regenerative cells can include stem cells and macrophages. In some embodiments, the regenerative cells can include stem cells and M2 macrophages. In some embodiments, the regenerative cells can include pericytes and endothelial precursor cells. In some embodiments, the regenerative cells can include platelets. Preferably, the regenerative cells comprise stem cells and endothelial precursor cells. In some embodiments, the regenerative cells can include regulatory cells such as Treg cells. In some embodiments, the regenerative cells are adipose-derived. Accordingly, some embodiments provide methods and compositions for mitigating or reducing burn progression with adipose-derived regenerative cells, e.g., that include adipose-derived stem and endothelial precursor cells.

[0076] In some embodiments, the regenerative cells are not cultured prior to use. By way of example, in some embodiments, the regenerative cells are for use following isolation from the tissue of origin, e.g., bone marrow, placenta, adipose tissue, skin, eschar tissue, endometrial tissue, adult muscle, cornea stroma, dental pulp, Wharton’s jelly, amniotic fluid, umbilical cord, and the like.

[0077] In some embodiments, the regenerative cells are cultured prior to use. For example, in some embodiments, the regenerative cells are subjected to“limited culture,” i.e., to separate cells that adhere to plastic from cells that do not adhere to plastic. Accordingly, in some embodiments, the regenerative cells are “adherent” regenerative cells. An exemplary, non-limiting method of isolating adherent regenerative cells from adipose tissue are described e.g, in Zuk, et al. (2001). Exemplary, non-limiting method of isolating adherent regenerative cells from bone marrow are described, e.g, Pereira (1995) Proc. Nat. Acad. Sci. USA 92:4857-4861, Castro-Malaspina et al. (1980), Blood 56:289-30125; Piersma et al. (1985) Exp. Hematol. 13:237-243; Simmons et al., 1991, Blood 78:55-62; Beresford et al, 1992, J. Cell. Sci. 102:341-3 51; Liesveld et al. (1989) Blood 73: 1794-1800; Liesveld et al., Exp. Hematol 19:63-70; Bennett et al. (1991) J. Cell. Sci. 99: 131-139), ET.S. Patent No. 7056738, each of which is hereby expressly incorporated by reference in its entirety. In some embodiments, the regenerative cells are cultured for more than 3 passages in vitro. For example, in some embodiments, the regenerative cells are cultured for 4, 5, 6, 7, 8, 9, 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

37, 38, 39, 40, or more passages in vitro.

[0078] The regenerative cells described herein can be cultured according to approaches known in the art, and the cultured cells can be used in several of the embodied methods. For example, regenerative cells can be cultured on collagen-coated dishes or 3D collagen gel cultures in endothelial cell basal medium in the presence of low or high fetal bovine serum or similar product, as described in Ng, et al, (2004), Microvasc. Res. 68(3):258-64, hereby expressly incorporated by reference in its entirety. Alternatively, regenerative cells can be cultured on other extracellular matrix protein- coated dishes. Examples of extracellular matrix proteins that may be used include, but are not limited to, fibronectin, laminin, vitronectin, or collagen IV. Gelatin or any other compound or support, which similarly promotes adhesion of endothelial cells into culture vessels may be used to culture regenerative cells, as well.

[0079] Examples of basal culture medium that can be used to culture regenerative cells in vitro include, but are not limited to, EGM, RPMI, Ml 99, MCDB131, DMEM, EMEM, McCoy’s 5 A, Iscove’s medium, or modified Iscove’s medium, or any other medium known in the art to support the growth of blood endothelial cells. In some embodiments, the regenerative cells are cultured in EGM-2MV media. Examples of supplemental factors or compounds that can be added to the basal culture medium that could be used to culture regenerative cells include, but are not limited to, ascorbic acid, heparin, endothelial cell growth factor, endothelial growth supplement, glutamine, HEPES, Nu serum, fetal bovine serum, human serum, equine serum, plasma- derived horse serum, iron-supplemented calf serum, penicillin, streptomycin, amphotericin B, basic and acidic fibroblast growth factors, insulin-growth factor, astrocyte conditioned medium, fibroblast or fibroblast-like cell conditioned medium, sodium hydrogencarbonate, epidermal growth factor, bovine pituitary extract, magnesium sulphate, isobutylmethylxanthine, hydrocortisone, dexamethasone, dibutyril cyclic AMP, insulin, transferrin, sodium selenite, oestradiol, progesterone, growth hormone, angiogenin, angiopoietin- 1 , Del-l, follistatin, granulocyte colony-stimulating factor (G-CSF), erythropoietin, hepatocyte growth factor (HGF) /scatter factor (SF), leptin, midkine , placental growth factor, platelet-derived endothelial cell growth factor (PD-ECGF), platelet-derived growth factor-BB (PDGF-BB), pleiotrophin (PTN), progranulin, proliferin, transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF), interleukin-3 (IL-3), interleukin 7 (IL-7), interleukin-8 (IL-8), ephrins, or matrix metalloproteinases (such as MMP2 and MMP9), or any other compound known in the art to promote survival, proliferation or differentiation of endothelial cells.

[0080] Further processing of the cells may also include: cell expansion (of one or more regenerative cell types) and cell maintenance (including cell sheet rinsing and media changing); sub-culturing; cell seeding; transient transfection (including seeding of transfected cells from bulk supply); harvesting (including enzymatic, non-enzymatic harvesting and harvesting by mechanical scraping); measuring cell viability; cell plating (e.g., on microtiter plates, including picking cells from individual wells for expansion, expansion of cells into fresh wells); high throughput screening; cell therapy applications; gene therapy applications; tissue engineering applications; therapeutic protein applications; viral vaccine applications; harvest of regenerative cells or supernatant for banking or screening, measurement of cell growth, lysis, inoculation, infection or induction; generation of cell lines (including hybridoma cells); culture of cells for permeability studies; cells for RNAi and viral resistance studies; cells for knock-out and transgenic animal studies; affinity purification studies; structural biology applications; assay development or protein engineering applications.

