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1. (WO2014140930) COMPOSITIONS AND METHODS FOR ENHANCING THE THERAPEUTIC POTENTIAL OF STEM CELLS
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COMPOSITIONS AND METHODS FOR ENHANCING THE THERAPEUTIC POTENTIAL OF STEM CELLS

FIELD OF THE INVENTION

The invention relates to stem cell compositions and methods.

BACKGROUND OF THE INVENTION

Stern cell therapy is a promising approach in bone marrow, skin, heart, and corneal transplantation, graft versus host disease, hepatic and renal failure, lursg injury, rheumatoid arthritis, treatment of autoimmune diseases such as Crohn's disease, ulcerative colitis, multiple sclerosis, lupus and diabetes; prevention of allograft rejection, neurological disorders and cardiovascular medicine.

Mesenchymal stem cells (MSCs) and/or progenitor or precursor cells can be isolated from a variety of tissues, such as bone marrow, skeletal muscle, dental pulp, bone, umbilical cord and adipose tissue. MSCs are gaining increasing interest as a potential source for therapeutic approaches in regenerative medicine. Stern cells can self-renew and have the capacity to differentiate into specific cell types or can be therapeutically used for the production and secretion of soluble factors that will promote tissue regeneration, In particular, adult stern/progenitor cells from abundant autologous origin have the additional advantages over embryonic stem ("ES") cell types given their great availability, low tutuorgenicity and allowing for the avoidance of controversial ethical issues associated with ES cells.

To facilitate an ability to efficiently repair or regenerate defective or diseased organs aud tissues, donor stern/progenitor cells should possess desirable therapeutic properties, for example, minimal side effects, ability to integrate into host tissue, differentiation into desired cell lineages, paracrine effects, regulation of tissue remodeling and activation of endogenous repair/regeneration mechanisms.

MSCs have been, employed in numerous preelimcal studies m rodents, rabbits and baboon monkeys among others, for bone marrow, s.idn, heart, and corneal transplantation, graft versus host disease, hepatic and renal failure, lung injury, multiple sclerosis, rheumatoid arthritis, diabetes and lupus diseases. Preliminary results from some of these studies have led to human clinical trials that are currently being carried out. These trials involve treatment of autoimmune diseases such as Crohn's disease, ulcerative colitis, multiple sclerosis and type I diabetes melnrus; prevention, of allograft rejection; and enhancement of die survival of bone niarrow and kidney grafts; and treatment of resistant graft ve rsus best disease,

A major compoBent of morbidity and mortality attributable to cardiovascular disease occurs as a eoaseque:nce of the partial or complete blockage of vessels carrying blood in the coronary and/or persphersd vasculature. When such vessels are partially occluded, lack of blood flow causes ischemia to the muscle tissues supplied by such vessel, consequently inhibiting nruscie contraction, and/or per ftmciioa. Total occlusion of blood flow causes necrosis of the nruscle tissue.

Peripheral artery disease (PAD) affects 8 million Americans, It is a result of narrowed arteries is the legs. The most common symptoms of PAD are cramping, pain or tiredness when walking. However, some FAD patients can. lose the ability to walk and may suffer from amputations.

In some individuals, blood vessel, occlusion, is partially compensated by natural processes, in which new vessels are formed (a process ofien referred to as "ajjgiogenesis" ) and small vessels are enlarged (termed "arieriogenesis") to replace the ftmction of the impaired vessels. These new conduits may facilitate restoration of blood flow to the deprived tissue, thereby constituting '"natural bypasses" around the occluded vessels.

However, some individuals with impaired angiogenesis are oBable to generate sufficient collateral vessels to adequately compensate for the diminished blood flow caused by cardiovascular disease. There is a need for new therapeutic approaches m treat ischemic diseases.

Various studks have evaluated the therapeutic effect of MSCs in preclinical animal models and demonstrated great clinical potential. However, there remain majnr .q««stkms concerning optimal MSC dosage, route of administration, and MSC cell fate following ififusion.

As such, there is a great need for optimised therapeutic MSC compositions .ami methods of using them, to ameliorate the symptoms of various, diseases in general -audi in the treatment of vascular disease in particular.

Walker et al., Nat Comtuun. Author manuscript; available m 2010, generally disclose that treating human piuripoiem stem (hPS) cells such as human embryonic stem (hES) and induced plnripotem stem (MFS) ceils with the inhibitor of non-ranscte myosin 11 (N.MlI), blebbisiaik. enhances the survival of hPS ceils under clonal density and suspension conditions, U.S. Pub, No. 20100216181 discloses cultivation of pluripotent stem cells in a medium that is free of serum and feeder cells using blebbistatin as survival factor, increased numbers of cells are generated by eukivatbg the cells in sptuaer flasks or bioresetors. Published PCX application WO2012062819 discloses a method for controlling binding of cells to a substrate using bkbbistatin as a promoter for adhesion of cells to which the ceils usually have no or limited affinity.

However, none of these references teach or suggest the unexpectedly superior results observed when treating patients with a composition containing stem ceils and a non-muscle myson 11 antagonist.

SUMMARY OF THE INVENTION

One aspect: of the invention relates to a method of treating a vascular disease comprising administering to a patient in need thereof, an effective amount of a composition comprising a population, of ceils aad non-niusck myosin 11 antagonist, thus treating the vascular disease.

In oue embodiment, the vascular disease is s result of diabetes. In another embod.tm.eat, the vascular disease is a resuk of athemsc!erosis. In a further embodiment, she vascular disease is peripheral vascular disease (PVD), lis still another embodiment, the vascular disease is peripheral artery disease (FAD). In still a iniher emtellment. the vascular disease is associated with m ankle brachial pressure index of about or less than. 0.9. In yet another cmbodiffista, the vascular disease is associated with an aitkle brachial pressure index of about or less than 0.7. In still another entbodiment, the vascular disease has resulted in critical limb ischemia, in another embodiment the vascular disease .has resulted in skin ulcerations or gangrene. In a further embodiment, the composition results its or enhances anglogenesis, la still another embodiment, the composition results in or enhances angiogenesis by at least about 20 percent compared to control.

