WO2014091373A1

WO2014091373A1 - Cellular and molecular therapies for peripheral vascular disease

Info

Publication number: WO2014091373A1
Application number: PCT/IB2013/060681
Authority: WO
Inventors: Paul Shiels; Wayne Davies
Original assignee: The University Court Of The University Of Glasgow
Priority date: 2012-12-11
Filing date: 2013-12-05
Publication date: 2014-06-19

Abstract

The present invention relates to compositions and methods for the treatment of peripheral vascular disease (PVD). In particular, the invention provides compositions and methods drawn to critical limb ischemia, and related diseases, disorders or conditions, based on the use of pathfinder cells, extracellular secretomes thereof, and/or associated microRNAs.

Description

CELLULAR AND MOLECULAR THERAPIES FOR

PERIPHERAL VASCULAR DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Application Serial

No. 61/735,797 filed on December 11, 2012, the disclosure of which is incorporated herein by reference.

BACKGROUND

[0002] Peripheral vascular disease (PVD) is generally characterized by partial or complete obstruction of vasculature outside the heart or brain, and can result from

atherosclerosis, inflammatory processes leading to stenosis, embolism, or thrombus formation, among others. Peripheral artery disease (PAD) is a form of PVD in which there is a partial or total blockage of arterial blood supply to various internal organs and/or limbs. Risk factors for PAD include elevated blood cholesterol, diabetes, smoking, hypertension, inactivity, and obesity. About 5% of people over the age of 50 are believed to suffer from PAD. Symptoms of PAD depend upon the location and extent of the blocked arteries. The most common symptom of PAD is intermittent claudication, manifested by pain (usually in the calf) that occurs while walking and dissipates at rest. Over time, as the severity of PAD increases, symptoms appear after a shorter duration of exercise. When PAD becomes more severe, symptoms may include pain and cramps at night, pain or tingling in the feet or toes, pain that is worse when legs are elevated and dissipates when legs are dangled (e.g., over the side of the bed), and ulcers that do not heal. PAD can ultimately reach a stage of critical limb ischemia (CLI), which is generally characterized by skin sores that do not heal, ulcers, gangrene, and/or infections in the extremities. In rare cases, amputation may be necessary. In general, an individual who is affected by PAD has an increased potential for developing arterial disease of the arteries within the heart and the brain. Indeed, according to the American Heart Association, people with peripheral arterial disease have four to five times more risk of heart attack or stroke.

[0003] PVD (e.g., PAD) can be treated by lifestyle alterations, medications, angioplasty and related treatments, or surgery. Although these therapies alleviate symptoms, and may even improve survival, none can reverse the disease process and directly repair the lasting damage. Thus, the cure of diseases, disorders or conditions associated with PVD (e.g., PAD and/or CLI) remain a major unmet medical need.

SUMMARY OF THE INVENTION

[0004] The present invention provides, among other things, cellular and molecular therapies for treatment of peripheral vascular disease (PVD) and related diseases, disorders and conditions. The present invention encompasses the discovery that multipotent cells isolated from various adult tissues, also known as "pathfinder cells," showed significant therapeutic effects in an animal hind limb ischemia (HLI) disease model, which is a model of mild ischemia and critical limb ischemia (CLI), indicating that pathfinder cells may be particularly useful in treating peripheral vascular diseases, disorders, and/or conditions.

[0005] Thus, in various embodiments, the present invention provides methods and compositions for treating or curing PVD and related diseases, disorders and conditions based on pathfinder cells and/or extracellular secretomes such as associated microvesicles and/or microRNAs.

[0006] In various embodiments, the present invention provides a method for treating peripheral vascular disease comprising a step of administering a population of cells (e.g., pathfinder cells), or extracellular secretomes thereof, to an individual suffering from a peripheral vascular disease characterized by partial or complete blockage of blood flow to one or more tissues outside the heart and brain, wherein the population of the cells is originated from an adult tissue and further wherein the population of the cells, or the extracellular secretomes thereof, is administered in a therapeutically effective amount to the individual such that at least one symptom or feature of the peripheral vascular disease is reduced in intensity, severity, or frequency, and/or has delayed onset. In some embodiments, the one or more tissues outside the heart and brain comprise one or more limbs of the individual.

[0007] In some embodiments, the peripheral vascular disease is a peripheral artery disease. For example, in certain embodiments, the peripheral artery disease is critical limb ischemia. In some embodiments, the peripheral vascular disease is an acute ischemia. In some embodiments, the peripheral vascular disease is a chronic ischemia.

[0008] In various embodiments, the cells (e.g., pathfinder cells), or the extracellular secretomes thereof, induce (e.g., promote) and/or increase angiogenesis and/or vascularization in the one or more tissues outside the heart and brain. In some embodiments, the cells decrease and/or delay cell death in the one or more tissues outside the heart and brain. For example, in some embodiments, the cell death occurs via apoptosis. In some embodiments, the cell death occurs via necrosis.

[0009] In certain embodiments, the cells (e.g., pathfinder cells), or the extracellular secretomes thereof, increase and/or enhance cell survival in the one or more tissues outside the heart and brain.

[0010] In various embodiments, the therapeutically effective amount of the cells, or the extracellular secretomes thereof, is sufficient to decrease partial or total blockage of blood flow to the one or more tissues outside the heart and brain.

[0011] In various embodiments, the therapeutically effective amount of the cells, or the extracellular secretomes thereof, is sufficient to decrease or delay tissue damage in the one or more tissues outside the heart and brain.

[0012] In various embodiments, the therapeutically effective amount of the cells, or the extracellular secretomes thereof, is sufficient to prevent further progress of disease condition in the one or more tissues outside the heart and brain.

[0013] In various embodiments, the therapeutically effective amount of the cells, or the extracellular secretomes thereof, is sufficient to improve function of the one or more tissues outside the heart and brain.

[0014] In certain embodiments, the therapeutically effective amount ranges from approximately 1 x 10⁶ to 3 x 10⁸ cells per kg body weight per dose. [0015] In some embodiments, the cells are administered to an individual intravenously, intra-arterially, intramuscularly, subcutaneously, cutaneously, intradermally, intrapleurally, intra- orbitally, intranasally, orally, intra alimentrally, and/or colorectally.

[0016] It will be appreciated that the cells may be administered in any appropriate regimen. For example, in various embodiments, the cells are administered daily. In some embodiments, the cells are administered weekly. In some embodiments, the cells are

administered biweekly. In some embodiments, the cells are administered monthly.

[0017] In various embodiments, the cells are originated from an adult tissue that is distinct from the diseased tissue. In some embodiments, the cells are originated from an adult tissue that is from a species different from that of the individual.

[0018] In some embodiments, the adult tissue is a human adult tissue. In some embodiments, the adult tissue is a non-human adult tissue.

[0019] In various embodiments, the adult tissue may be selected from the group consisting of pancreas, kidney, breast, lymph node, liver, spleen, myometrium, placenta, tonsils, peripheral blood, cord blood, bone marrow, and combination thereof.

[0020] In certain embodiments, the cells are first cultivated in a cell culture medium under conditions and time sufficient for cell proliferation. In some embodiments, cell culture medium is a Matrigel free culture medium comprising serum. In certain embodiments, the cells are first treated to reduce a telomeric attrition rate before the cultivating step.

[0021] In some embodiments, the population of cells are substantially homogenous. In certain embodiments, at least 50%, at least 80%, or at least 90% of the population of cells express one or more markers selected from the group consisting of CD24, c-myc, HLA class 1 ABC, ICAM3, Nestin, Nanog, Oct4, Integrin a2 + bl, Ngn3, and CD130. In certain

embodiments, the cells do not express at least one of the following markers: CD34, CD 105, VCAM1, CXCR2, CD44, CD73 and ICAMl.

[0022] In various embodiments, the present invention provides a method further including administering a pro-angiogenic agent in combination with the cells. In certain embodiments, the pro-angiogenic agent is or comprises vascular endothelial growth factor (VEGF).

[0023] In certain embodiments, the present invention provides a method further including a step of performing a vascular or endovascular procedure on an affected tissue of an individual. In some embodiments, such procedure is performed on one or more limbs of the individual.

[0024] In this application, the use of "or" means "and/or" unless stated otherwise. As used in this application, the term "comprise" and variations of the term, such as "comprising" and "comprises," are not intended to exclude other additives, components, integers or steps. As used in this application, the terms "about" and "approximately" are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0025] Other features, objects, and advantages of the present invention are apparent in the detailed description, drawings and claims that follow. It should be understood, however, that the detailed description, the drawings, and the claims, while indicating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings are for illustration purposes only not for limitation.

[0027] Figure 1 depicts exemplary blood flow measurements up to 49 days after induction of hindlimb ischemia. [0028] Figure 2 depicts exemplary limb necrosis scores up to 49 days after induction of hindlimb ischemia.

[0029] Figure 3 depicts exemplary limb functional scores up to 49 days after induction of hindlimb ischemia.

DEFINITIONS

[0030] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0031] Acute: As used herein, the term "acute" when used in connection with tissue damage and related diseases, disorders, or conditions, has the meaning understood by any one skilled in the medical art. For example, the term typically refers to a disease, disorder, or condition in which there is sudden or severe onset of symptoms. In some embodiments, acute damage is due to an ischemic or traumatic event. Typically, the term "acute" is used in contrast to the term "chronic."