[0081] In some embodiments, methods for isolating regenerative useful in the embodiments described herein can include positive selection (selecting the target cells), negative selection (selective removal of unwanted cells), or combinations thereof. In addition to separation by flow cytometry as described herein and in the literature, cells can be separated based on a number of different parameters, including, but not limited to, charge or size (e.g., by dielectrophoresis or various centrifugation methods, etc.).

[0082] By way of example, the regenerative cells useful in the methods of therapy disclosed herein may be identified by different combinations of cellular and genetic markers. For example, in some embodiments, the regenerative cells express CD90. In some embodiments, the regenerative cells do not express significant levels of lin. In some embodiments, the regenerative cells do not express significant levels of ckit. In some embodiments, the regenerative cells are CD90+/lin-/ckit-/CD45-. In some embodiments, the regenerative cells express STRO-l. In some embodiments, the regenerative cells express STRO-l and CD49d. In some embodiments, the regenerative cells express STRO-l, CD49d, and one or more of CD29, CD44, CD71, CD90, C105/SH2 or SH3. In some embodiments, the regenerative cells express STRO-l, CD49d, and one or more of CD29, CD44, CD71, CD90, C105/SH2 or SH3, but express low or undetectable levels of CD106.

[0083] In some embodiments, the regenerative cells express one or more of STRO-l, CD49d, CD13, CD29, SH3, CD44, CD71, CD90, or CD105, or any combination thereof. By way of example only, in some embodiments, the regenerative cells express each of do not express significant levels of CD31 , CD34, CD45 or CD 104 and do not express detectable levels of CD4, CDS, CD11, CD14, CD16, CD19, CD33, CD56, CD62E, CD106 or CD58.

[0084] In some approaches, the regenerative cells are CD 14 positive and/or CD1 lb positive.

[0085] In some embodiments, the cells are depleted for cells expressing the markers CD45(+). In some embodiments, the cells are depleted for cells expressing glycophorin-A (GlyA). In some embodiments, the cells are depleted for CD45(+) and GlyA(+) cells.

[0086] Negative selection of cells, e.g., depletion of certain cell types from a heterogeneous population of cells can done using art-accepted techniques, e.g., utilizing micromagnetic beads or FACS cell sorting, or the like. In some embodiments, the regenerative cells are CD34+.

[0087] In some embodiments, the regenerative cells are not cryopreserved. In some embodiments, the regenerative cells are cryopreserved. For example, in some embodiments, the regenerative cells include cryopreserved cells, e.g., as described in Liu, et al. (2010) Biotechnol Prog. 26(6): 1635-43, Carvalho, et al. (2008) Transplant /‘roc. ;40(3 ) : 839-41 , International Patent Application Publication No. WO 97 039104, WO 03/024215, WO 2011/064733, WO 2013/020492, WO 2008/09063, WO 2001/011011, European Patent No. EP0343217 Bl, each of which is hereby expressly incorporated by reference in its entirety.

Secretions or Conditioned Media from Regenerative Cells

[0088] Some embodiments provide compositions that include conditioned media, or media that contains secretions from regenerative cells. In some embodiments, regenerative cells can be cultured in tissue culture media, e.g, serum-free media, or the like, for a period of time. The culture supernatant can then be collected by methods known to those skilled I the art, e.g., centrifugation at 2,000 x g for 10 minutes. In some embodiments, following the collection of the supernatant, the secretions or conditioned media can be further treated by including an optional ultracentrifugation step (e.g, 100,000 x g (4°C) for 70 minutes). Non-limiting examples of methods to obtain secretions or conditioned media from regenerative cells are described in U.S. Patent Application Publication No. US20150147409A1, Bangh, et al. (2014) Molecular Therapy 22 4, 862-872, Pawitan, et al (2014), Biomed Research Int’l, art. ID 965849 and references cited therein, Park, et al.(20l0) Biomed Res. 3l(l):27-34.. Jones, et al. (2012) Stem Cells Devel, 21(15): 2817-2826, each of which is hereby expressly incorporated by reference in its entirety.

[0089] In some embodiments, the regenerative cells are cultured in EGM, EGM2, RPMI, M199, MCDB131, DMEM, EMEM, McCoy’s 5A, Iscove’s medium, or modified Iscove’s medium, or any other medium known in the art to support the growth of stem and progenitor. In some embodiments, the cells are cultured for 2, 5, 10, 12, 24, 36, 48, 96, or more hours, or any number in between.