In yet another eotbodiment, a portion of the population of cells comprises phtripotent cells. In still a further embodiment, the nhsripotect cells are induced phuipotent stem cells. In another embodirnent, a portion of the population of cells comprises mnitipotent cells, in still, another embodiment, the rmdtlpotent cells are moltipotem stromal cells or mesetKhyrnal stem cells. In yet a further esibodimsnt, the ma!tipoteot cells are derived horn kdueed pluripotent stem cells. In soil another embodiment, the composition comprises a plmnnaceotically acceptable carrier.

in a farther embodiment, the .limb function, in the patient improves by about or at least 2-3 grades compared to the same pattern not receiving the composition. In another embodiment the limb blood flow in the patient irrrpnoves to about or at least 65-85% of norrrral or entreated limb blood flow, in yet a farther embodintent, the isebemie damage in the patient improves by about or at least 2, 3 or 4 grades compared to the same patient not receiving the composition. In yet a Ilirther embodiment, the ischemic damage in the patient improves at a faster rate (or aeeciarsied rate) compared to the same patient not tecelviug the composition.

In yet a further embodiment, the eornposiliori is admMstered intravenously. In still another esnbodtmcnt, the composition is administered intramuscularly, in another embodiment, the composition is administered intramuscularly at or in proximityl

In still another embodiment the non-muscle myosin. 11 antagonist knocks down non-masde myosin II gene and/or protein expression. in yet a further embodiment the non-museie myosin II antagonist is an siRNA. targeted to kaoek dowa non-anisek myosin II gene and/or protein expression. Irs an. alternative, embodiment, the non-nmsc-le myosin II antagonist is an. DMA vector encoding an siRNA, miRNA or anti-sense RNA targeted to knock down non- muscle myosin II gene and/or protein expression. In still another embodiment, the non-muscle myosin Il antagonist is aa Angiotensin II receptor antagonist or aagiotensin receptor blockea In anoihsr embodiment the non-muscle myosin II antagonist is an angiotenshvoonverting- enzyme (ACE) inhibitor. In a further anbodimeat, the non-muscle myosin II antagonist is 2,3-butanediona-2-monoxime (B.DM), In still a further embodiment, the non-mnscle myosin It antagonist is biebbistutin or analog, derivative or variant mereof; in still a further embodiment the noo-masek myosin II antagonist is a pyrrohdiaorie derivative. In yet a further embodiment, the non-muscle myosin II antagonist is a molecule from the class of pyrrolidmones,

Another aspect of the invention relates to a kit comprising: a population of multipotent celts; a non-muscle myosin II antagonist: a pharmaceutically aeceplable carrier; and optionally, instructions for administering these to a patient diagnosed with disease which is amenable, to treatment using stem cell thereapy. In one endxsdbnent the disease is a cardiovascular disease. In another embodiment, the disease is peripheral artery disease,

la one embodiment, the cells are MSCs. In another embodiment, the non-muscle myosin II antagonist is blebbistatin. In another embodiment the cardiovascular disease is peripheral artery disease. In a farther embodiment, the cells are MSCs, the nommuscis myosin il antagonist is bkbbsstatia and the cardiovascular disease is. peripheral artery disease. In still another embodiment, the kit farmer comprises a cardiovascular drag- in a further embodiment the kit further comprises a stent. In still another embodiment, the kit further comprises a dnsg-ehiting stent, in one embodiment, the items comprising the kit are In separate containers. To another embodiment, the population of multipotent ceils: the non-muscle myosin II antagonist; and the pharmaceutically acceptable carrier are in the same container, e,g., a syringe or automated adndnistration device. Th

artery disease. The kit may have cells that are MSCs, The .non-mnsole mynsin II atttagoaist is Ideally olebbistatin. and the disease is a peripheral artery disease. The kit may forther comprises a cardiovascular drug, and/or a stent and/or a drug-cluting stent. The stems may be in separate containers, in the same container or, in a syringe.

Another aspect of the invention relates to a method of reducing the rate of amputation in a peripheral artery disease patient treated with a composition comprising MCs, comprising contacting the MSCs with a non-muscle myosin II antagonist prior to the treatment, in one embodiment, the non-muscle myosin II antagonist is blehhistatim In another embodiment, the rate is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more.

Another aspect of the invention relates to a stem cell composition for treating a disease amenable to treatment with Stem cell therapy comprising a population of maultipotent cells; and a non-muscle myosin, II antagonist, in. one embodiment of this aspect of the invention, the cells are MSCs. in another embodiment, the non-muscte myosin II antagonist is blebbistatin.

Another aspect of the in vention relates to a method of making a stem cell composition for treating a disease amenable to treatment with stem cell therapy comprising a population of multipotent cells; an a non-muscle myosin II antagonist; wherein the population of mukipotem cells and the NM II antagonist are Incubated together for period of time prior to administration to a patient.

A further aspect of the invention is directed to a method of accelerating the healing of a disease amenable to treatment with stem cell therapy comprising a treating a patient with a-composition comprising a population of multipotent cells; and a non-muscle myosin H antagonist; wherein the composition results in healing of the disease taster than the- same composition lacking the a non-muscle myosin II antagonist.

In certain embodiments of these aspects, the cells are MSCs, in other embodiments, the NM II antagonist is blebbistatin, In yet further embodiments, the disease are a cardiovascular disease.

BRIEF DESCRIPTION OF THE FIGURES

A more complete understanding of the present invention may be obtained by reference to the accompanying drawings, when considered In conjunction with the subsequent detailed description. The embodiments illustrated in the drawings are intended only to exemplify the invention and should not he construed as limiting the invention to the illustrated embodiments.

Figures 1 A-B show Blood Flow (%) over Time; The median lis the group was computed across all valid animals. Blebbisiatin (BB) accelerates and enhances blood How regeneration by mesenchymal stem cell implantation, Stem cells administered in the presence of blebbistatin (1 A, Group 4, diamond) showed raster and enhanced regeneration in blood flow in animals compared to stem ceils that have been pre-treated with blebbistatin but were ImplatHed in die absence of blebbistatin (1 A, Group 5, solid black square). Media control containing blebbistatin (1 A, Group s, circle) showed marginally recovery effects as well as MSCs, which picked up on recovery at later time points (1 A, Group 3, triangle), fatrajtunscular application of MSCs showed consistently superior cfteets over Intravenous application. The data show that blebbistatin (BB) alone had no effect on the regeneration of blood flow but blebbistatin enhances and/or supports the regenerative potential of MSCs when applied together with stem cells.

Figure 2A-B show Limb Function. (%) over Time: The median, in the group was computed across all valid animals. The data show mat blebbistatin (BB) presence with MSCs supports fast improvement in limb function. Despite the blood perfusion improvement in other treated groups, limb functional deterioration and limb necrosis inerease was observed from day 28 after the treatment Group 3 exhibited larger occurrence of amputations and larger distribution between animals. The data show that intravenous treatment (2B) was somewhat less effective and caused more complications compared to the local intramuscular treatment regime (Figure 2A). Mice that have received MSCs implanted in the presence of blebbistatin (2A, Group 4, open square) showed constant and fast improvement of limb function consistently over time after implantation. The same tendency was detected in mice with

to a lower amount (2A, Group 5, triangle). In contrast, the application, of MSCs alone showed slight improvement until day 20 and limb function worsened, at later time points. Media controls exhibited marginal effects only. Note, that a negative slope means improvement of the limb function. Again, intramucular application of MSCs (2A) showed consistently superior effects over intravenous application (2B). Application of MSCs in the presence of blebbistatin outperforms the regenerative potential of MSCs, This application shewed the fastest improvement in regenerative potential.