[0032] Adult: Cells useful for the present invention (e.g., pathfinder cells) may be originated from an adult tissue. As used herein, the term "adult" when used to describe source tissues is not meant herein to imply that the tissues must be obtained from an adult individual, but rather, that the tissues are themselves fully developed or differentiated, rather than being in an embryonic or undifferentiated form. Thus the term is used herein to embrace adult tissues including, but are not limited to: tissues of pancreas, kidney, breast, lymph node, liver, spleen, myometrium, peripheral blood, cord blood, and bone marrow, and combinations thereof. In some embodiments, the tissues useful for the present invention are not embryonic tissues. In some embodiments, the tissues useful for the present invention are not mesenchymal tissues. Accordingly, in some embodiments, cells useful for the present invention are not embryonic cells, e.g., embryonic stem cells. In some embodiments, cells useful for the present invention are not mesenchymal cells, e.g., mesenchymal stem cells. [0033] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, at any stage of development. In some embodiments, "animal" refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.

[0034] Approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0035] Autologous and non-autologous: As used herein, the term "autologous" means from the same organism. In the context of the present application, the term is used to mean that the population of cells and/or microvesicles referred to as "autologous" to each other do not contain any material which could be regarded as allogenic or xenogenic, that is to say derived from a "foreign" cellular source. As used herein, the term "non-autologous" means not from the same organism.

[0036] Cell culture: As used herein, the term "cell culture" or its equivalents shall mean one or more cells cultivated in a controlled environment. A cell culture typically pertains to an isolated collection of cells in a defined medium under controlled conditions. However, in certain circumstances, a cell culture may contain a single cell. Typically, a single cell culture is obtained from a larger culture by dilution. In other embodiments, a cell culture contains two or more cells. For such a cell culture, the culture may consist of cells of a significantly pure population. For example, in some embodiments, cells in a culture are clonal in nature. Clonal cells are derived from a single parental cell and typically contain identical genetic make-up. In some embodiments, however, at least one of the cells in a culture contains at least one genetic mutation. Alternatively, a culture may be characterized as a mixed cell culture, which contains multiple cell types. In some embodiments, a mixed cell culture is prepared by design. In other embodiments, a mixed cell culture is a result of a contamination. In the context of cell cultures, the terms "grown" "cultivated" "maintained" and the like, are used interchangeably herein, unless specifically stated otherwise.

[0037] Chronic: As used herein, the term "chronic," when used in connection with tissue damage or related diseases, disorders, or conditions has the meaning as understood by any one skilled in the medical art. Typically, the term "chronic" refers to diseases, disorders, or conditions that involve persisting and/or recurring symptoms. Chronic diseases, disorders, or conditions typically develop over a long period of time. The term "chronic" is used in contrast to the term "acute." In some embodiments, a chronic disease, disorder, or condition results from cell degeneration. In some embodiments, a chronic disease, disorder, or condition results from age-related cell degeneration.

[0038] Control: As used herein, the term "control" has its art-understood meaning of being a standard against which results are compared. Typically, controls are used to augment integrity in experiments by isolating variables in order to make a conclusion about such variables. In some embodiments, a control is a reaction or assay that is performed

simultaneously with a test reaction or assay to provide a comparator. In one experiment, the "test" (i.e., the variable being tested) is applied. In the second experiment, the "control," the variable being tested is not applied. In some embodiments, a control is a historical control (i.e., of a test or assay performed previously, or an amount or result that is previously known). In some embodiments, a control is or comprises a printed or otherwise saved record. A control may be a positive control or a negative control. In some embodiments, a control is also referred to as a reference.

[0039] Critical Limb Ischemia: As used herein, the term "critical limb ischemia" or

"CLI" generally refers to a condition characterized by restriction in blood or oxygen supply to the extremities (e.g., hands, feet, legs) of an individual that may result in damage or dysfunction of a tissue in the extremities. Critical limb ischemia may be caused by any of a variety of factors, such as peripheral artery disease (PAD), and may cause severe pain, skin ulcers, or sores, among other symptoms, and in some cases leads to amputation. Critical limb ischemia may be characterized by vasoconstriction, thrombosis, or embolism in one or more extremities. Any tissue in an extremity that normally receives a blood supply can experience critical limb ischemia.

[0040] Crude: As used herein, the term "crude," when used in connection with a biological sample, refers to a sample which is in a substantially unrefined state. For example, a crude sample can be cell lysates or biopsy tissue sample. A crude sample may exist in solution or as a dry preparation.

[0041] Derivative thereof: As used herein, the term "derivative thereof," when used in connection with microvesicles or cells, refers to a fraction or extract (especially those containing RNA and/or DNA and/or protein) of the original microvesicle or population of cells which retains at least some biological activity (especially the ability to induce differentiation and/or the ability to provide therapeutic benefit) of the original. The term also includes complexed, encapsulated or formulated microvesicles or cells (for example, microvesicles that have been encapsulated, complexed or formulated to facilitate administration). Examples of derivatives include lysates, lyophilates and homogenates.

[0042] Dysfunction: As used herein, the term "dysfunction" refers to an abnormal function. Dysfunction of a molecule (e.g., a protein) can be caused by an increase or decrease of an activity associated with such molecule. Dysfunction of a molecule can be caused by defects associated with the molecule itself or other molecules that directly or indirectly interact with or regulate the molecule.

[0043] Extracellular secretomes: As used herein, extracellular secretomes of pathfinder cells refer to any isolates derived from pathfinder cells according to the invention, including, but not limited to, any microvesicles including exosomes and/or microRNAs isolated from

Pathfinder cells and microvesicles.

[0044] Functional: As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized. [0045] Functional derivative: As used herein, the term "functional derivative" denotes, in the context of a functional derivative of a nucleotide sequence (e.g., microRNA), a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence. A functional derivative or equivalent may be a natural derivative or is prepared synthetically. Exemplary functional derivatives include nucleotide sequences having substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the nucleic acids (e.g., microRNAs) is conserved.

[0046] Inducer. As used herein, the term "inducer" refers to any molecule or other substance capable of inducing a change in the fate of differentiation of a cell to which it is applied.

[0047] In vitro: As used herein, the term "m vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0048] In vivo: As used herein, the term "m vivo" refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0049] Ischemia: As used herein, the term "ischemia" (also spelled "ischaemia") typically refers to a restriction in blood or oxygen supply that may result in damage or dysfunction of a tissue. Ischemia may be caused by any of a variety of factors, such as factors in blood vessels, a blood clot, a severe drop in blood pressure, an increase in compartmental pressure, and/or trauma. The term "ischemia" as used herein also refers to local anemia in a given part of a body or tissue that may result, for example, from vasoconstriction, thrombosis, or embolism. Any tissue that normally receives a blood supply can experience ischemia.

[0050] Isolated: As used herein, the term "isolated" refers to a substance and/or entity that has been ( 1 ) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated. In some embodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, the term "isolated cell" refers to a cell not contained in a multi-cellular organism.

[0051] microRNA: As used herein, the term "microRNAs (miRNAs)" refers to post- transcriptional regulators that typically bind to complementary sequences in the three prime untranslated regions (3' UTRs) of target messenger RNA transcripts (mRNAs), usually resulting in gene silencing. Typically, miRNAs are short ribonucleic acid (RNA) molecules, for example, 21 or 22 nucleotides long. The terms "microRNA" and "miRNA" are used interchangeably.

[0052] Microvesicle As used herein, the term "microvesicle" refers to a membranaceous particle comprising fragments of plasma membrane, which may or may not be derived from the plasma membrane. Microvesicles described in the present invention therefore may include membranes that are originated from various cell compartments, including the outer membrane (e.g., the plasma membrane) and intracellular membranes of various cellular organelles, such as ER, Golgi, nucleus, etc. Typically, microvesicles have a diameter (or largest dimension where the particle is not spheroid) of between about 10 nm to about 5000 nm (e.g., between about 50 nm and 1500 nm, between about 75 nm and 1500 nm, between about 75 nm and 1250 nm, between about 50 nm and 1250 nm, between about 30 nm and 1000 nm, between about 50 nm and 1000 nm, between about 100 nm and 1000 nm, between about 50 nm and 750 nm, etc.). Typically, at least part of the membrane of the microvesicle is directly obtained from a cell (also known as a donor cell). Microvesicles suitable for use in the present invention may originate from cells by membrane inversion, exocytosis, shedding, blebbing, and/or budding. Depending on the manner of generation (e.g., membrane inversion, exocytosis, shedding, or budding), the microvesicles contemplated herein may exhibit different surface/lipid characteristics.

Alternative names for microvesicles include, but are not limited to, exosomes, ectosomes, membrane particles, exosome-like particles, and apoptotic vesicles. As used herein, an abbreviated form "MV" is sometime used to refer to microvesicle.