Scaffolds

[0090] In some embodiments, the regenerative cells and/or conditioned media disclosed herein can be administered to a subject with a scaffold. In some embodiments, the scaffold can be a skin substitute, e.g, a biological or synthetic skin substitute. Exemplary skin substitutes useful in the embodiments disclosed herein include, but are not limited to, cell-containing skin substitutes such as EPICEL^® skin graft (Genzyme Biosurgery, MA, USA); CELLSPRAY^® skin graft (Avita Medical, Perth, Australia), MYSKIN™ skin graft (CellTran Ltd., Sheffeild, UK), LASERSKIN^® skin graft (Fidia Advanced Biopolymers, Abano Terme, Italy); RECELL^® skin graft (Avita Medical, Perth, Australia), ORCEL^® skin graft (Ortec Int’l, GA, USA), APLIGRAFT^® skin graft (Organogenesis, MA, USA), or POLY ACTIVE^® skin graft (HC Implants BV, Leiden, Netherlands), and the like. Exemplary non-cellular skin substitutes useful in the embodiments disclosed herein include, but are not limited INTEGRA^® (Integra NeuroSciences, NJ, USA) scaffold; ALLODERM^® scaffold (LifeCell Corp., NJ, USA), HYALOMATRIX PA^® scaffold (Fidia Advanced Biopolymers, Abano Terme, Italy), DERMAGRAFT^® scaffold (Advanced BioHealing, CT, USA), TRANSCYTE^® (Advanced BioHealing, CT, USA), HYALOGRAFT 3D™ scaffold (Fidia Advanced Biopolymers, Abano Terme, Italy), or DERMAMATRIX^® scaffold (Synthes, CMF, PA, USA), and the like. The skilled person will readily appreciate skin substitutes (whether cellular or acellular) developed in the future are useful in the embodiments disclosed herein. Various skin substitutes useful in the embodiments disclosed herein are described in US Patent Application Publication number U.8. 201 1 /0245929, hereby expressly incorporated by reference in its entirety.

[0091] Other, non-limiting examples of scaffolds and matrices useful in the embodiments disclosed herein include PURAPLY^® collagen dressing (Organogensis, Inc. MA, USA), ALLEVYN^® matrix (Smith & Nephew, Hull, UK), ACTICOAT^® matrix (Smith & Nephew, Hull, UK), CICA-CARE^® matrix (Smith & Nephew', Hull, UK), DURA-FIBER^® matrix (Smith & Nephew', Hull, UK), INTRASITE^® matrix (Smith & Nephew, Hull, UK), IODOSORB^® matrix (Smith & Nephew, Hull, UK), OPSITE^® matrix (Smith & Nephew', Hull, UK), PROFORE^® matrix (Smith & Nephew, Hull, UK), CUTINOVA^® matrix (Smith & Nephew, Hull, UK), JELONET^® matrix (Smith & Nephew', Hull, UK), BIOBRANE^® matrix (Smith & Nephew', Hull, UK) FORTAFLEX^® bioengineered collagen matrix (Organogenesis, MA, USA), or FORTAGEN^® collagen construct (Organogenesis, MA, USA), and the like.

[0092] Accordingly, m some embodiments, the regenerative ceils and/or conditioned media are combined with a biocompatible matrix such as a mesh, a gauze, a sponge, a monophasic plug, a biphasic plug, a paste, a putty, a wrap, a bandage, a patch, a mesh, or a pad. In some embodiments, the biocompatible matrix can be resorbable, porous, or both resorbable and porous. Biocompatible matrices useful in the embodiments disclosed herein can include one or more of the following: proteins, polysaccharides, nucleic acids, carbohydrates, inorganic components or minerals, or synthetic polymers; or mixtures or combinations thereof. For example, in some embodiments, the biocompatible matrix can include one or more of a polyurethane, e.g. , NOVO SORB™ biocompatible polyurethane matrices, a siloxane, a polysiloxane, a collagen, a glycosaminoglycan, oxidized regenerated cellulose (ORC), an ORC: collagen composite, an alginate, an alginatexollagen composite, a ethylene diamine tetraacetic acid (EDTA), a poiy(laetie~co-glyco!itic acid (PLGA), a carboxymethylcellulose, a granulated col!agen-glyeosaminog!ycan composite, methylcellulose, hydroxypropyl methyleellu!ose, or hydroxyethyl cellulose algimc acid, po!y(a- hydroxy acids), poly(!actones), po!y(amino acids), poly(anhydrides), poly (orthoesters), poly(anhydride-co-imides), poly(orthocarbonates), palyfa-hydroxy alkanoates), poly(dioxanones), poly(phosphoesters), poly(L-lactide) (PLLA), poly(D,L- lactide) (PDLLA), polyglycolide (PGA), poly (lactide-co-gly col ide (PLGA), poly(L-lactide- co-D, L-lactide), poly(D,L-3actide-co-trimethylene carbonate), polyhydroxybutyrate (PUB), poly(e-caprolactone), poly(5-valerolactone), poiy(y-butyrolactone), poly(caprolactone), polyacrylic acid, polycarboxylic acid, poly(allyiamine hydrochloride), poly(diallyldimethylammonium chloride), poly(ethyieneirnine), polypropylene furnarate, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene, polymethylmethacrylate, carbon fibers, poly(ethylene glycol), polyethylene oxide), polyvinyl alcohol), poiy(vmylpyrrolidone), poly(ethyloxazoline), poly (ethylene oxide)- co-poly(propylene oxide) block copolymers, polyethylene tereplithalatejpoiyamidearabic gum, guar gum, xantham gum, gelatin, chitm, chitosan, chitosan acetate, chitosan lactate, chondroitin sulfate, N,O-carboxymethyl chitosan, a dextran, fibrin glue, glycerol, hyaluronic acid, sodium hyaiuronate, a cellulose, a glucosamine, a proteoglycan, a starch, lactic acid, a pluronic, sodium glycerophosphate, glycogen, a keratin, or a silk, or one or more composites thereof, or one or more mixtures thereof, or one or more combinations thereof. In some embodiments, the composition comprises a calcium phosphate. [0093] In some embodiments, the biocompatible matrix may comprise a collagen. In certain embodiments, the biocompatible matrix comprises a Type I collagen, a Type II collagen, a Type III collagen, a Type IV collagen, a Type V collagen, a Type VI collagen, a Type VII collagen, or a Type VIII collagen, or combinations thereof. Moreover, the collagen can comprise bovine collagen, human collagen, porcine collagen, equine collagen, or avian collagen, or combinations thereof. In certain embodiments, the collagen comprises bovine Type I collagen or human Type I collagen. In some embodiments the collagen is m combination with other materials (e.g., chondroitin 6 sulfate) and/or is supplemented with materials that provide barrier function (e.g., a silicone backing vapor barrier). One example of a composite collagen-containing graft is INTEGRA^® (Integra NeuroSciences, NJ, USA) scaffold.