Figure 3A-B show Ischemic Severity Score (%): The median in tire group was computed across all valid, animals, MSCs applied in conjunction, with blebbiststin impede severity in Ischemic Score, Analysis of the ischemia severity score over time show consistently better results for Group 4 and 5 when applied intramuscularly; although media controls ranged within the scatter. Note that MSCs (Group 3) showed a high variance in Score for both intramuscular sad intravenous applications as Group 5 for the intravenous application only. Again, intramuscular application of MSCs is shown in 3A; intravenous application is shown in 3B.

Figure 4 shows the distribution of box plot slopes for Blood Flow (%), computed as the linear trend front surgery to Day 35 lor Blood Flow (%). The single number lor each annual represents its change on an endpoint over time. These plots provide a convenient method for presenting many groups on a single graph. Exemplary, the evatnahoo of blood Bow (%) is depicted as slopes boxplots by group in order to point out the low variation (indicated by the small box sizes) within controls and five best performing group in the study BMMSC in DMEM + BB for intramuscular as well as intravenous application.

Figure 5 shows blebbistatin impedes severe complications during the healing process. A major problem in pre-clinical studies is often the high drop-out rate of animals due to treatment related failure (amputations after day 14 post surgery). or unrelated to treatment (e.g. death during anesthesia, or amputaions before day 14 post surgery). Only a Sew animals had to be excluded from statistical analysis due to the latter reason. However, those animals that showed severe side effects (amputation) were ranked and the

statistical analyses, Data dearly demonstrate that blebbistatln (Groasps 4 and 8) drastically improves regenerative potential of MSCs and stand out by strikingly low rates of severe complications,

DETAILED DESCRIPTION OF THE !INYENTION

Described herein are MSG compositions and methods that allow for Wastes' healing and etihaaced regeneration of diseased or damaged assises while strongly reducing complications during healing, fit particular, it is disclosed herein that Inventive MSCs compositions have great regenerative potential in a validated animal model In particular, it has been, found that bkhbistatin dramatically and unexpectedly accelerate and enhances the therapeutic potentitsl of MSCs and redoees severe complications in healing.

The use of the word %r or "an" when used in conjunction with die term "com prising" In the claims and/or the specification may mean "one." but it is also consistent with the meaning of "one or more," "at least one," and ""one or more than one."

Throughout, this application, the term "about" is used to indicate that a value, includes the inherent variation of error for the device, the .method being employed, to determine the value, or the variation that exists among the study subjects. Typically the term is meant to encompass approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, $%, 9%, 1.0%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 1 7%, 18%, 19% or 20% variability depending on the situation.

The use of the term "or" in the claims is used to mean "and/or unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/orf

As used in this specification asid claim(s), the words "comprising" (and any form of comprising, such as "comprise" and ''comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "comnmhtg" (and any fonn of containing, such as "contains"

or open-ended md do not exclude additional, untecited elements or method steps, k is contemplated that any embodiment, discussed, in ibis specification can be implemented with respect to any method or composition of the Invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

'"Treating," "treat" or "treatment" is referred to herein as administration of a substance to a subject with the purpose to cure, alleviate, relieve, remedy, prevent, or ameliorate a disorder, symptoms of the disorder, a disease state secondary to (he disorder, or predisposition toward the disorder. An. "effective amount" Is an amount of the substance dial is capable of producing a medically desirable result as delineated herein in a treated subject The medically desirable result may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an. indication of or feels an effect).

"Disease amenable to treatment with stem cell therapy" as re&rred to herein means any procedures, conditions, disorders, aliments and/or illnesses which can be treated by the adtTooistration of stem cells. Such diseases include but are not limited to bone marrow, skin, heart, and corneal transplantation, graft versus host disease, hepatic and renal failure, lung injury, rheumatoid arthritis, treatment of autoimmune diseases such as Crohn's disease, ulcerative colitis, multiple sclerosis, inpus and diabetes; prevention of allograft rejection, neurological disorders and cardiovascular medicine; as well as Acute lymphoblastic leukemia (ALL), Acute myeloid, leukemia. (AML), Burkitt's lymphoma. Chronic myeloid .leukemia (CML), Juvenile myelomonoeytic lenkenua (JMMI), Non-Hodgfcin's lyn-usboma Hodgkitvs lymphoma, Lymphomatold granulomatosis, Mysiodysplastie syndrome (MBS), Chronic snyelomonocyhc leukemia (CMML), Bone Marrow Failure Syndromes, Ams-gakaryocyiic thrombocytopenia. Autoimmune neutropenia (severe). Congenital dyserydiarpoietic anemia, Cyclic neutropenia, Diaroond-Slackfan anemia, Evan's syndrome, Faneoni anemia, Glaoxmamfs disease. Juvenile derraaiornyositis, Kostmansvs syndrome, Red cell aplasia, Schwachman syndrome. Severe aplastic anemrs. Congenital sideroblastic anemia, Thrombocytopenia with absent radius (TAR. syndrome). Dyskeratosis congenita!, Blood Disorders, SicMe-cell anemia (hemoglobin SS), HbSC disease. Sickle po Thalassemia, thalassemia major (hydrops retails), hMhalassemia major (Coo