[0053] Pathfinder cells: As used herein, the term "pathfinder cells" refers to cells that have the capacity to induce or stimulate tissue repair, regeneration, remodeling, reconstitution, or differentiation. Typically, pathfinder cells induce or stimulate tissue or repair, regeneration, remodeling, reconstitution or differentiation without being a source of new tissue themselves. In some embodiments, pathfinder cells are also referred to as "progenitor cells." As used herein, an abbreviated form "PC" is sometime used to refer to a pathfinder cell. Typically, pathfinder cells are originated from adult tissues, including primary cells directly isolated from adult tissues or cells grown from primary cells in a cell culture system. The term "adult" when used to describe tissues is not meant herein to imply that the tissues must be obtained from an adult individual, but rather, that the tissues are themselves fully developed (e.g., post-natal), rather than being in embryonic form or derived from a fetus. Thus, it is specifically contemplated that pathfinder cells may be obtained from any developed individual (whether a child, adolescent, or adult), but are generally not obtained from embryos or fetuses. Pathfinder cells typically have one or more characteristics as described herein, such as expression of microRNAs, cell surface markers, and production of microvesicles. For further detail on pathfinder cells, see, PCT/IB11/02048 and PCT/IB11/02028, each of which is incorporated herein by reference in its entirety.

[0054] Peripheral vascular disease: As used herein, the term "peripheral vascular disease" or "PVD" refers to a disease, disorder or condition caused by partial or complete obstruction of blood vesicles (e.g., arteries) located outside the heart and brain (e.g., not within the coronary, aortic arch vasculature, or brain). As used herein, the term, "peripheral artery disease" or "PAD" refers to a form of PVD in which there is partial or total blockage of arteries that provide blood supply to one or more tissues located outside the heart and brain (e.g., not within the coronary, aortic arch vasculature, or brain) such as internal organs and/or limbs.

[0055] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre and post natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term "subject" is used herein interchangeably with "individual" or "patient." A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0056] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0057] Suffering from: An individual who is "suffering from" a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and/or condition.

[0058] Susceptible to: An individual who is "susceptible to" a disease, disorder, and/or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

[0059] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0060] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to any agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect. In some embodiments, a therapeutic agent of the invention refers to a peptide inhibitor or derivatives thereof according to the invention.

[0061] Tissue: As used herein, the term "tissue" refers to an aggregation of

morphologically similar cells and associated intercellular matter acting together to perform one or more specific functions in the body. In some embodiments, tissues fall into one of four basic types: muscle, nerve, epidermal, and connective. In some embodiments, a tissue is substantially solid, e.g., cells within the tissue are strongly associated with one another to form a solid. In some embodiments, a tissue is substantially non-solid, e.g, cells within the tissue are loosely associated with one another, or not at all physically associated with one another, but may be found in the same space, bodily fluid, etc. For example, blood cells are considered a tissue in non-solid form.

[0062] Transdifferentiation As used herein, the term "transdifferentiation" refers to a process in which a non-stem cell transforms into a different type of cell, or an already differentiated stem cell creates cells outside its already established differentiation path.

Typically, transdifferentiation include de- and then re-differentiation of adult cell types (or differentiated cell types).

[0063] Treating: As used herein, the term "treat," "treatment," or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0064] The present invention provides, among other things, improved compositions and methods for the treatment of peripheral vascular disease (PVD) and related diseases, disorders or conditions based on the use of pathfinder cells and/or extracellular secretomes thereof, such as associated microvesicles and/or microRNAs. [0065] Various aspects of the invention are described in detail in the following sections.

The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of "or" means "and/or" unless stated otherwise.

I. Cells

[0066] Cells suitable for the present invention are typically multipotent or pluripotent and can induce tissue repair, regeneration, remodeling, reconstruction, reprogramming, or transdifferentiation in vivo and/or in vitro. Typically, the present invention utilizes cells originated from an adult tissue. Such cells are also referred to as "pathfinder cells" (e.g., PCs) or "progenitor cells." In this application, the terms "cells" (used as a therapeutic agent as described herein), "pathfinder cells" and "progenitor cells" are used interchangeably. In some

embodiments of the invention, a population of cells suitable for the present invention is substantially free from mesenchymal stem cells. In certain embodiments of the invention, a population of cells suitable for the present invention does not contain mesenchymal stem cells. In some embodiments of the invention, a population of cells suitable for the present invention is substantially free from embryonic stem cells. In certain embodiments of the invention, a population of cells suitable for the present invention does not contain embryonic stem cells.

[0067] The present inventor has provided evidence that pathfinder cells may induce or stimulate host tissue repair, regeneration, remodeling, reconstitution, differentiation or transdifferentiation without being a source of new tissue themselves. See, e.g., PCT/IB 11/02048, entitled "Cell and Molecular Therapies", filed on August 12, 2011 and PCT/IB 11/02028, entitled "Therapeutic Uses of Microvesicles and Related MicroRNAs", filed on August 12, 2011, the entire contents of each of which are incorporated herein by reference. Without wishing to be bound by any theory, it is contemplated that pathfinder cells may induce changes within target tissue or cells to convert them into active repair mode by providing microRNAs and/or other components (e.g., membrane associated polypeptide, transcription factors, etc.) that will regulate expression of genes relating to, e.g., increased cell mobility, tissue remodeling and

reprogramming, growth, angiogenesis, cell adhesion and cell signaling, etc. [0068] As shown in the Examples, pathfinder cells led to increased blood flow and reduced limb necrosis, among other things, in a hind limb ischemia model of PVD. Without wishing to be bound by any theory, it is contemplated that pathfinder cells and/or extracellular secretomes thereof, such as associated microvesicles and/or microRNAs from different tissues, cell types or organisms can induce, enhance, or increase vascularization or angiogenesis, increase or enhance cell survival, and/or decrease or delay cell death.

[0069] In some embodiments, cells suitable for the present invention may be originated from an adult tissue that may or may not be the same as the target tissue. In some embodiments, cells suitable for the present invention may be originated from autologous or non-autologous adult tissues. In some embodiments, cells suitable for the present invention may be originated from adult tissues obtained from different species. For example, cells suitable for the present invention may be originated from human or non-human adult tissues.

[0070] In some embodiments, cells suitable for the present invention may have any desirable origin, including endothelial, mesothelial, and ectothelial origins. Thus, suitable cells include those found in a gland, an organ, muscle, a structural tissue, etc. Suitable cells may be heterologous (or non-autologous) or autologous relative to recipient. For example, suitable cells may be derived from a tissue the same as or different than the recipient tissue (e.g., a diseased or damaged tissue to be treated). In some embodiments, cells may be derived from a different organism (i.e., non-autologous). For example, a cell may be a porcine pancreatic cell, while the recipient is a human.

[0071] Cells suitable for the present invention may be isolated from any of a variety of tissue types, including, but not limited to, pancreas, kidney, lymph node, liver, spleen, myometrium, blood cells (including cells from peripheral blood and cord blood), placenta, tonsils, and/or bone marrow. Cells suitable for the present invention (e.g., pathfinder cells) may be grown in culture as previously described (see, for example, WO2006/ 120476 and

WO2009/136168, the entire contents of which are incorporated by reference).

[0072] Suitable cells may also be in any stage of their individual cellular age (e.g., stage or phase), ranging from just separated from their progenitor cell to a senescent or even dead cell. Thus, cells may include pre-apoptotic cells, or a cell committed to apoptosis. Suitable cells of the present invention may also be in any phase of the cell cycle. In some embodiments, a population of cells suitable for practicing the inventive methods provided herein may contain synchronized cells, such that a majority of cells in the population are in the same cell cycle phase. In some embodiments, a population of suitable cells comprises cells in two or more phases of the cell cycle.

[0073] Furthermore, it is contemplated that suitable cells also include non-diseased and diseased cells, wherein diseased cells may be affected by one or more pathogens and/or conditions. For example, a diseased cell may be infected with a virus, an intracellular parasite, or bacterium. In other examples, a diseased cell may be a metabolically diseased cell (e.g., due to genetic defect, due to an enzyme, receptor, and/or transporter dysfunction, or due to metabolic insult), a neoplastic cell, or cell that has one or more mutations that render the cell susceptible to uncontrolled cell growth. Similarly, cells may be native (e.g., obtained by biopsy), cultured (e.g., native, or immortalized), or treated. For example, cells may be chemically and/or mechanically treated, resulting in a cell that exhibits a cell-specific stress response. In some embodiments, suitable cells may be treated with a natural or synthetic ligand to which the cell has a receptor or otherwise complementary structure. In some embodiments, a cell may also be treated with a drug or compound that alters at least one of a metabolism, cell growth, cell division, cell structure, and/or secretion.

[0074] In some embodiments, suitable cells are recombinant cells. For example, recombinant cells may contain one or more nucleic acid molecules introduced by recombinant DNA technology. All known manners of introducing nucleic acids are deemed suitable for use herein (e.g., viral transfection, chemical transfection, electroporation, ballistic transfection, etc.). Where the nucleic is a DNA, it is contemplated that the DNA may be integrated into the genome of the cell, or that the DNA may reside as extrachromosomal unit within the cell. Such DNA may be employed as a template for RNA production, which may have regulatory and/or protein encoding function. Similarly where the nucleic acid is an RNA, such RNA may be used as a regulatory entity (e.g., via antisense or interference) and/or as a protein encoding entity. As used herein, nucleic acids encompass all known nucleic acid analogs (e.g., phosphorothioate analogs, peptide nucleic acid analogs, etc.) [0075] In some embodiments, cells are obtained from freshly isolated or stored materials

(e.g., biological fluids, tissues, organs, etc.). When cells are obtained from stored materials, such storage may include storage at a reduced temperature (e.g., 4 °C) or even storage in frozen form. Similarly, cells may also be obtained from an in vitro source, and most typically from cell or tissue culture (see the Cell Culture Condition section below), or even organ culture.