[0094] In some embodiments, the regenerative cells and/or conditioned media can be combined with a HELISTAT® absorbable collagen hemostatic sponge (Integra Life

Sciences, NX USA); a HELITENE absorbable collagen hemostatic agent (Integra Life Sciences, NT, USA); Matrix Collagen Particles™ wound dressing (Collagen Matrix, Inc., NJ, USA); Matrix Collagen Sponge™ wound dressing (Collagen Matrix, Inc., NJ, USA); OASIS'”¹ wound matrix (Smith & Nephew', Hull, UK); BIOBLANKET™ surgical mesh (Kensey Nash, Corp.); ZIMMER™ collagen repair patch (Zimmer, Inc., Svvisdon, UK); PROMOGRAN™ matrix wound dressing (Systagenix, MA, USA), or FIBROCOL PLUS^®' collagen dressing (Systagenix, MA, USA), or the like. Yet other scaffolds and grafts useful in the embodiments disclosed herein are described in U.S. Patent No. 6,979,670, 7,972,631 ,

7,824,71 1 , and 7,358,284U.S. Patent Application Publication No. 201 1/0091515, each of which is hereby expressly incorporated by reference in its entirety.

[0095] In some embodiments, the regenerative cells and/or conditioned media are combined with a tissue scaffold, e.g., unprocessed adipose tissue, platelet rich plasma, or other tissue. Mixture of regenerative cells with tissue to form a fortified scaffold (e.g., a cell- enriched fat graft) useful in the embodiments described herein is disclosed, for example, in U.S. Patent No. 7651684, and Kakudo, et ai. (2013) Journal of Translational Medicine 11 :254, each of which is hereby expressly incorporated by reference in its entirety. Combination Therapy

[0096] As explained in further detail below, some embodiments provide for treatment or therapy of subjects utilizing a combination therapy, e.g., one or more additional additives (e.g., pharmaceutical agents, biologic agents, or other therapeutic agents) in addition to the regenerative cells and/or conditioned media as described herein.

[0097] In some embodiments, the one or more additional“agents” described above can be administered in a single composition with the regenerative cells and/or conditioned media. In some embodiments, the one or more additional“agents” can be administered separately from the regenerative cells and/or conditioned media. For example, in some embodiments, one or more additional agents can be administered just prior to, or just after, administration of the regenerative cells and/or conditioned media. As used herein, the term“just prior” can refer to within 15 minutes, 30 minutes, an hour, 2 hours, 3 hours, 4 hours, 5 hours, or within a range defined by any two of the aforementioned time points. Likewise, the phrase“just after administration” can refer to within 15 minutes, 30 minutes, an hour, 2 hours, 3 hours, 4 hours, 5 hours, or within a range defined by any two of the aforementioned time points.

[0098] Additional agents useful in combination therapy in the methods described herein include, for example, growth factors, cytokines, platelet rich plasma, steroids, non steroidal anti-inflammatory agents, anti-bacterial or anti-fungal agents, as well as, other agents known in the art to have beneficial effects in treatment of a burn or wound.

1) Growth Factors. Cytokines, and Hormones

[0099] In some embodiments, subjects can be administered one or more growth factors, cytokines or hormones, including combinations thereof, in addition to the regenerative cells and/or conditioned media disclosed herein. For example, in some embodiments, growth factors are administered concomitantly with, prior to, or following the administration of the regenerative cells and/or conditioned media. Non-limiting examples of growth factors useful in the embodiments disclosed herein include, but are not limited to, angiogenin, angiopoietin-l (Ang-l), angiopoietin-2 (Ang-2), brain-derived neurotrophic factor (BDNF), Cardiotrophin- 1 (CT-l), ciliary neurotrophic factor (CNTF), Del-l, acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), follistatin, ganulocyte colony-stimulating factor (G-CSF), glial cell line-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), scatter factor (SF), Interleukin-8 (IL-8), leptin, midkine, nerve growth factor (NGF), neurotrophin-3 (NT-3), Neurotrophin-4/5, Neurturin (NTN), placental growth factor, Platelet-derived endothelial cell growth factor (PD-ECGF), Platelet- derived growth factor-BB (PDGF-BB), Pleiotrophin (PTN), Progranulin, Proliferin, PBSF/SDF-l, Transforming growth factor-alpha (TGF-alpha), Transforming growth factor- beta (TGF-beta), Tumor necrosis factor-alpha (TNF-alpha), Vascular endothelial growth factor (VEGF), vascular permeability factor (VPF), or erythropoietin (see, e.g., Tobalem, et a. (2012) Br. J. Surg. 99(9): 1295-1303), and the like.

2) Anti-Inflammatory Agents

[0100] In some embodiments, subjects are administered one or more anti inflammatory agents, in addition to the regenerative cells and/or conditioned media as disclosed herein. As used herein, the term “anti-inflammatory agent” refers to any compound that reduces inflammation, and includes, but is not limited to steroids, or non steroidal anti-inflammatory drugs, or other biologies that have been demonstrated to have an anti-inflammatory effect.