intermedia, E-βo thalassemia. Ε-β+ thalassemia, Metabolic Disorders, Adfcnisfeykodystrophy Gaucbei's disease (infantile), Metachromatic leukodystrophy^ Krabbe disease (globoid ceil leukodystrophy), Ganther disease, Hermansky-Pudlali: syndrome, Hurler syndrome, Hurler- Scheie syndrome. Hunter syndrome, Saafilippo syndrome, Maioteaux-Lamy syndrome, Mucolipidosis Type II, III, Alpha mannosidosis, Niemann Pick Syndrome, type A and B, Sandhoff Syndrome, Tay-Saehs Disease, Batten disease (inherited neuronal ceroid lipofuscinosis), Lesoir-Nyhan disease, Immunodeficiencics, Ataxia telangiectasia. Chrome granulomatous disease. DlGeorge syndroms. IKK gamma deficiency, Imnaune dysregnlatlou polyendocririeopaihy, X-linked Mucolipidosis, Type 11, Myelokathexis Xdioked immunodeficiency. Severe combined immunodeficiency. Adenosine deaminase deficiency, Wiskott-Aldnch syndrome, X-liaked agammaglobulinemia, X-imked lympboproliferative disease, Omeno's syndrome, Reticular, dysplasia, Thymic dysplasia. Leukocyte adhesion deficiency. Other Osteopetrosis, Laogerhans cell histiocytosis, Hemophagocyoc lymphohistiocytosfe, Acute & Chronic Kidney Disease, Alzheimer's disease, Anti-Aging, Arthritis, Asthma, Cardiac Stem Cell Therapy, Cerebral Marction (Stroke), Cerebral Palsy (Stoke), Chronic Obstructive Pulmonary Disease (COPD), Congestive Heart Failure, Diabetes Meilnus (Type 1 II). Fibromyalgia, Immune Deficiencies, ischemic Heart Disease, Lupus, Multiple Sclerosis, Myocardial Infarction, Osteoarthritis, Osteoporosis, Parkinson's Disease, Peripheral Arterial Disease, Rheumatoid Arthritis, Stem Cell Therapy in Plastic Surgery, Traumatic Brain injury and Neurological Diseases,

"Vascular or cardiovascular disease" as referred to herein is characterised by clinical events including clinical, symptoms and clinical signs. Clinical symptoms are those experiences reported by a patient that indicate to the clinician the presence of pathology. Clinical signs are those objective findings on physical or laboratory examinations that indicate to the clinician the presence of pathology, Cardiovascular disease" includes both "coronary artery disease" and ''peripheral vascular disease," both terras being defined below. Clinical symptoms in cardiovascular disease include chest pain, shortness of breath, weakness, fainting spells, alterations in consciousness, extremity pairs, paroxysmal nocturnal dyspnea, transient ischemic attacks and other such phenomena experienced by the patient Clinical signs in cardiovascular disease include such findings as EKG abnormalities, altered per

abnormal heart sounds, rales and wheezes, jugular venous distention;, neurological. alterations and other such findings discerned by the clinician., Clinical symptoms and clinical signs ears combine lit a cardiovascular disease such as a myocardial infarction (Mi) or a stroke (also termed & "cerebrovascular accident" or "CVA"), where the patient will report certain phenomena (symptoms) and die clinician will perceive other phenomena (signs) all indicative; of m underlying pathology. "Cardiovascular disease" dietaries those diseases related to the cardiovascular disorders of fragile plaque disorder, occlusive disorder arid stenosis. For example, a cardiovascular disease resulting from a fragile plaque disorder, as that term is defined below, can be termed a "fragile plaque disease." Clinical events associated with fragile plaque disease include those signs and symptoms where the rapture of a fragile plaque with subsequent acute thrombosis or with distal emholizadon are hallmarks. Examples of fragile plaque disease include certain strokes and myocardial infarctions. As another example, a cardiovascular disease resulting from an occlusive disorder cars be termed as "occlusive disease." Clinical events associated with occlusive disease include those signs and symptoms where the progressive occlusion of an artery affects the amount of circulation, that reaches a target tissue. Progressive arterial occasion may result in progressive ischemia that may ultimately progress to tissue death if the amount of circulation is insufficient to maintain live tissues. Signs sod symptoms of occlusive disease include claudication, pain while resting, angina, and gangrene, as well as physical and laboratory findings indicative of vessel stenosis arid decreased distal perfusion. As yet another example-, a cardiovascular disease: resulting from restenosis can be termed m in-sient stenosis disease, in-stent stenosis disease includes the signs and symptoms resulting from the progressive blockage of an arterial stent that has been positioned as part of a procedure like a percutaneous transluminal angioplasty, where the presence of the stent is intended to help hold the vessel in Its newly expanded configuration. The clinical events that accompany in-stent stenosis disease are those attributable to the restenosis of the reconstructed artery,

A "coronary artery disease" ("CAD") refers to a vascular disorder relating to the blockage of arteries serving the heart. Blockage can occur suddenly, by mechanisms such as plaque rapture or embolization. Blockage can occur progressively, with narrowing of the artery via myointimal hyperplasia, and plaque Ibnnation. Those clinical s:

from the blockage of sterns serving the Mart, are mmiiestations of coronary artery disease. Manifestations of coronary artery disease include angina, ischemia, myocardial infarction, cardiomyopathy, congestive heart failure, arrhythmias and aneurysm formation. It h understood that fragile plaque disease in the coronary circulation is associated with arterial thrombosis or distal embolization that manifests Itself as a myocardial infarction. It is understood that occlusive disease in the coronary circulation is associated with arterial stenosis accompanied by anginal symptoms, a condition commonly seated with pharmacological briervefttiorjs and with angioplasty.

"Peripheral Vascular Disease" or ("PVD") is a cardiovascular disease resulting from the blockage of tire peripheral (i.e., nou-eorouary) arteries. Blockage can occur suddenly, fey mechanisms such as plaque rupture or embolization, as occurs in fragile plaque disease, Blockage can occur progressively, with narrowing of the artery via rnyomtirnal hyperplasia and plaque formation, as in occlusive disease. Blockage can be complete or partial Those clinical signs and symptoms resulting tern the blockage of peripheral arteries are manifestations of peripheral, vascular disease. Manifestations of peripheral vascular diseases include, inter alia, claudication, ischemia, intestinal angina, vascular-based renal insufficiency, transient ischemic attacks, aneurysm fonuatiosr, peripheral embolization and stroke, ischemic cerebrovascular disease Is a type of peripheral vascular disease.

Upon suspicion of PVD, the ankle brachial pressure index (ΑΒΡI/AΒI) may be determined. When the blood pressure readings in the ankles are lower frjau that in. the arms, blockages in the arteries: which provide Mood fiom the heart to the ankle may be suspected,. An ABI ratio of about or less than 0.9 consistent with PVD; values of ABI about or below (18 indicate moderate disease and about or below 0,5 imply severe ischemic disease. An ABI ratio of about or less than 0,5 or 0,4 may he used as a threshold for diagnosis.

It is possible for conditions which stiffen the vessel walls ( such as calcifications that occur in the se tting of chronic diabetes) to produce false negatives usually, hut not al ways. Indicated by abnormally high ABIs (about or > 1.3). Such results and suspicions merit torihar investigation, and higher level studies. If ABIs are abnormal the next step is g

ultrasound examination to look at site and extern of atherosclerosis. Other imaging may be performed by angiography, where a catheter is inserted into the common femora! artery and selectively guided to the artery in question. While injecting a radiodense contrast agent an X- my may be taken. Any flow limiting stenoses found in the x-ray can. be identitled and treated. by atherectomy, angioplasty or sienting. Muitlslioe computerized tonography (CT) scanners may provide direct Imaging of the arterial system as an alternative to angiography. CT may facilitate evaluation of the aorta, and lower iitnb arteries without She tseed for an angiogram's arterial injection of contrast agent.