[0076] Cells may be isolated, for example, according to methods previously developed by the present inventor and available in the art, or variations thereof. See, e.g., Shiels et al. (2009) Stem Cells Dev., 18(10): 1389-98 and International Patent Publications WO2006/120476 and WO2009/136168, the entire contents of which are incorporated by reference. For example, a tissue or a part of a tissue (e.g., pancreatic ducts) can be isolated from an adult animal and minced. The minced tissue can be seeded in a suitable culture medium and incubated. In some embodiments, pathfinder cells will emerge as a confluent monolayer after a period of time (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or more) in culture. Pathfinder cells in a monolayer can be harvested and optionally washed in a solution such as PBS (phosphate-buffered saline).

[0077] Suitable cell culture conditions are described below. In embodiments in which pathfinder cells are obtained from cell cultures, they may be of any passage number so long as they can still have one or more properties that confer therapeutic benefit. Thus, pathfinder cells of low (e.g., less than about 10 passages), medium (e.g., between to about 10 to about 20 passages), or high (e.g., more than about 20 passages (e.g., about 30, 40, 50, 60, 70, 80, 90, 100, or more than 100)) passage number may be used in various embodiments of the invention.

Cell Culture Conditions

[0078] In some embodiments, pathfinder cells are obtained from a cell culture that had been established from freshly isolated or stored materials. For example, cells may be cultured in a liquid medium that contains nutrients for the cells and are incubated in an environment where the temperature and/or gas composition is controlled. As will be appreciated by one of ordinary skill in the art, specific cell culture conditions may vary depending on the type of cells used. Cell culture conditions for pathfinder cells have been described. See, e.g., International Patent Publication WO2006/120476.

[0079] An exemplary suitable medium for culture of pathfinder cells is CMRL 1066 medium (Invitrogen) supplemented with fetal bovine serum (e.g., at 10%). In some

embodiments, media is supplemented with glutamine or glutamine-containing mixtures such as GLUTAMAX™ (Invitrogen) and/or with antibiotics (e.g., amphotericin, penicillin, and/or streptomycin).

[0080] In some embodiments, cells are grown such they are attached on a surface. In some such embodiments, cells are grown as a monolayer on the surface. In some embodiments, cells are grown until they are confluent, i.e., until they cover the entire surface on which they are growing and there is nowhere else on the surface for cells to grow. In some embodiments, cells are grown until they are close to but not yet at confluence, i.e., until they cover most of the surface on which they are growing, but there is still some room for cells to grow. In some embodiments, cells are grown until they are approximately or more than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more confluent, wherein x% confluent is defined as coverage of approximately x% of the growing surface. In some embodiments, cells are grown until they are approximately 50-99% (e.g., 60-99%, 70-99%, 75-99%, 80-99%, 85-99%, 90-99%, or 95-99%) confluent.

[0081] In some embodiments, cells are grown on a substrate that may affect one or more properties of the cell, such as microvesicle production rate, cell proliferation rate, or miRNA expression pattern. In some embodiments, cells are grown on a nonwoven substrate such as a nonwoven fabric comprised of fibers. As used herein, the term "nonwoven fabric" includes, but is not limited to, bonded fabrics, formed fabrics, or engineered fabrics, that are manufactured by processes other than, weaving or knitting. In some embodiments, the term "nonwoven fabric" refers to a porous, textile-like material, usually in flat sheet form, composed primarily or entirely of fibers, such as staple fibers assembled in a web, sheet or batt. The structure of the nonwoven fabric may be based on the arrangement of, for example, staple fibers that are typically arranged more or less randomly. Nonwoven fabrics can be created by a variety of techniques known in the textile industry. Various methods may create carded, wet laid, melt blown, spunbonded, or air laid nonwovens. Exemplary methods and substrates are described in U.S. Application Publication No. 20100151575, the entire teachings of which are incorporated herein by reference. The density of the nonwoven fabrics may be varied depending upon the processing conditions. In one embodiment, the nonwoven fabrics have a density of about 60 mg/mL to about 350 mg/mL.

[0082] In some embodiments, the nonwoven substrates are biocompatible and/or bioabsorbable. Examples of suitable biocompatible, bioabsorbable polymers that could be used include polymers selected from the group consisting of aliphatic polyesters, poly( amino acids), copoly( ether-esters), polyalkylene oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amine groups, poly(anhydrides), polyphosphazenes, and blends thereof.

[0083] In some embodiments, the aliphatic polyesters are homopolymers and/or copolymers of monomers selected from the group consisting of lactide (which includes lactic acid, D-,L- and meso lactide), glycolide (including glycolic acid), epsilon-caprolactone, p- dioxanone (l,4-dioxan-2-one), trimethylene carbonate (l,3-dioxan-2-one), alkyl derivatives of trimethylene carbonate, delta-valerolactone, beta-butyrolactone, gamma-butyrolactone, epsilon- decalactone, hydroxybutyrate (repeating units), hydroxyvalerate (repeating units), 1 ,4-dioxepan- 2-one (including its dimer 1,5,8, 12-tetraoxacyclotetradecane-7, 14-dione), l,5-dioxepan-2-one, 6,6-dimethyl-l,4-dioxan-2-one and polymer blends thereof. In another embodiment, aliphatic polyesters which include, but are not limited to homopolymers and/or copolymers of lactide (which includes lactic acid, D-,L- and meso lactide), glycolide (including glycolic acid), epsilon- caprolactone, p-dioxanone (l,4-dioxan-2-one), trimethylene carbonate (l,3-dioxan-2-one) and combinations thereof.

[0084] In some embodiments, the aliphatic polyesters are homopolymers and/or copolymers of monomers selected from the group consisting of lactide (which includes lactic acid, D-,L- and meso lactide), glycolide (including glycolic acid), epsilon-caprolactone, p- dioxanone (l,4-dioxan-2-one), trimethylene carbonate (l,3-dioxan-2-one) and combinations thereof. In yet another embodiment, the aliphatic polyesters are homopolymers and/or copolymers of monomers selected from the group consisting of lactide (which includes lactic acid, D-,L- and meso lactide), glycolide (including glycolic acid), and p-dioxanone (1,4-dioxan- 2-one) and combinations thereof. Non-limiting examples of suitable fabrics include those that comprise aliphatic polyester fibers, e.g., fibers that comprise homopolymers or copolymers of lactide (e.g., lactic acid D-. L- and meso lactide), glycolide (e.g., glycolic acid), epsilon- caprolactone, p-dioxanone (l,4-dioxan-2-one), trimethylene carbonate ( l,3-dioxan-2-one), and combinations thereof. For example, suitable fabrics may contain poly(glycolide-co-lactide) (PGA/PLA); poly(lactide-co-glycolide) (PLA/PGA); 1 ,3 propanediol (PDO), and/or blends thereof.

[0085] In some embodiments, cells are grown on a solid surface that has been textured in a particular way so as to confer special properties to the surface (e.g. , repulsion or attraction of certain substances, reduced adsorption of proteins, etc.), which in turn may influence behavior of cells on such surfaces. For example, cells may be grown on a nano-textured surface

("nanosurface"). See, e.g., US 7,597,950; Sun et al. (2009) "Combining nanosurface chemistry and microfluidics for molecular analysis and cell biology," Analytica Chimica Acta, 650(1 ):98- 105; the entire contents of each of which are herein incorporated by reference. Nanosurfaces and other textured surfaces may be generated, for example by any of a variety of methods known in the art, including sanding, chemical etching, sandblasting, and/or dewetting.

[0086] In some embodiments, cells are grown in suspension.

[0087] In some embodiments, cells are grown in a Matrigel free culture system in the presence of serum. Pathfinder cells have been shown to be able to grow in such systems. See, e.g., International Patent Publication WO 2006/120476.

[0088] In some embodiments, cells are subjected for at least part of the time to one or more conditions that enhance their growth, e.g., such that increased numbers of cells can be obtained from a culture. In some embodiments, enhancement of cell growth is accomplished by modulating accessibility of telomeres in cells. For example, cells may be first treated to reduce a telomeric attrition rate prior to cell culture. In some embodiments, to achieve reduced telomeric attrition rate, components of the telomere complex and/or factors that associate with it may be targeted by an agent that causes their downregulation or otherwise impairs their function. For example, cells may be exposed to inhibitory RNAs such as small inhibitory RNAs (siRNAs) to inhibit expression a gene encoding a protein or other gene product that forms part of the telomere complex or associates with the telomere complex. In some embodiments, RAFl expression is inhibited with the use of a RAFl specific siRNA. In some embodiments, STAUl expression is inhibited with the use of a STAUl specific siRNA. For a fuller discussion of methods for enhancing cell growth that may be used in accordance with the present invention, see, e.g., U.S. provisional application entitled "Enhanced Cell Growth by the Modulation of Telomere

Accessibility" filed on even date, the entire contents of which are herein incorporated by reference.

[0089] Various growth media may be used to culture cells. Growth medium generally refers to any substance or preparation used for the cultivation of living cells. In some embodiments, the growth medium is renal growth medium. In some embodiment the growth medium is Dulbecco's Modification of Eagle's medium (DMEM). In some embodiments, cells are grown in media that does not contain serum (e.g., serum-free). In some embodiments, cells are grown for at least a period of time in media that has been depleted of microvesicles from media components. For example, media containing fetal bovine serum may be depleted of bovine microvesicles. Alternatively or additionally, commercially available medium that is depleted of microvesicles (e.g., bovine microvesicles) is used.