[0101] Accordingly, in some embodiments, steroids are administered concomitantly with, prior to, or following the administration of the regenerative cells and/or conditioned media. Non-limiting examples of steroids useful in the embodiments disclosed herein include, but are not limited to, progestegens, e.g., progesterone, and the like; corticosteroids, e.g., prednisone, aldosterone, cortisol, and the like, androgens, e.g., testosterone, and the like, or estrogens.

[0102] Other anti-inflammatory agents useful in the embodiments disclosed herein include, for example, antibodies that inhibit action of TNF-a, IL-6 (see, e.g., Sun, et al. (2012) Repair and Regeneration, 20(4): 563-572), or anti-TNF conjugates, Sun, et al. (2012) Wound Repair Regen. 20(4): 563-572, and the like. These anti-inflammatory agents have been demonstrated to exhibit beneficial effects in burn recovery.

[0103] Non-steroidal anti-inflammatory drugs useful in the embodiments disclosed herein include propionic derivatives; acetic acid derivatives; biphenylcarboxylic acid derivatives; fenamic acid derivatives; or oxicams. Examples of anti-inflammatory actives include without limitation acetaminophen, diclofenac, diclofenac sodium and other salts, ibuprofen and its salts acetaminophen, indomethacin, oxaprozin, pranoprofen, benoxaprofen, bucloxic acid, or elocon; or mixtures thereof.

3) Anti-Oxidants

[0104] In some embodiments, the methods and compositions disclosed herein include administration of one or more anti-oxidants in addition to the regenerative cells and/or conditioned media. Antioxidants useful in the embodiments disclosed herein include, but are not limited to, N-acetylcysteine, cureumarin, galactomannan, pyruvate and other aipha-ketoacids, tliioglycoi!ate vitamin A and derivatives, including retinoic acid, retinyl aldehyde, retin A, retinyl palmitate, adapaiene, and beta-carotene; vitamin B (panthenol, provitamin B5, panthenic acid, vitamin B complex factor); vitamin€ (ascorbic acid and salts thereof) and derivatives such as ascorbyl pal itate; vitamin D including calcipotriene (a vitamin D3 analog) vitamin E including its individual constituents alpha-, beta-, gamma-, delta-tocopherol and cotrienols and mixtures thereof and vitamin E derivatives including vitamin E palmitate, vitamin E linolate and vitamin E acetate; vitamin K and derivatives; or vitamin Q (ubiquinone) or any combination thereof.

4) Platelet-Containins Fluids

[0105] In some embodiments, subjects are administered platelet rich plasma, in addition to the regenerative cells and/or conditioned media disclosed herein. For example, in some embodiments, a platelet containing fluid is administered concomitantly with, prior to, or following the administration of the regenerative cells and/or conditioned media. In some embodiments, the regenerative cells and/or conditioned media as disclosed herein are combined with a synergistically effective amount of platelet-containing fluid.

[0106] As used herein, the term“platelet-containing fluid” refers to any fluid, either biological or artificial, which contains platelets. Non-limiting examples of such fluids include various forms of whole blood, blood plasma, platelet rich plasma, or concentrated platelets in any medium, or the like, derived from human and non-human sources. For example, in some embodiments, the platelet-containing fluid refers to blood, platelets, serum, platelet concentrate, platelet-rich plasma (PRP), platelet-poor plasma (PPP), plasma, or fresh frozen plasma (FFP), and the like.

[0107] The term "PRP as used herein refers to a concentration of platelets greater than the peripheral blood concentration suspended in a solution of plasma. Methods for isolating PRP useful in the embodiments disclosed herein are known in the art. See, e.g., US Patent No. 8557535, International Patent Application Publication No. WO 09/155069, U.S. Patent Application Publication No’s, US20100183561, US20030060352, US20030232712, US20130216626, US20130273008, US20130233803, US20100025342, European Patent No. EP1848474B1, each of which is hereby expressly incorporated by reference in its entirety. Platelets or PRP can suspended in an excipient other than plasma. In some embodiments, the platelet composition can include other excipients suitable for administration to a human or non-human animal including, but not limited to isotonic sodium chloride solution, physiological saline, normal saline, dextrose 5% in water, dextrose 30% in water, or lactated ringer’s solution and the like. Typically, platelet counts in PRP as defined herein range from 500,000 to 1 ,200,000 per cubic millimeter, or even more. PRP may be obtained using autologous, allogeneic, or pooled sources of platelets and/or plasma. PRP may be obtained from a variety of animal sources, including human sources. In preferred embodiments, PRP according to the invention is buffered to physiological pH.

Methods of Administration

[0108] In some embodiments, the methods disclosed herein include administering a therapeutically effective amount of a composition comprising regenerative cells and/or conditioned media to a subject. As used herein, the term“therapeutically effective amount” refers to an amount sufficient to improve endothelial cell function. Determination of the exact dose of regenerative cells for the embodiments disclosed herein is well within the skill in the art.

[0109] The amount and frequency of administration of the compositions can vary depending on, for example, what is being administered, the state of the patient, and the manner of administration in therapeutic applications, compositions can be administered to a patient suffering from a burn or wound (e.g., a subject that has been identified as having a partial thickness burn and/or a full thickness burn or that is in need of a graft), in an amount sufficient to relieve or least partially mitigate burn or wound progression. The compositions can also be administered to a patient receiving a graft (e.g., a subject that has a debrided wound or burn) in an amount sufficient to improve survival of the graft, once administered to the patient. The dosage is likely to depend on such variables as the type and extent of the burn or wound graft, as well as, the age, weight and general condition of the particular subject, and the route of administration. Effective doses can be extrapolated from dose- response curves derived from in vitro or animal model test system.