Peripheral artery disease (PAD) is a form or peripheral vascular diseases (PVD) in which there are partial or total, blockage of blood supply to a limb, usually the leg, leading to impaired blood flow and hypoxia in tire tissue. When PAD advances it reaches the stage of critical limb ischemia (CLl) with skin ulcerations; gatrgrene and potentially unavoidable amputations. Hind limb ischemia animal models have been rssed to evaluate various therapeutic approaches addressing stem cell, transplantation.

The current FAD standard of care includes snKddng cessation, reduction in cholesterol antiplatelet agents, treatment of diabetes, treatment of high blood pressure, ACE-inhibitors, exercise, and eilosiazoL Additional treatment options can involve stents, e.g.. drag-ekning stents. Such, as paciiiaxel-eluting stents. The methods and compositions disclosed, herein are all envisaged to be provided with such treatment options. See for example, Burns et al., BMJ, 2003 March 15; 326(7389): 584-588

Peripheral artery disease is commonly divided in the Fontaine stages, introduced by Rene Fontaine in 1054 tor ischemia: 1 ) Mild pain when walking (claudication), incomplete blood vessel obstruction: 2) Severe pain when walking relatively short distances (inlermittenl claudication), pain, triggered, by walking "after a distance of > 150 ra in stage li-a and alter <150 m in stage II-b"; 3) Fain while resting (rest pain), mostly in the feet, increasing when the limb is raised; 4) Biological tissue loss (gangrene) and difficulty walking. The compositions sad methods disclosed herein are envisaged to result in the improvement of peripheral artery disease by improving the Fontaine Stage of a patient by about

over a period of at least or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 hours, days, weeks or months from the initiation of treatment or compared to control.

Alternatively, classification of peripheral artery disease by Ruthereford consists of six stages: 1) Mild claudication; 2) Moderate claudicatino; 3) Severe claudication: 4) Ischemic pain at rest; 5) Minor tissue loss; and 6) Major tissue loss. The compositions and methods disclosed herein are envisaged to result in the improvement of peripheral artery disease by improving the Rutherford Stage of a patient by about or at least 1 , 2, 3. 4, 5 or 6 Stages over a period of at least or about 1, 2, 3. 4, 5, b, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 31 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36 hours, days, weeks or months from the initiation of treatment or compared to control

In one embodiment of the invention the composition, claimed herein results m or enhances angiogenesis.

Ideally, the composition results in or enhances angiogenesis by at least 20 percent compared to control.

In one embodiment of the invention the composition claimed herein comprises a pharmaceutically acceptable carrier.

"Assessment of limb function" is referred to herein as a relative measurement of the effectiveness of the methods and compositions disclosed herein. Alternatively, limb function grades are; "(F for Dextog th e toes to resist gentle itaction of the tail, "1" for plantar flexion., "2" for dragging but no plantar flexion, and "3" for dragging of foot. The compositions and methods disclosed herein are envisaged to result in the improvement in limb function of a patient by . about or at least 1 , 2, 3 or 4 grades over s period of at least or about 1 , 2, 3, 4, 5, 6, 7, 8 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 , 27, 28, 24, 30, 31 , 32, 33, 34, 35, 36 hours, days, weeks or months from the initiation of treatment or compared to control.

"Assessment of Ischemic damage" cm be used to evaluate various therapeutic approaches addressing stem cells transplantation. In an embodiment ischemic damage can he expressed as a morphological grade. The morphological grades are; "0" for substantial, absence of necrosis, is for necrosis substantially limiting to toes (toes loss), "2" for necrosis extending to a dorsum pedis (foot loss), "3" tor necrosis extending to a crus (force loss), and "4" for necrosis extending to a thigh (total bind-lmib loss). The composhtion and methods disclosed herein, are envisaged to result m the reduction of isensmio damage in a patient by about or at least 1 , 2, 3 or 4 grades over a period of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 20, 27, 28, 29, 30, 3 1 , 32, 33, 34, 35, 36 hours, days, weeks or months from the inibation of treatment or compared to control.

"Assessment in limb blood flow" is referred to herein as m additional relative measurement of the effectiveness of the methods and compositions disclosed teds. Preisrably, blood flow in legs from both sides is measured, e.g., with, a noo-contact Laser Doppier, before and after treatment. The compositions and methods disclosed herein arc envisaged so result in the improvement, in limb blood flow of a patient to about or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 1 3 , 14, 15, 16, 17, 18, 19, 30 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 1 , 32, 33, 34, 35, 30, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 89, 90, 91, 92, 93, 94, 95, 96, 97, 9S, 99, 09,5 or 100 percent of normal blood perfusion levels over a period of at least or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 1 7, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 hours, days, weeks or months Horn, the initiation of treatment or compared to control.

"Reducing the rate of amputation in a peripheral artery disease patienf refer to the rate of FAD patients requiring amputation of limbs, digits and/or tissues due to e.g„ infection or necrosis, There are standard rates of amputation associated with treatment with MSCs which, can be readily determined. Preferably, the treatment of patients with a composition which, includes a combination of MSCs and an NM II antagonists reduces the rates of such amputation by about or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36. 37, 3

47, 48, 49, 50, 51 , 52, 53, 54, 55, 54 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 80, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 100, 125, 150, 175, 200, 250, 300, 350, 400, 500 or more percent relative to a standard (e.g., standard of eare) or control (e.g., MSC treatment or NM 11 antagonist treatment aiooo but nest m combination).

"Cardiovascular Disease Drugs" as defined herein refers to medicaments useful for the managmtsnt of diabetes, hypertension (e.g., ACE-inhibitors), cholesterol (e.g., statins), and antiplatelet drugs (e.g. , aspirin, clopidogrel), anti -eoagnlants (e.g., warfarin), diuretics and beta-blockers. Additionally, cilostazol or pentoxifylline are cardiovascular diabetes drugs. The term "agiogenesis" is defined as a physiological process involving the growth of new blood vessels from pre-eslstmg vessels. Vaseulogenesis is the term used for spontaneous blood-vessel formation, and intussusception is the term for new blood, vessel formation by Splitting off existing ones, Angiogeoesis is a normal process in growth and development, as well as m wonnd healing. If is also a fuodamental step in the transition of tumors trom a dormant state to a malignant state. Angiogenesis is said to be taking place when there is about or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 1 8, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 51, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 40, 47, 48, 49, 50. 51, 52. 53, 54, 55, 56. 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 08, 69, 78, 71 , 72, 73, 74, 7S, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 100, 125, 150, 175, 200, 250, 300, 350, 400, 500 or snore percent or snore increase in the growth of new blood vessels in a given area when compared with the same tissue prior to angiogenesis or to a standard (e.g., standard of care) or control (e.g., MSC treatment or NM II antagonist treatment alone but not io combination).