[0090] In some embodiments, cells are grown at or about 37 °C. In some embodiments, cells are grown in the presence of at or about 5% C0₂. In some embodiments, cells are grown under room air oxygen conditions. In some embodiments, cells are grown under conditions where the oxygen pressure is less than or equal to 5% 0₂. In some embodiments, cells are grown in conditions of normal oxygen (e.g., about 5% 0₂). In some embodiments, cells are grown in hypoxic conditions (e.g., low oxygen such as < 5%, < 4%, < 3 %, < 2%, or < 1% 0₂).

[0091] In some embodiments, cells are grown under serum starvation conditions. As used herein, the term "serum starvation" includes, but is not limited to, serum depletion, serum- free medium or conditions. Various serum starvation conditions are known in the art and can be used to practice the present invention. In some embodiments, cells may be grown under serum starvation conditions for about 6, about 12, about 18, about 24, about 30, about 36, about 42, about 48 hours, or longer. In some embodiments, cells may be grown under conditions where the serum concentration is less than or equal to 10%, less than or equal to 9%, less than or equal to 8%, less than or equal to 7%, less than or equal to 6%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1.5%, less than or equal to 1%, or less than or equal to 0.5%. In some embodiments, cells may be grown under conditions where the serum concentration is 0% (i.e., serum is absent). In some embodiments, cells may be grown under conditions where the serum concentration is decreased in a step-wise manner over time. For example, in some embodiments, cells may be grown under conditions where the serum concentration is between about 2% to about 11% (e.g., about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 11%) and is subsequently reduced in one or more steps to a serum concentration between about 0% to about 5% (e.g., about 0%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, or 5%).

Cell differentiation

[0092] In certain embodiments, pathfinder cells may differentiate into one or more cell types in the cell culture and the differentiated cell types are used in therapeutic applications in accordance with the invention. In some embodiments, pathfinder cells are differentiated fully to a defined cell type, including, but not limited to, pancreatic cells, neuronal cells, cardiovascular cells, epithelial cells, hepatocytes, muscle cells, retinal cells, hair follicle cells, and kidney cells.

[0093] In some embodiments, pathfinder cells are differentiated into an "intermediate" cell type that is less pluripotent than a pathfinder cell, yet has greater potency than at least most fully differentiated cells. For example, a pathfinder cell may be differentiated into one of several progenitor cells of the immune system (e.g., lymphoid progenitor cells or myeloid progenitor cells) which are less pluripotent than hematopoietic stem cells yet not fully differentiated.

[0094] In some embodiments, the cell type into which pathfinder cells are differentiated are tailored for the specific therapeutic indication for which the pathfinder cells are being used. For example, in some embodiments in which pathfinder cells are being used to stimulate repair in an individual with damage to the liver, pathfinder cells are differentiated into hepatocytes before administering to the individual. [0095] Methods of differentiating pathfinder cells are known in the art and are described, for example, in WO 2009/136168. Typically, pathfinder cells are cultured in a growth medium ("differentiation media") distinct from the growth medium in which pathfinder cells were maintained before differentiation ("maintenance media"). Differentiation media may vary depending on intended cell type, and may, for example, have a composition known to be suitable for growth of a particular cell type. In some embodiments, differentiation media contains one or more elements (e.g., growth factors, serum, additives, etc.) that are not present in maintenance media. In some embodiments, differentiation media lack one or more elements (e.g., growth factors, serum, additives, etc.) that are present in maintenance media.

[0096] Alternatively or additionally, other culture conditions are altered as compared to culture conditions used to isolate and/or maintain pathfinder cells. For example, pathfinder cells may be grown at a lower cell density than they are typically grown during maintenance. Without wishing to be bound by any particular theory, growth at lower cell densities affect the growth properties of the cells in a culture, because their ability to signal to one another is altered when the cell density in the plate is lower. As another non-limiting example, pathfinder cells may be grown on a different kind of surface (e.g., substrate) as was used to grown them for maintenance purposes. Such surfaces may help alter properties of the pathfinder cells and thereby help induce differentiation.

Biomarkers

[0097] In some embodiments, cells suitable for the present invention bear one or more characteristic biomarkers (e.g., protein or nucleic acid). Pathfinder cells may express one or more markers including, but not limited to, CD24, c-myc, HLA class 1 ABC, ICAM3, Nestin, Nanog, Oct4, Integrin oc2 + βΐ, Ngn3, and CD 130. In some embodiments, pathfinder cells may express two, three, four, five, six, seven, eight, or all of the markers described herein. In some embodiments, pathfinder cells express one or more markers typically associated with stem cells, such as, for example, Nestin. In some embodiments, pathfinder cells suitable for the present invention express Oct 4, Nanog, and c-myc. [0098] In some embodiments, pathfinder cells suitable for the present invention are characterized with the absence of certain biomarkers. In some embodiments, pathfinder cells suitable for the present invention do not express at least one of the following markers: CD34, CD105, VCAMl, CXCR2, CD44, CD73 and ICAMl. In some embodiments, pathfinder cells do not express two, three, four, five or six of the following markers: CD34, CD 105, VCAMl, CXCR2, CD44, CD73 and ICAMl. In some embodiments, pathfinder cells suitable for the invention do not express any of the above biomarkers.

[0099] In some embodiments, pathfinder cells have the biomarker profile: CD24⁺, c- myc⁺, HLA class 1 ABC⁺, ICAM3⁺, Nesting Nanog⁺, Oct4⁺, Integrin oc2 + βΓ, Ngn3⁺, CD130⁺, CD34 , CD 105^", VCAMl , CXCR2 , CD44 , CD73 and ICAMl .

[0100] In some embodiments, biomarkers may be used to isolate, enrich, and/or monitor the growth of cells suitable for the present invention. For example, biomarkers may be used to prepare, isolate, or obtain a population of substantially homogeneous cells for therapeutic uses. As used herein, a population of substantially homogeneous cells refers to a population with at least 30% (e.g., at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) of the cells expressing one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more) identical biomarkers that are typically expressed on pathfinder cells. In some embodiments, suitable biomarkers include, but are not limited to, CD24, c-myc, HLA class 1 ABC, ICAM3, Nestin, Nanog, Oct4, Integrin a2 + bl, Ngn3, and CD 130. Additionally or alternatively, a population of substantially homogeneous cells refers to a population with at least 30% (e.g., at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) of the cells that do not express one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more) biomarkers that are typically not expressed on pathfinder cells. Such biomarkers include, but are not limited to, CD34, CD105, VCAMl, CXCR2, CD44, CD73 and ICAMl.

[0101] Additionally or alternatively, nucleic acids such as the microRNAs described below may be used as biomarkers to isolate, enrich or monitor the growth of pathfinder cells and to obtain a population of substantially homogeneous cells for therapeutic uses. II. MicroRNAs

[0102] In some embodiments, cells useful for the present invention, such as pathfinder cells, comprise one or more specific microRNAs. As used herein, "pathfinder cell microRNAs" include those microRNAs that are present in pathfinder cells derived from one or more tissues. "Pathfinder cell-specific microRNAs" are a subset of pathfinder cell microRNAs and include those microRNAs that are present in pathfinder cells but not other comparable cell types derived from the same tissue, such as differentiated cells of the tissue.

[0103] The terms "pathfinder cell microRNAs" and "pathfinder cells-specific

microRNAs" encompass microRNAs isolated or purified from pathfinder cells as well as microRNAs having the same sequence synthesized using recombinant or chemical techniques. For example, microRNA molecules may be generated by in vitro transcription of DNA sequences encoding the relevant molecule. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7, T3, or SP6. As used herein, the term "microRNAs (miRNAs)" refers to post-transcriptional regulators that typically bind to complementary sequences in the three prime untranslated regions (3 ' UTRs) of target messenger RNA transcripts (mRNAs), usually resulting in gene silencing. Typically, miRNAs are short ribonucleic acid (RNA) molecules. For example, microRNAs may be approximately 18-25 nucleotides long (e.g., approximately 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides long).

[0104] It is contemplated that pathfinder cell microRNAs, individually or in combination, may be used to induce or stimulate repair of tissues, cell growth, tissue regeneration, remodeling, reconstruction, differentiation and/or transdifferentiation, among other functions. It is further contemplated that pathfinder cell microRNAs can stimulate repair of tissues that are damaged from trauma or other acute diseases, disorders, or conditions.

III. Extracellular Secretomes

[0105] As used herein, extracellular secretomes of pathfinder cells refer to any isolates derived or secreted from pathfinder cells according to the invention, including, but not are limited to, exosomes isolated from Pathfinder cells, microvesicles, their contents, and/or or other factors secreted or produced by pathfinder cells. Various methods of isolating or enriching microvesicles known in the art may be used to practice the present invention. As used herein, the terms "isolation" or "isolating" in conjunction with microvesicles are interchangeably used with the terms "enrichment" or "enriching," and refer to one or more process steps that result in an increase of the fraction of microvesicles in a sample as compared to the fraction of microvesicles in the obtained biological sample. Thus, microvesicles may be purified to homogeneity, purified to at least 90% (with respect to non-microvesicle particulate matter), at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, or at least 20% (or even less). For example, physical properties of microvesicles- may be employed to separate them from a medium or other source material. For example, microvesicles may be separated on the basis of electrical charge (e.g., electrophoretic separation), size (e.g., filtration, molecular sieving, etc.), density (e.g., regular or gradient centrifugation), Svedberg constant (e.g., sedimentation with or without external force, etc.).