[0110] In some embodiments, at least 1 x 10² regenerative cells is a therapeutically effective amount. In some embodiments, at least 1 x 10³ regenerative cells is a therapeutically effective amount. In some embodiments, at least 1 x 10⁴ cells is a therapeutically effective amount. In some embodiments, at least 1 x 10⁵ regenerative cells is a therapeutically effective amount. In some embodiments, at least 1 x 10⁶ regenerative cells is a therapeutically effective amount. In some embodiments, at least 1 x 10⁷ regenerative cells is a therapeutically effective amount. In some embodiments, at least 1 x 10⁸ regenerative cells is a therapeutically effective amount. In some embodiments, at least 1 x 10⁹ regenerative cells is a therapeutically effective amount. In some embodiments, at least 1 x 10¹⁰ regenerative cells is a therapeutically effective amount.

[0111] In some embodiments, the regenerative cells comprise at least 0.05% stem cells. For example, in some embodiments, the regenerative cells comprise at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50%, or more, stem cells. That is, in some embodiments, at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50%, or more, of the nucleated cells within the regenerative cell population are stem cells or an amount that is within a range defined by any two of the aforementioned percentages.

[0112] Compositions administered according to the methods described herein can be introduced into the subject by, e.g., by intravenous, intra-arterial, intradermal, intramuscular, intra-lymphatic, intranodal, intramammary, intraperitoneal, intrathecal, retrobulbar, or intrapulmonary (e.g., term release); by oral, sublingual, nasal, anal, vaginal, or transdermal delivery, or by surgical implantation at a particular site. The introduction may consist of a single dose or a plurality of doses over a period of time. In such cases the plurality of introductions need not be by the same mechanism. For example, in some embodiments introduction at one time might be in the form of a topical spray of the regenerative cells whereas at another time the introduction may be regenerative cells combined with an fat graft, e.g., an autologous fat graft. Vehicles for cell therapy agents are known in the art and have been described in the literature. See, for example Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publ. Co, Easton Pa. 18042) pp 1435-1712, incorporated herein by reference. Sterile solutions are prepared by incorporating the regenerative cells that are in the required amount in the appropriate buffer with or without various of the other components described herein.

[0113] In some embodiments, the regenerative cells and/or conditioned media disclosed herein are formulated for injection. Accordingly, in some embodiments, the compositions disclosed herein are formulated for intravenous, intraarterial, intradermal, intramuscular, intraperitoneal, intrasternal, subcutaneous, intranodal and intra-lymphatic injection, infusion, or placement. In some embodiments, the compositions disclosed herein are formulated for intra-lymphatic delivery.

[0114] In some embodiments, the regenerative cells and/or conditioned media are formulated for administration in multiple doses, e.g., in multiple injections. In some embodiments, the regenerative cells disclosed herein are administered via one or multiple intravenous injections. For example, in some embodiments, the regenerative cells and/or conditioned media are administered via a single intravenous infusion over a period of 1 min, 2 min, 3 min, 4 min, 5 min, 10 min, 30 min, 45 min, 1 h, 2 h, or longer or within a range defined by any two of the aforementioned time points.

[0115] In some embodiments, the regenerative cells and/or conditioned media disclosed herein are administered by applying the cells to a scaffold as discussed elsewhere herein (e.g., including but not limited to biocompatible synthetic or non-synthetic matrices, such as skin substitutes), and applying the scaffold seeded with the regenerative cells to the subject. In some embodiments, a scaffold (e.g., including but not limited to biocompatible synthetic or non-synthetic matrices, such as skin substitutes) is applied to the subject, and the regenerative cells disclosed herein are applied onto the scaffold.

[0116] In some embodiments, the compositions including the regenerative cells and/or conditioned media disclosed herein are administered within 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, 1 h, 2h, 3h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 24 h, 36 h, 48 h, 60 h, 1 week, 2 weeks, or less or within a range defined by any two of the aforementioned time points. In some embodiments, the regenerative cells and/or conditioned media are administered serially over a period of time ( e.g ., wherein the subject can be administered regenerative cells in a single or in a plurality of doses each time). For example, in some embodiments, the regenerative cells and/or conditioned media described herein can be administered every 12 hours, every day, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, every month, or more or within a range defined by any two of the aforementioned time points. The frequency of treatment may also vary. The subject can be treated one or more times per day (e.g , once, twice, three, four or more times) or every' so-many hours (e.g., about every 2, 4, 6, 8, 12, or 24 hours or within a range defined by any two of the aforementioned time points). The time course of treatment may be of varying duration, for example, for two, three, four, five, six, seven, eight, nine, ten or more days. For example, the treatment can be twice a day for three days, twice a day for seven days, twice a day for ten days or within a range defined by any two of the aforementioned time points. For example treatment can be repeated weekly, bimonthly or monthly, and the periods of treatment can be separated by periods in which no treatment is given. The treatment can be a single treatment or can last as long as the life span of the subject (e.g., many years).

[0117] As disclosed herein, the regenerative cells and/or conditioned media can be provided to the subject, or applied directly to damaged tissue of the subject (e.g., to a wound or ulcer, a site of edema, or the like), or in proximity to the damaged tissue, without further processing or following additional procedures to further purify, modify, stimulate, or otherwise change the cells after isolation from the tissue of origin. For example, the cells obtained from a patient may be provided back to said patient without culturing the cells before administration. In several embodiments, the collection and processing of adipose tissue, as well as, administration of the regenerative cells is performed at a patient's bedside. In a preferred embodiment the regenerative cells are extracted from the tissue of the person into whom they are to be implanted, thereby reducing potential complications associated with antigenic and/or immunogenic responses to the transplant. However, the use of cells extracted from or derived from another individual is also contemplated. EXAMPLES

Example 1 : Adipose-Derived Regenerative Cells Produce Secretions that Improve Viability of Endothelial Cells.