"Accelerating healing" refers to the increased speed at which a disease amenable to stent cell therapy shows signs of improvement (with respect to a well excepted qualitative or poaotifative morphological or functoinal measurement associated with the disease in question) io response to therapy with the compositions disclosed herein, Preferably, the speed of healing increases by about or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, I 5, 1.0, 1 7, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36,

46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 100, 125, 150. 1 75, 200, 250, 300, 350, 400, 500 or more percent a standard (e.g., standard of care) or control (e.g., MSC treatment or KM II antagonist treatment alone hut not in combination).

"Patient" as used herein refers to a mammalian subject diagnosed with or suspected of having or developing cardiovascular disease. Exemplary patients may be humans, apes, dogs, pigs, cattle, cats, horses, goats, sheep, rodents sod other mammalians that can benefit from stem cell therapies.

"Adminsstering" is referred to herein as providing the compositions of the invention to a patient By way of example artd not limitation, composition, administration, e,g,, injection, may he performed by tntrgvenons (ί,ν.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d,) injection, intraperitoneal (i.p,) injection, or intramuscular (Lot.) injection, One or more such routes may be employed. Parenteral administration can be, for example, by bolus injection er by gradual periston over iims. Alternatively, or concurrently, administration may be by the oral route. Additionally, adrnkitsfraikm may also be by surgical deposition of a bolus or pellet of cells, or positioning of a medical device, e.g,, a stent, loaded with cells, Preferably, the compositions of the invention are administered at the site of ischemic cardiovascular disease, e.g. at the site or sear (e.g., about or at least 1 , 2, 3, 4, 5, 6, 7. $, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 millimeters from) the a of the ischemic cardiovascular disease lesion (e.g,, vascular stenosis/bbekage, necrotic tissue or site of gangrenous infection). Administration may be mtravenoys, intramuscular, at or near a site of a disease-associated lesion and/or intrtiumseukir at or in proximity to the site of ischemic damage.

"A patient in need thereof is referred to herein as a patient diagnosed wish or suspected of having cardiovascular disease. In one embodiment, the patient has or is likely to develop peripheral artery disease.

" Tofipotency " is referred to hereht as the ability of a single cell to divide and/produce all the differentiated cells in an organism, including
extra-embrynoc tissues. Totipoten cells include nporm and zygotes. In some organisms, cells can dedifferentiate arsd regain totlpoteaey.

"Pluripotency" is referred to herein ss the potential to differentiate into my of the three germ layers; endoderm. (intenor stomach liming gastrointestinal tract, the lungs), mesoderm (muscle, bose, blood, urogenital), or ectoderm (epidermal tissues and nervous system).

"'Plunpoteat stem cells" include natural pluripotent stem cells and induced plunpoteut stern cells, They can give rise to my fetal or adult cell type. However, atone they generally cannot develop into a fetal or adult organism because they lack the potential to contribute to extra- embryonic tissue, such as the placenta.

"Induced pluripotem stem ceils" or ("IPS cells" ) are similar to natural pluripotent stem cells, such as embtyomc stem (ES) cells, in many aspects, such as the expression of certain, stem cell genes and/orteios, chromatin, methylallon patterns, doubling time, emhryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability. Induced plurrpotenl cells may be derived from for example, adult stomach, liver, skin, cells and blood cells. IPS cells may be derived by transaction, of certain stem celh-assoeiated genes into non-plunpotent cells, such as adult fibroblasts. In certain embodiments, transteetion may be achieved through viral vectors, such as retroviruses, for example, Transiected genes can include, but arc not limited to, master transcriptiontd regulators Oet-3/4 (PooSf1) and Sox2 Oct-4, Nanog and Lm28 transgenes. Sub-populations of transfeeted cells may begin to become morphologically and biochemically similar to pbiripotent stem cells, and can be isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection.


Multipotency" is referred to herein a assmuitipotent progenitor cells which have the potential to give rise to multiple ceil types, but a number ofiiaeages mors limited than a pluripotent stem ceil, for example, a'muitipoterst stem cell is a hematopoietic cell that can. develop Into several types of blood sells, but cannot de velop into brain cells or other ς

"Mesenchymal stem cells or Multipotent. Stromal Cell (both referred to as "MSCs") are referred to herein, as being multipotent stromal cells that can differentiate into a variety of cell types, including but not limited to: osteoblasts (bone cells), chondrocytes (cartilage cells), and adipocytes (fat cells).

In one embodiment, mesenchymal stem, cells are obtained from bone marrow, In another one embodiment mesenchymal stem cells are obtained from developing tooth bud of the mandibular third molar In another embodiment, MSCs are obtained from amniotic fluid, In another embodiment MSCs may he obtained irom the umbilical cord, tissue, e.g., from Wharton' s jelly and the umbilical cord blood. In a further embodiment MSCs are isolated from adipose tissue. Alternatively, MSCs are isolated from Wharton's jelly. In another embodiment, MSCs are derived from IPS cells as, for example, described in Guiliani et al., "Human, mesenchymal stem cells derived from induced pluripotsnt stem cells dp.waregukte NK cell cytolytic machinery," Blood, July 29 201 1.

MSCs preferably have a small cell body with a few cell processes ihat-are long and thin. The cell body may contain, a large;, round nucleus with a prominent nucleolus, which is surrounded by finely dispersed chromatin particles, giving the nucleus a clear, appearance. The remainder of the cell body contains a small amount of go!gi. apparatus, rough, endoplasmic reticulum, mitochondria, and polyribosomes, MSCs which are long and this,, are widely dispersed and the adjacent extracellular matrix is populated 'by a few reticular fibrils but is devoid of the other types of collagen fibrils.

Cell attachment is not a homogeneous process but rather occurs through · particular zones, so-called focal, adhesion zones. Once attached, cells exert tension by means of interna! motor proteins. Thus, the cytoskeleton may become organised and functions in a variety of processes Including transport scaffolding and cell survival Upon de-attaching cells, these may quickly constrict as a function of the internal tension build up by .motor proteins and the previously organised iatemal structures (cytoskeletal and other) can be disturbed. If not allowed to re-attach to surfaces, such cells may initiate cell death programs which have been described as attachment-dependent apepotosis/anoikis.