[0106] In some embodiments, microvesicles are isolated or purified by centrifugation

(e.g., ultracentrifugation). It will be appreciated that various centrifugation conditions (e.g., speed, centrifugal force, centrifugation time, etc.) may be used in order to obtain a desired fraction of isolated or purified microvesicles. For example, in some embodiments, a sample may be centrifuged at a fairly low centrifugal force (e.g., approximately 16,000 x g) sufficient to pellet larger microvesicles (e.g., approximately 1000 nm or more). In some embodiments, a sample (e.g., the resulting supernatant from the initial low speed spin) may be centrifuged at a higher centrifugal force (e.g., approximately 120,000 x g) sufficient to pellet microvesicles of a smaller size (e.g., less than 1000 nm). In some embodiments, a microvesicle preparation prepared using this method may contain substantially small particles, for example, particles with a size ranging from about lOnm to 1000 nm (e.g., about 50-1000 nm, 75-1000 nm, 100-1000 nm, 10-750 nm, 50-750 nm, 100-750nm, 100-500 nm). In some embodiments, such small particles are also referred to as exosomes, exosome-like vesicles, and/or membrane particles. In some embodiments, such fraction is referred to as exosome fraction.

[0107] In some embodiments, microvesicles are isolated or purified by precipitation. It will be appreciated that various precipitation conditions may be used in order to obtain a desired fraction of isolated or purified microvesicles. For example, various kits are available for exosome precipitation, such as ExoQuick and Exo-Quick-TC (available from System Biosciences, Mountain View, California) and may be used in accordance with the present invention.

[0108] Alternatively, or additionally, isolation may be based on one or more biological properties, and may employ surface markers (e.g., for precipitation, reversible binding to solid phase, FACS separation, specific ligand binding, non-specific ligand binding such as annexin V, etc.). In yet further contemplated methods, the microvesicles may also be fused using chemical and/or physical methods, including PEG-induced fusion and/or ultrasonic fusion.

[0109] In some embodiments, microvesicles are obtained from conditioned media from cultures of microvesicle-producing cells.

Synthetic Microvesicles

[0110] In some embodiments, microvesicles suitable for the present invention may be synthetically produced. Synthetic microvesicles typically include one or more membrane components obtained from a donor cell. In some embodiments, synthetic microvesicles include at least one microRNA described herein. For example, synthetic microvesicles may be prepared by disintegration of a donor cell (e.g., via detergent, sonication, shear forces, etc.) and use of the crude preparation or an at least partially enriched membrane fraction to reconstitute one or more microvesicles. In some embodiments, exogenous microRNAs may be added to microvesicles.

[0111] Further detailed description of microvesicles and uses thereof are provide in

PCT/IBl 1/02028 entitled "Therapeutic Uses of Microvesicles and Related MicroRNAs" filed on August 12, 2011, the entirety of which is incorporated herein by reference.

IV. Therapeutic Applications

[0112] In some embodiments, the present invention provides methods of using pathfinder cells, cells differentiated from pathfinder cells, extracellular secretomes (e.g., microvesicles) and/or microRNAs associated therewith for treatment of peripheral vascular disease (PVD) and related diseases, disorders and conditions. While not wishing to be bound by a particular theory or hypothesis, it is contemplated that pathfinder cells may induce changes within target tissue or cells to convert them into active repair mode by providing microRNAs and/or other components (e.g., membrane associated polypeptide, transcription factors, etc.) that will regulate expression of genes relating to, e.g., increased cell mobility, tissue remodeling and reprogramming, growth, angiogenesis, cell adhesion and cell signaling, etc. It is further contemplated that pathfinder cells will typically not be part of the new tissue or cells. Thus, according to the present invention, pathfinder cells, extracellular secretomes, or microRNAs from different tissues, cell types or organisms may be used.

[0113] Thus, suitable pathfinder cells, extracellular secretomes or microRNAs can be derived from autologous cells (i. e. , cells from the same individual as the patient) or non- autologous cells (i.e., cells from a different individual as the patient) or both. In some embodiments, pathfinder cells are derived from tissue that is the same as the diseased tissue. For example, in methods of treating critical limb ischemia (CLI) disease, pathfinder cells may be taken from healthy limb cells from the same or different individual being treated. In some embodiments, pathfinder cells are derived from tissue that is different than the diseased tissue.

[0114] In some embodiments, methods of treatment comprise one or more steps that are performed in vitro or ex vivo to induce cells ("recipient cells") to differentiate or

transdifferentiate into a desirable cell type. Such recipient cells can then be transferred into a patient.

[0115] Without wishing to be bound by any particular theory, the inventors have proposed that pathfinder cells may act on other cells at least partially through microvesicles. (See PCT/IB 11/02028, entitled "Therapeutic Uses of Microvesicles and Related MicroRNAs", filed on August 12, 2011, the entire contents which is incorporated herein by reference).

According to this theory, microvesicles produced by pathfinder cells contain factors (e.g., microRNAs) that confer desirable properties on recipient cells (i. e. , cells that receive

microvesicles, their contents, and/or or other factors secreted or produced by pathfinder cells).

[0116] In some embodiments, provided methods comprise co-culturing pathfinder cells and recipient cells ex vivo and then transferring recipient cells into an patient. In some embodiments, recipient cells are transferred back into the same individual from whom recipient cells were obtained. For example, pathfinder cells can be co-cultured with bone marrow cells obtained from a patient for a period of time ex vivo to allow transfer of stimulatory factors (e.g., microvesicles, their contents, and/or other factors secreted or produced by pathfinder cells), then bone marrow cells may be transferred back into the individual.

[0117] In some embodiments, recipient cells are tested for expression of specific biomarkers such as certain proteins and/or microRNAs after co-culturing with pathfinder cells before transfer into a patient.

[0118] In certain embodiments, methods of treatment comprise a step of administering to a patient in need of treatment a therapeutically effective amount of one or more microRNAs as described herein. miRNAs may be used in the absence or presence of cells.

[0119] In certain embodiments, methods and compositions of the present invention are used to stimulate repair of tissues and/or cells that have are damaged by ischemia caused from a peripheral vascular disease, disorder or condition. In some embodiments, methods and compositions of the present invention are used to stimulate repair of damaged tissue in an acute condition resulting from ischemia. By way of non-limiting example, methods and compositions of the present invention may be used to peripheral vascular diseases, such as peripheral artery disease (PAD) and critical limb ischemia (CLI).

Peripheral Vascular Disease

[0120] Among other things, methods and compositions of the present invention are used to treat or ameliorate peripheral vascular disease. As used herein, the term "peripheral vascular disease" or "PVD" refers to a disease of the blood vessels located outside the heart and the brain.

[0121] In some embodiments, treatment refers to partially or completely alleviation, amelioration, relief, inhibition, delaying onset, reducing severity and/or incidence of peripheral artery disease in a subject. As used herein, the term, "peripheral artery disease" or "PAD" refers to a form of PVD in which there are partial or total blockage of arteries that provide blood supply to internal organs and/or limbs. In some embodiments, treatment refers to partially or completely alleviation, amelioration, relief, inhibition, delaying onset, reducing severity and/or incidence of critical limb ischemia in a subject. As used herein, the term "critical limb ischemia" or "CLI" generally refers to a condition characterized by restriction in blood or oxygen supply to the extremities (e.g., hands, arms, feet, legs) of an individual that may result in damage or dysfunction of a tissue in the extremities. Critical limb ischemia may be caused by any of a variety of factors, such as peripheral artery disease (PAD), and may cause severe pain, skin ulcers, or sores, and in some cases leads to amputation. Critical limb ischemia may be characterized by vasoconstriction, thrombosis, or embolism in one or more extremities. Any tissue in an extremity that normally receives a blood supply can experience critical limb ischemia.

[0122] Thus, in some embodiments, treatment refers to improved blood flow in a subject suffering from a peripheral vascular disease, disorder or condition. It will be appreciated that blood flow can be measured using any available methods and/or instrumentation. For example, in some embodiments, blood flow is measured using a laser Doppler. It will be appreciated that blood flow can be measured at any appropriate time before and/or after treatment. For example, in some embodiments, blood flow is measured at one or more of day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 21, day 28, day 35, day 42, or day 49 of treatment. In some embodiments, blood flow measurements are expressed as a ratio of blood flow in the diseased and/or damaged tissue compared to that in a normal tissue. In some embodiments, blood flow in a diseased and/or damaged tissue is more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, or more than 65% as compared to a normal tissue in the same individual. In some embodiments, blood flow in the diseased and/or damaged tissue is increased by, on average, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more per week.

[0123] In some embodiments, treatment refers to reduced or prevented necrosis (e.g., increased ischemic score) in diseased and/or damaged tissue. For example, in some

embodiments, necrosis is determined by macroscopic evaluation of ischemic severity in a diseased and/or damaged tissue. It will be appreciated that necrosis can be determined by any appropriate method. For example, in some embodiments, morphological grades for necrotic areas are assigned, such as those disclosed in Goto et al. (Tokai J Exp Clin Med, 31(3): 128, 2006). Exemplary morphological grades for necrotic area in mice are shown in Table 1 below.