[0118] The effect of ADRCs-CM on serum starved HUVEC viability was determined using the Resazurin assay and a co-culture transwell assay.

[0119] In the experiments described herein, adipose-derived regenerative cells (“ADRCs”) were isolated from human adipose tissue by [using the Celution® CRS/800. Following isolation, the ADRCs were used to prepare conditioned media as described below.

[0120] In the experiments described herein, two types of culture supernatant (fresh and short) from adipose-derived regenerative cells (ADRCs) were collected as “CM’S”.

[0121] For“fresh CM,” 5x10⁶ of human ADRCs were cultured with l2ml or 6ml (“dense”) of 2% FBS endothelial basal medium-2 (EBM2) in 75cm² flask. After 48 hours, supernatants were collected and floating cells were removed by centrifugation (400G, 5min).

[0122] For“short CM,” 5x10⁶ of human ADRCs were cultured with endothelial growth medium (EGM2) for 2 days. On day 2, EGM2 media was replaced with 0.25% BSA EBM2 media (6 or l2mL/75cm² flask). Floating cells were washed with PBS1X and were added to the culture. Culture supernatants without floating cells were collected after 48 hours as short CM. CM were stored at -80°C prior to use.

[0123] For cell viability assays, Passage 3 to 5 of human umbilical vein endothelial cell (HUVECs; Lonza) were used. HUVECs (3x10⁴ cells/well) were seeded in a 96 well plate (Black plate, clear bottom: Corning) and cultured with EGM2. Next, confluent HUVECs were starved with EBM2 containing 0.25% BSA. After 48hr, starvation medium was replaced with“fresh CM’,“dense fresh CM’,“short CM’, or“dense short CM’ for an additional 24hrs. The next day, the Resazurin (SIGMA) dye solution was added to each well at a final concentration of 12.5pl/ml and cells were incubated for 4 hours at 37°C.

[0124] Cell viability in HUVECs was measured by determination of the metabolic reduction of resazurin to resorufin. The reduction of resazurin was determined by recording fluorescence intensity using a microplate reader at excitation/emission wavelengths of 544/590nm (SpectraMax Gemini XS, Molecular Devices). [0125] The remaining adherent HUVECs were washed by PBS after the Resazurin assay. Next, the cells were incubated with the Cell Stain solution (Millipore). Cells were easily observed under a microscope (40X) and pictures of stained cells were collected.

[0126] Results: As shown in Figure 1A, a significant decrease in resazurin metabolism was observed with the EBM2 media compared to positive control EGM2. Addition of both fresh and short-CM increased resazurin metabolism compared to EBM2 control, respectively (p<0.0l, P<0.00ln=6). Interestingly, concentration of the ADRC-CM (dense CM) further increased resazurin metabolism by 26% and 43% compared to control (r<0.001, n=6).

[0127] To confirm these data, HUVECs were stained and observed under a microscope at 40X. As shown in Figure IB, the number of adherent cells is increased in HUVECs treated with ADRCs-CM compared to control EBM2. Data shown are representative of four independent experiments.

Example 2: Adipose-Derived Regenerative Cells Improve Viability of Endothelial Cells

[0128] A co-culture transwell assay was used to observe the effect of ADRCs on HUVECs viability. For these experiments, freshly isolated ADRCs [2.5 x 10⁵ cells (“high ADRC”) or .05 x 10⁵ cells“low ADRC”)] were seeded into the upper chamber of the insert (Corning, 0.4mhi) with 700m1 of EBM2 containing 2% FBS for 24 hrs. HUVECs were cultured in a 12 well plate (Corning) and starved for 48hrs in 800m1 of 0.25% BSA EBM2.

[0129] Following starvation, the co-culture was initiated by placing the inserts containing cultured ADRCs onto the HUVECs. ADRCs and HUVECs were co-cultured for 24hours and HUVEC viability was determined using the rezasurin assay.

[0130] As shown in Figure 2, the co-culture system of ADRCs and HUVECs revealed indirect cell to cell action through the release of growth factors /cytokines and exosomes. Using the Resazurin assay, ADRCs were able to significantly increase HUVECs viability compared to control EBM2 in a dose-dependent manner (n=6, p<0.05). Data shown are representative of three independent experiments.

[0131] Together, the data from Examples 1 and 2 demonstrate the cytoprotective role of adipose-derived regenerative cells and their secretions on endothelial cells, and show that adipose-derived regenerative cells can function to improve viability of endothelial cells. The co-culture system also reveals indirect cell to cell action through the release of growth factors /cytokines and exosomes. Using the Resazurin assay, ADRCs were able to significantly increase HUVECs viability compared to control EBM2 in a dose-dependent manner (n=6, p<0.05)

Example 3. Adipose-derived Regenerative Cells Improve Endothelial Function by Promoting Endothelial Cell Migration.

[0132] To determine whether ADRCs influence HUVEC migration, a Transwell Migration System was used, in which HUVECs were allowed to migrate toward freshly isolated ADRCs.

[0133] Migration activity of HUVECs was evaluated by using Colorimetric Transwell Cell Migration Assay (Millipore). Briefly, 1 x 10⁵ HUVECs were seeded into the upper chamber of the 0.1% gelatin coated- insert (transwell plates are 6.5 mm in diameter with 8 pm pore filters; Corning Costar, Cambridge, MA), with 300 pL of 0.25% BSA EBM2.