"Myosins" include a family of ATP-dependent motor proteins and are generally known tor their role in muscle contraction and their involvement in a wide range of other eukaryotis motility processes. They are generally responsible for actin-based motility Generally, myosin II (also known as conventional myosin) is the myosin type responsible for producing muscle contraction in muscle cells.

The major cytoskeletal motor protein responsible for generating cell tension is non-muscle myosin II (referred to as myosin II). "Non-Muscle Myosin II or "NM II" is an actin -binding protein that has actin cross-linking and contractile properties and is regulated in part through the phosphorylation of its light and heavy chains. Like muscle myosin II, NM II molecules are comprised of three pairs of peptides: two heavy chains of 230 kDa, two 20 kDa regulatory light chains (RLCs) that regulate NM II activity and two 1 7 kDa essential light chains (ELCs) that stabilize the heavy chain structure. Although these myosins are referred to herein as "non-muscle" myosin lIs to distinguish them from their muscle counterparts, they are also present in muscle cells, where they have distinct functions during skeletal nmscle development and diffrentiation, as well as in the maintenance of tension in smooth muscle.

Generally, three mammalian NM II isoforms have both overiapning and unique properties. The two globular head domains of NM II contain a binding site for hoth ATP and actm and they are followed by neck regions, each of which binds the two functionally different light chains, The neck domain acts as a lever arm to amplify head rotation while the chemical energy of ATP is converted into die mechanical movement of the myosin head. This neck domain Is followed by a long α-helical colled coil, which tonus an extended rod-shaped domain that effects dimemation between the heavy chains and terminates in a relatively short non-helical tail. The rod domains of KM H self-associate to form bipolar filaments (anti-parallel arrays of myosin molecules), which are smaller ihm those found m cardiac and skeletal, muscle.

NMI II acts to integrate processes that drive cell migration and adhesion, It is also an important end point on which many signaling pathways converge, largely through Rho GTFases. NM II itself is tightly regulated at different levels, including at the love

assembly and disassembly, aetia bissiing and ATPase and mo-tor activity. The regulation of the actio cytoskeleton by NM II controls multiple interrelated processes, such as migration, cellcell and cell-matrix adhesion, cell differentiabon, tissue morphogenesis mid development. The spat.k«eroporai regulation of NM II by sisbcelhdar localization end activation, of its regulating Mnases in different ceils and tissues Iras important ramifications so compiling the NM II function.

The .regulation of Mg2+-ATP hydrolysis and filament formation of NM 11 involves the reversible phosphorylation of specific amino adds present m the pair of 20 kDa EX.Cs and the heavy chains. The function of the BLC pair is to stabilize the NMHC and there is. no evkleace that they undergo reversible phosphorylation. See also Vieerite-Man^anares et al, Nat Rev MM Cell Biol 2009 November: KK1 1): 778-790,

"Non-Muscle Mysosin II antagonist" refers to an agent that directly or indirectly inhibits, blocks or re verses the activity of Myosin II as it occurs is nature or in lire diseased or damaged tissues of patients suffering item cardiovascular disease, Such an agent cm. be a small molecule (e,g,, hkhblstatin); nucleic acid including r&NA, mRNA, iRNA, miRNA, siRNA (see e.g., Ma et al., Mol. Biol Cell November 15, 2010 vol, 2 i no. 22 3952-3962 or Kim el al J Biol Cheer. 2012 Aug 1.0;287(33):27345- 58), anti-sense RNA, DMA, RNA/DNA hybrid, nfooxyme, RNA, aptatner, (all or any of which may include synthetic or non-naniral oneleotide bases): peptides; or proteins e.g., antibodies, antibody fragments, antibody-dike molecules or single chain antibodies, as welt as phosphatases aad. kinases. For example, NM II antagonist can. be an siRNA targeted to knock down the expression of the NM 0. encoding mRNA. or the mRNA of a gene associated with the up-regulation of NM II; or a vector encoding such an siRNA.

Preferably, die NM II antagonists include Angiotensin II Type 1 Receptor Antagonists, (eg., the nou-peptide AT I receptor antagonist FK-739 sec Fuji et al., Hypertension, 1999;33:973-980) as well as Angiotensin II receptor antagonists, also known as angiotensin: receptor bloetes (ARBs), ATI -receptor antagonists or sartans, are a group of phnmiaceutieals which modulaie the renin-angsoteussn-a!dosterone system. Their mam

byperteusion (high blood, pressure), diabetic nephropathy (kidney damage due to diabetes;) and congestive heart failure. Losartan, srbssarian, olmesasian, eaodesartan and valsarian include the tetrazole group (a ring whh .tour nitrogen and one carbon); Losaslan, irbesartan, obnesartan, candesartaa, aad iebnisartau include one or mo imidazole groups.

Non-Mosck Mysosin II antagonists also include ACE inhibitors. An ACE inhibitor (or angiotensin-converting-eazyme inhibitor) is a medication pharmacentieal drug used primarily for the treatment of high blood pressure (hypeoeosion) and weak heart muscie (congestive besrf failure). Exemplary ACE inhibitors include cspiopril, sofenopril, enalapril ramiprih quinapril, perindopril, lisinopril, benazepril, Imidapril, zoknopril, trandolapril, fosinopril, easofcimns and laetokinin% as as lire lactoiripeptides Val-Pro-Pro and IIe-Pro-Pro produced by the probiotie Lactobacillus hdvetkus or derived fkms .casein have been shown to have ACE-inhibiting asd antihypertensive functions,

Non-Muscle Mysosin II antagonists also include 2.3-butanedione-2-monoxime (ΒDΜ) aod inhibitors of myosin II adenosine triphosphatase (ATPase) activity.

One NM If antagonist is bkbbistatm (a 1-pheayl-1 -2-pyrrolidiaooe derivative) which is a specific pharmacologic inhibitor of skeletal muscle and nonmuscle myosin U adenosine triphosphatase (ATPase) activity. Blebbistatin is highly active stnaii mokeu!e that inhibits ceil hkbhing. This compound is eell-permsam, benign, and, importantly, readily reversible. It has been described as a survival factor in stem cell e¾lmres. it has also been described as a promoter for adhesion of cells to a substrate to which the cells usually have no or only low affinity (WO20I2062819), e.g. as for coating of Implanted, medico! devices such as pace inskars, hip or bone implants or stents.

Biehhistatiu is shown below:

In some embodiments, the term "blebbistatin" includes a racemic mixture. In some embodiments, the term blebhistatin refers to R-blebbistatin. In some embodiments, the term blebbistatin refers to S-blebbistatin.