Table 1:

[0124] In some embodiments, morphological grades for necrotic areas are decreased by about 0.1, 0.2, 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, 1.6, 1.7, 1.8, 1.9, 2.0 or more grades. In some embodiments, morphological grades for necrotic areas are decreased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more.

[0125] In some embodiments, treatment refers to improved limb function. It will be appreciated that limb function can be measured using any appropriate methods and/or instrumentation. For example, in some embodiments, limb function is determined by a semiquantitative assessment of impaired use of an ischemic limb (see, e.g., Stabile, et al. Circulation 108(2):205, 2003). Exemplary assessment of limb function in mice are provided in Table 2 below. It will be appreciated that assessment of limb function in humans correlates with that of mice. Table 2:

[0126] In some embodiments, grades for limb function necrotic areas are decreased by about 0.1, 0.2, 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, 1.6, 1.7, 1.8, 1.9, 2.0 or more grades. In some embodiments, grades for limb function are decreased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more.

V. Pharmaceutical compositions

[0127] In certain embodiments, the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of cells and/or microRNAs for the treatment of various diseases, disorders or conditions described herein. Provided compositions typically comprise a therapeutically effective amount of cells (e.g., pathfinder cells, cells differentiated from pathfinder cells, and/or microRNAs associated with pathfinder cells) and a pharmaceutically acceptable carrier. In some embodiments, provided are pharmaceutical compositions comprising a therapeutically effective amount of cells and/or microRNAs for the treatment of peripheral vascular disease (e.g, peripheral artery disease and/or critical limb ischemia).

Preparation of compositions comprising cells

[0128] Various methods of preparing cells from biological materials and/or from cell cultures are known in the art and may be used to practice the present invention. In some embodiments, cells are enriched from their source material or culture. As used herein, the terms "isolation" or "isolating" in conjunction with cells are interchangeably used with the terms "enrichment" or "enriching," and refer to one or more process steps that result in an increase of the fraction of cells in a sample or solution as compared to the fraction of cells in the material or culture from which it was derived.

[0129] Pathfinder cells (and/or cells that are differentiated from pathfinder cells) can be enriched or purified to homogeneity, to at least 90% (with respect to non-pathfinder cells), at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, or at least 20% (or even less). For example, physical properties of pathfinder cells may be employed to separate them from a medium or other source material. For example, cells may be separated on the basis of size (e.g., filtration, molecular sieving, etc.), density (e.g., regular or gradient centrifugation), or Svedberg constant (e.g., sedimentation with or without external force, etc.). In some embodiments, cells are isolated or purified by centrifugation.

[0130] In some embodiments, the percentage of desired cells (e.g., pathfinder cells and/or cells differentiated from pathfinder cells) in a population of cells is assessed by methods based on one or more distinctive biological properties or set of biological properties of those cells. Especially suitable assessment methods may employ surface markers (e.g., for precipitation, reversible binding to solid phase, (fluorescence-activated cell sorting) FACS analyses or sorting, specific ligand binding, non-specific ligand binding such as annexin V, etc.). In some embodiments, cells having a particular set of characteristics (e.g., cell surface biomarker profile) are selected for using cell separation methods known in the art (e.g., marker-based cell separation methods). Thus, in some embodiments, a composition of cells comprising mostly or only cells of a desired type (e.g., pathfinder cells and/or cells differentiated from pathfinder cells) is obtained.

[0131] In some embodiments, cells are reconstituted in a suitable solution (such as pharmaceutically acceptable diluent or carrier) such that the cells are present in the solution at a known concentration. In some embodiments, the known concentration is chosen for convenience for dosing. In some embodiments, the known concentration is a concentration at which cells are known to be stable (e.g., retain viability and/or their therapeutic properties) for at least a period of time under clinically approved storage conditions. Provided compositions may also contain minor amounts of wetting agents, emulsifying agents, and/or pH buffering agents. Provided compositions can take any of a variety of solid, liquid, or gel forms, including solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Non-limiting examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Compositions will generally contain a

therapeutically effective amount of cells and/or microRNAs, optionally in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.

[0132] Formulations are typically adapted to suit the mode of administration. For example, compositions for intravenous administration may be formulated as solutions in sterile isotonic aqueous buffer. Such compositions may also include a solubihzing agent and/or a local anesthetic such as lidocaine (also known as lignocaine, xylocaine, or xylocard) to ease pain at the site of injection.

[0133] As further example, compositions for topical and/or local use may be formulated, for example, as a lotion or cream comprising a liquid or semi-solid oil-in-water or water-in-oil emulsion and ointments. Such compositions may also comprise a preservative.

[0134] Compositions for delivery to the eye include may be formulated, for example, as eye drops that comprise the active ingredient in aqueous or oily solution and eye ointments that may be manufactured in sterile form. Compositions for delivery to the nose may be formulated, for example, as aerosols or sprays, coarse powders to be rapidly inhaled, or nose drops that comprise the active ingredient (e.g., cells and/or microRNAs) in aqueous or oily solution.

Compositions for local delivery to the buccal cavity may be formulated, for example, as lozenges that comprise the active ingredient in a mass generally formed of sugar and gum arabic or tragacanth, and pastilles that comprise the active ingredient in an inert mass (for example of gelatine and glycerine or sugar and gum arabic). Flavoring ingredients may be added to lozenges or pastilles.

[0135] Aerosol and spray formulations may comprise, for example, a suitable pharmaceutically acceptable solvent (such as ethanol and water) or a mixture of such solvents. In some embodiments, such formulations comprise other pharmaceutical adjuncts (such as non- ionic or anionic surface-active agents, emulsifiers, and stabilizers) and/or active ingredients of other kinds. Aerosol and spray formulations may be mixed with a propellant gas, such as an inert gas under elevated pressure or with a volatile liquid (e.g., a liquid that boils under normal atmospheric pressure below customary room temperature, for example from -30 to +10 °C).

Routes of administration and dosage regimens

[0136] In methods of treatment or of inducing tissue repair, remodeling or differentiation in vivo of the present invention, cells, miRNAs, or a pharmaceutical composition thereof, will generally be administered in such amounts and for such a time as is necessary or sufficient to achieve at least one desired result. For example, cells or miRNAs can be administered in such amounts and for such a time that it ameliorates one or more symptoms of a disease, disorder, or condition; prolongs the survival time of patients; or otherwise yields clinical benefits.

[0137] A dosing regimen according to the present invention may consist of a single dose or a plurality of doses over a period of time. Administration may be, e.g. , one or multiple times daily, weekly (or at some other multiple day interval), biweekly, monthly, or on an intermittent schedule. Typically an effective amount is administered. The effective amount of cells, microRNAs, or a pharmaceutical composition thereof, will vary from subject to subject and will depend on several factors (see below).

[0138] Cells, microRNAs, or pharmaceutical compositions thereof, may be administered using any administration route effective for achieving the desired therapeutic effect. Both systemic and local routes of administration may be used in accordance with methods of the invention. Suitable routes of administration include, but are not limited to, intravenous, intraarterial, intramuscular, subcutaneous, cutaneous {e.g., topical), intradermal, intracranial, intrathecal, intrapleural, intra-orbital, intranasal, oral, intra-alimentary {e.g., via suppository), colorectal {e.g., via suppository), and intra-cerebrospinal.

[0139] Depending on the route of administration, effective doses may be calculated according to, e.g., the body weight and/or body surface area of the patient, the extent of damaged or diseased tissue, etc.. Optimization of the appropriate dosages can readily be made by one skilled in the art, e.g., by a clinician. The final dosage regimen is typically determined by the attending physician, considering various factors that might modify the action of the cells, miRNAs, or pharmaceutical compositions thereof (collectively referred herein as "drug"), e.g., the drug's specific activity, the severity of tissue damage and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any present infection, time of administration, the use (or not) of other therapies, and other clinical factors.

[0140] In some embodiments, dosing is given by number of cells per body weight of the individual to which cells are administered. In some embodiments, approximately lx 10⁶ to 3 x 10⁸ cells per kg per dose are administered. In some embodiments, approximately 1 x 10⁶ to approximately 3 x 10 8 , approximately 1 x 107 to approximately 3 x 108 , or approximately 3 x

10 7 to approximately 3 x 108 cells are administered per kg per dose. In some embodiments, dosing is not calculated based on body weight and/or dosing is not calculated based on number of cells administered. For example, in some embodiments, a standard unit may be developed based on effectiveness in a particular measure for a particular condition. Alternatively or additionally, in some embodiments, recommended dosages are developed for adult and/or child individuals without regard to body weight.

EXEMPLIFICATION

Example 1 - Stimulation of repair and/or vascularization by PCs in a hind limb ischemic model

[0141] The present Example demonstrates that pathfinder cells (PCs) can stimulate repair in a model of hind limb ischemia.

Hind Limb Ischemia Model

[0142] A stable hind limb ischemia model has been described previously and is generally characterized by uniform ischemic damage useful for examining the effect of various therapies (Goto, et al. Tokai J Exp Clin Med, 31(3): 128 2006; Kang Y, et al. PLoS One. 2009;4(l):e4275)). The hind limb ischemia model in mice used in this example involves two ligations of the proximal end of the femoral artery and its dissection between the two ligatures. The surgery causes obstruction of the blood flow and subsequently leads to severe ischemic damage (Goto, et al.). In this experiment, healthy adult female Balb/c mice were used. Hind limb ischemia was induced in mice using protocols previously described. Balb/c female mice were maintained on a standard diet with water available ad libitum. Mice were anesthetized and an incision was made on the inguinal area. The femoral artery was closed with 4-0 silk thread and the mouse was allowed to recover.