[0134] Freshly isolated ADRCs (either lxl 0⁵ or 0.3x10⁵ in 600pl of 2% FBS EBM_2;“ADRCS” or“1/3 ADRCs”, respectively) were seeded in the bottom chamber.

[0135] HUVECs were allowed to migrate for 16 hours at 37°C in an atmosphere containing 5% C0₂. The filters were then rinsed with PBS, fixed and stain per manufacturer’ s instructions .

[0136] The upper surfaces of the filters were scraped with cotton swabs to remove the non-migrating cells. The number of migrating cells attached to the lower surfaces of the filters was counted in four random high power fields (x400 magnification) under an Axiovert microscope (Zeiss).

[0137] As shown in Figure 3, ADRCs increased HUVEC migration compared to control EBM2 in a dose-dependent manner (n=2 or 3, p<0.05 and p<0.0l). These data demonstrate that regenerative cells, e.g., adipose-derived regenerative cells, can improve endothelial function by promoting endothelial cell migration.

Claims

WHAT IS CLAIMED IS:

1. A method of improving endothelial cell function, comprising:

identifying a subject in need of improvement of endothelial cell function; and administering an amount of a composition comprising regenerative cells sufficient to improve endothelial cell function to said subject.

2. The method of claim 1, wherein improving endothelial cell function comprises enhancing or preserving endothelial cell viability.

3. The method of claim 1 or 2, wherein improving endothelial cell function comprises improving leakiness of the endothelial cell barrier.

4. The method of any one of claims 1-3, wherein improving endothelial cell function comprises mitigating or dampening improper endothelial cell activation

5. The method of any one of claims 1-4, wherein improving endothelial cell function comprises enhancing endothelial cell migration.

6. The method of any one of the preceding claims, wherein the regenerative cells comprise a heterogeneous population of cells.

7. The method of claim 6, wherein the heterogeneous population of regenerative cells comprises stem and progenitor cells.

8. The method of any one of the preceding claims, wherein the regenerative cells are derived from a tissue selected from the group consisting of: bone marrow, placenta, adipose tissue, skin, eschar tissue, endometrial tissue, adult muscle, corneal stroma, dental pulp, Wharton’s jelly, amniotic fluid, and umbilical cord.

9. The method of claim 8, wherein the regenerative cells are adipose-derived regenerative cells.

10. The method of any one of claims 1 -9, wherein the regenerative cells have not been cultured prior to the administering step.

11. The method of any one of claims 1-10, wherein the regenerative cells are adherent cells.

12. The method of any one of claims 1 -9 or 11 , wherein the regenerative cells are cultured for at least 5 passages in tissue culture.

13. The method of any one of claims 1-12, wherein the regenerative cells are cryopreserved.

14. The method of any one of claims 1-13, wherein the composition comprises an additive.

15. The method of claim 14, wherein the additive is selected from the group consisting of cells, tissue, and tissue fragments.

16. The method of claim 14 or 15, wherein the additive comprises undigested adipose tissue.

17. The method of claim 10, further comprising the steps of: processing adipose tissue to separate regenerative cells comprising stem and progenitor cells from mature adipocytes and connective tissue.

18. The method of claim 17, wherein the processing comprises mechanically or enzymatically disaggregating adipose tissue to release the regenerative cells from mature adipocytes and connective tissue present in the adipose tissue.

19. The method of claim 18, wherein the processing comprises enzymatically disaggregating the adipose tissue with collagenase, a neutral protease, or both.

20. The method of any one of claims 1-19, wherein the subject has a condition selected from the group consisting of: angina pectoris, myocardial infarction, coronary artery disease, hypertension, cerbrovascular accidents, transient ischemic attacks, chronic obstructive pulmonary disease, chronic hypoxic lung disease, pulmonary hypertension, renal hypertension, chronic renal disease, microvascular complications of diabetes, vasoocclusive complications of sickle cell anemia, erectile dysfunction, systemic sclerosis, peripheral vascular disease, hypercholesterolemia, hypertension, atherosclerosis, septic shock, stroke, heart disease, insulin resistance, tumor growth, metastasis, venous thrombosis and adult respiratory distress syndrome.

21. A method of improving endothelial cell function, comprising:

identifying a subject in need of improvement of endothelial cell function; and administering a composition comprising secretions from regenerative cells cultured in vitro , in an amount sufficient to improve endothelial cell function to said subject.

22. The method of claim 21, wherein the regenerative cells are derived from a tissue selected from the group consisting of: bone marrow, placenta, adipose tissue, skin, eschar tissue, endometrial tissue, adult muscle, corneal stroma, dental pulp, Wharton’s jelly, amniotic fluid, and umbilical cord.

23. The method of claim 21 or 22, wherein the regenerative cells are adipose- derived regenerative cells.

24. The method of any one of claims 21-23, wherein the regenerative cells are grown in EGIVh media.

25. The method of claim 23, wherein the adipose-derived regenerative cells are grown in EGM2 media

26. The method of any one of claims 21-25, wherein the subject has a condition selected from the group consisting of: angina pectoris, myocardial infarction, coronary artery disease, hypertension, cerbrovascular accidents, transient ischemic attacks, chronic obstructive pulmonary disease, chronic hypoxic lung disease, pulmonary hypertension, renal hypertension, chronic renal disease, microvascular complications of diabetes, vasoocclusive complications of sickle cell anemia, erectile dysfunction, systemic sclerosis, peripheral vascular disease, hypercholesterolemia, hypertension, atherosclerosis, septic shock, stroke, heart disease, insulin resistance, tumor growth, metastasis, venous thrombosis and adult respiratory distress syndrome.