Transplantation of rat PDPCs

[0143] PCs were isolated and cultured from male rats as previously described, for example, in International Patent Publications WO 2006/120476 and WO 2009/136168, the entire contents of each of which are incorporated by reference. Individual vials of cryopreserved PCs were processed one group at a time for immediate injection into recipient animals. Each vial contained sufficient cells to inject 1.5 x 10⁵ - 1.5 x 10⁶ cells into each recipient animal. Each vial was thawed quickly in a 35 - 37° C water bath. All subsequent steps used prewarmed PBS, and cells were kept at 30 - 37°C until transfer into the recipient animals. Thawed cells were brought to 15 ml with prewarmed PBS, and were added dropwise with mixing to the thawed cells. Cells were then centrifuged for 10 minutes at 1100 rpm, and the supernatant was discarded carefully to remove DMSO. The cells were then resuspended with gentle mixing (for 4-5 seconds) in a fresh 1 to 5 mis of prewarmed PBS depending on the cell number. Cell concentrations and cell viability (exclusion of 0.2% trypan blue) were determined. Cells were centrifuged again for 5 to 10 minutes at 1100 rpm (depending on the volume), then brought to the appropriate volume for injection with prewarmed PBS. Cells were transported immediately in a container with warmed PBS to keep the cells at 30 - 37° C. Cells were gently mixed again immediately before injection: using mixing by hand (3-5 second mix) or by drawing up into the syringe using the plunger on the syringe (i.e. without a needle) prior to intravenous injection via the lateral tail vein. This was done until no cell clumps were visible. If cell clumps were difficult to remove in this way, a brief 5 second vortex step was used, and/or gentle passage through an 18 gauge needle. [0144] PCs were injected intravenously into the tail vein of mice on day 1, 24 hours after ischemia. Positive control mice were injected intramuscularly at two sites, the proximal and distal side of the surgical would, with VEGF. Negative control mice were injected with PBS intravenously into the tail vein. Table 3 provides animal group allocations.

Table 3.

Assessment of ischemia

Blood flow

[0145] Blood flow in legs from both sides of the animals were measured with a non- contact laser Doppler before surgery and on days: 1, 7 15, 21, and 28 post operation. Blood flow measurements were expressed as the ratio of the flow in the ischemic limb to that in the normal limb. Exemplary blood flow results are shown in Figure 1.

Macroscopic evaluation

Macroscopic evaluation of the ischemic limb was done once a week post operation by using morphological grades for necrotic area (Goto, et al. Tokai J Exp Clin Med, 31(3): 128 2006) as shown in Table 4. Table 4. Morphological grades for necrotic area

[0146] Exemplary ischemic scores are shown in Figure 2.

In vivo assessment of limb function

[0147] Semi-quantitative assessment of impaired use of the ischemic limb was performed once a week post-surgery using the scale shown in Table 5 (Stabile et al, Circulation 108(2):205 2003).

Table 5. Assessment of limb function

[0148] Limb function is graded as "Not applicable" in case of partial or full limb amputation. In such case blood flow measurements will not be included in the statistical analysis. Exemplary limb function analyses are scores are shown in Figure 3.

Tissue fixation and Gross Pathology

[0149] After angiography gross pathology was performed. The quadriceps muscles of the ischemic and control legs were removed, weighed, fixed in 4% buffered formalin for analysis. [0150] Taken together, these results demonstrate that PCs can stimulate repair in a tissue that is not the same as the tissue of origin of PCs. Furthermore, Pathfinder cells can alleviate damage to limbs that have undergone ischemic stress. These findings support a potential for PCs in treating vascular conditions that affect limbs such as critical limb ischemia and other peripheral vascular diseases.

EQUIVALENTS AND SCOPE

[0151] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments, described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[0152] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[0153] In the claims articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0154] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. It is also noted that the term "comprising" is intended to be open and permits the inclusion of additional elements or steps.

[0155] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and

understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0156] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention {e.g., any cell type; any neuronal cell system; any reporter of synaptic vesicle cycling; any electrical stimulation system; any imaging system; any synaptic vesicle cycling assay; any synaptic vesicle cycle modulator; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art. INCORPORATION OF REFERENCES

[0157] All publications and patent documents cited in this application are incorporated by reference in their entirety to the same extent as though the contents of each individual publication or patent document were incorporated herein.

Claims

What is claimed is:

1. A method for treating peripheral vascular disease comprising a step of: administering a population of cells, or extracellular secretomes thereof, to an individual suffering from a peripheral vascular disease characterized by partial or complete blockage of blood flow to one or more tissues outside the heart and brain, wherein the cells are originated from an adult tissue and further wherein the cells, or the extracellular secretomes thereof, are administered in a therapeutically effective amount such that at least one symptom or feature of the peripheral vascular disease is reduced in intensity, severity, or frequency, or has delayed onset.

2. The method of claim 1 , wherein the one or more tissues outside the heart and brain

comprise one or more limbs of the individual.

3. The method of claim 1, wherein the peripheral vascular disease is a peripheral artery disease.

4. The method of claim 3, wherein the peripheral artery disease is critical limb ischemia.

5. The method of any one of the preceding claims, wherein the peripheral vascular disease is an acute ischemia.

6. The method of any one of claims 1 -4, wherein the peripheral vascular disease is a chronic ischemia.

7. The method of any one of the preceding claims, wherein the cells induce and/or increase angiogenesis and/or vascularization in the one or more tissues outside the heart and brain.

8. The method of any one of the preceding claims, wherein the cells decrease and/or delay cell death in the one or more tissues outside the heart and brain.

9. The method of claim 8, wherein the cell death is apoptosis.

10. The method of claim 8, wherein the cell death is necrosis.

11. The method of any one of the preceding claims, wherein the cells increase and/or enhance cell survival in the one or more tissues outside the heart and brain.

12. The method of any one of the preceding claims, wherein the therapeutically effective amount of the cells, or the extracellular secretomes thereof, is sufficient to decrease partial or total blockage of blood flow to the one or more tissues outside the heart and brain.

13. The method of any one of the preceding claims, wherein the therapeutically effective amount of the cells, or the extracellular secretomes thereof, is sufficient to decrease or delay tissue damage in the one or more tissues outside the heart and brain.

14. The method of any one of the preceding claims, wherein the therapeutically effective amount of the cells, or the extracellular secretomes thereof, is sufficient to improve function of the one or more tissues outside the heart and brain.

15. The method of any one of the preceding claims, wherein the therapeutically effective amount ranges from approximately 1 x 10⁶ to 3 x 10⁸ cells per kg body weight per dose.

16. The method of any one of the preceding claims, wherein the cells are administered

intravenously, intra-arterially, intramuscularly, subcutaneously, cutaenously,

intradermally, intrapleurally, intra-orbitally, intranasally, orally, intra alimentrally, and/or colorectally.

17. The method of any one of the preceding claims, wherein the cells are administered daily.

18. The method of any one of claims 1-16, wherein the cells are administered weekly.

19. The method of any one of claims 1-16, wherein the cells are administered biweekly.

20. The method of any one of claims 1-16, wherein the cells are administered monthly.

21. The method of any one of the preceding claims, wherein the cells are originated from an adult tissue that is distinct from the diseased tissue.

22. The method of any one of the preceding claims, wherein the cells are originated from an adult tissue that is from a different species from the individual.

23. The method of any one of the preceding claims, wherein the adult tissue is a human adult tissue.

24. The method of any one of claims 1 -22, wherein the adult tissue is a non-human adult tissue.

25. The method of any one of the preceding claims, wherein the adult tissue is selected from the group consisting of pancreas, kidney, breast, lymph node, liver, spleen, myometrium, placenta, tonsils, peripheral blood, cord blood, bone marrow, and combination thereof.

26. The method of any one of the preceding claims, wherein the cells are first cultivated in a cell culture medium under conditions and time sufficient for cell proliferation.

27. The method of claim 26, wherein the cell culture medium is a Matrigel free culture

medium comprising serum.

28. The method of claim 15 or 27, wherein the cells are firs treated to reduce a telomeric attrition rate before the cultivating step.

29. The method of any one of the preceding claims, wherein the population of cells are

substantially homogenous.

30. The method of any one of the preceding claims, wherein at least 50%, at least 80%, or at least 90% of the population of cells express one or more markers selected from the group consisting of CD24, c-myc, HLA class 1 ABC, ICAM3, Nestin, Nanog, Oct4, Integrin a2 + bl, Ngn3, and CD130.

31. The method of any one of the preceding claims, wherein the cells do not express at least one marker selected from the group consisting of CD34, CD105, VCAM1, CXCR2, CD44, CD73 and IC AMI.

32. The method of any one of the preceding claims, further comprising administering a pro- angiogenic agent in combination with the cells.

33. The method of claim 32, wherein the pro-angiogenic agent is or comprises VEGF. The method of any one of the preceding claims, further comprising performing a vascular or endovascular procedure on the one or more limbs.