This disclosure encompasses blebbistatin variants, analogs and derivatives. Additional blebbistatin variants, analogs am! derivatives and Non-Masck Mysosia II antagonists suitable and envisaged for use herein are disclosed in U.S. Pafc. No, 20080021035. See for example also; Straight et al., Science 299 (5613): 1743--47; Kovfcs et al., (Aug 2004), J Biol Cham, 279 (34): 35557-63; Limoaze et al., (2004) J Muscle Res Cell Motsl. 25 (4-5): .337-41.

In certain embodiments, the therapeutic compositions disclosed herein contain or are associated with, at least one Non-Moscie Mysosin 11 antagonist whereby each at least one Non-Muscle Mysossn II antagonist is present in single dose at a concentration of about, at least or more than 0.1 , 0.2, 0.3, 0.4, 0.5, 0,6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1.1, 32, 1 3, 14, 15, 16, 17, 18, 19, 20. 21 , .22, 23, 24, 25, 24 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40. 41, 42, 43, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 02, 03, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89. 90. 91 , 92, 93, 94, 95, 96, 97, 98 or 99 eaeh times 1.0- 9 , 10-810-710-710 -610- 5 10-410-310-210- 1 molar per dose. Preferably, Non-Muscle Mysosin II antagonist is present in single dose at a concentration between about 1 -400, 2-60, 3-50, 4-40, 5-30, 6-20, 7- 15, 8-10, 2- 18, 3-16, 4 - 14, 5- 12, 640 or 7-8 μΜ.

In certain embodiments, the therapeutic compositions disclosed herein are administered in a dose about or at least or more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1,

20, 21 , 22, 23 or 24 times per day, week or month over a period of about or at least or more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 or 24 days, weeks or months.

in certain embodiments, the NM II antagonist is incubated with the cells of the composition, about, at least or more than 0.1, 0.2, 0.3, 0,4, 0.5, 0.6, 0.7, 0,8, 0.9, 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 1 3. 14 , 15, 16, 17, 1 8, 19, .20, 21 , 22, 23, 24, 25, 26, 27. 2.8, 29, 80, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63. 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81 , 82, 83, 84. 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 minutes, hours or days prior to administration to the patent.

Certain embodiments involve a kit comprising cells and an NM II antagonist Certain kit embodiments comprise (a) a container that contains at least one NM II antagonist; and (b) a container havmg stem cells. The NM II may be in solution or in lyophlized form. The kit may optionally incloude instructions for (i) use of the solution or (ii) reconstitution and/or use of the lyophilized formulation or (iii) using the contents to treat cardiovascular disease. The kit may further comprise one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, (v) a syringe, or (νϊ) an automated medical, device, In one embodiment, the container is selected from the group consisting of: a bottle, a vial, a syringe, a test tube, or a muti-use container, in another embodiment, the target peptide composition is lyophilixed.

A single dose of the therapeutic compositions described herein can contain at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, .14, 15, 16, 17, 1 8, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 , 44, 45. 46, 47, 48, 49, 50, 51, 52, 53 , 54, 55, 56, 57, 58, 59, 60, 61 , 6.2, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83. 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 each times 1, 10, 102, 103, 104, 105, 106, 107, 108, 109, 1010or more cells per kilogram of patient body weight la certain embodiments, a. dose eomalos about, at least or more man 1 -20, 2- 19, 3-18, 4-17, 5-16, 6- 15, 7-14, 8 - 13, 9-12 or 10-1 1 each times 106 stem cells.

In. certain embodiments, the addition of the NM II antagonist in the inventive compositions allow the clinician to provide a patient with a lower dose of stem cells than would be otherwise be needed to achieve the same therapeutic effect achieved with a population of stem cells lacking the NM II antagonist in certain embodiments, the addition of the NM II antagonist in the inventive compositioos allow the clinician to provide a patient with at least or about 5, 1 0, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90 or more percent less stem cells per dose than would be otherwise be needed to achieve the same therapeutic effect over a define period of time.

Disclosed herein are both methods of and compositions for treating cardiovascular disease; and methods of rcducing the complications (e.g., amputation) of treating cardiovascular disease when employing MSCs, Moreover, kits including cells, NM II antagonists, pharmaceutically acceptable carriers, and - optionally appropriate instructions, are disclosed.

The invention also relates to a method of redneing the rate of amputation in a peripheral artery disease patient treated with a composition comprising MSCs, comprising contacting the MSCs witn a non-muscle myosin II antagonist prior to the treatment. Ideally, the non-muscle myosin II antagonist is blehbistatin.

in some embodiments, the methods of treating can generally involve intramuscular injection of a dose of a composition of MSCs treated with an NM II antagonist saeh as- blebbisiatln at or near the site of the lesions arising from CVD such as PAD. Such injections generally result in an increase in limb function, blood flow, and ischemic symptoms. Moreover, the use of an NM II antagonist such as blebbistatin in the composition dramatically accelerates MSC-triggered regeneration of damaged tissues. Furthermore, application of blehbistatin with MSCs drastically reduces severe complications associated with PAD and/or the transplantation of MSCs to a patient suffering from PAD.

The methods and kits can also include additional cardiovascular drugs or medical devices such as for example stents (which may or may not be drug eluting).

stents may be loaded with MSCs which have been contacted with NM II antagonists prior so or during implantation.

Additional References:

Armstrong, L., Lako, M., Buckley, N., Lappin, T. R. J., Murpfey, M. J., Nolta. J. a, Pittenger M., et al. (2012). Editorial: Our top 10 developments in stem cell biology over the last 30 years. Stem cells (Dayton, Ohio), 30(1 ), 2-9, doi: l 0J 002/ste.rn,I00?

Ren. G. Chen X, Dsng F, Li W, Ren X, Zhang Y, Shi Y: Concise review: mesenhymal stem cells and translational Medicine: emerging issues. Stem Cells Trahsl Med. 2012 Jan; 1(1):51 -8. Review.

Masuzawa K, Jesmin S. Maeda S, Kaji Y, Oshika T, Zaedi S, Shimojo N, Yaji N, Miyauchi T, Goto K. A model of retinal ischremia-reperfuson injury in rats by subconjunctival injection of endothelin-1. Exp Biol Med (Maywood). 2006 Jan;231.(6); 1085-9.

Straight AF, Cheung A, Limoaze J, Chen I, Westwood NJ, Sellers JR, Mitchison TJ,

Dissecting temporal and spatial cuntrol of cytokinesis with a myosin II Inhibitor. Science, 2003 Mar 14;299(5613): 1743-7.

Walker A, Su H, Ccmti MA, Harb N, Adelstein RS, Sato N, Non-muscle myosin II regulates survival threshold of pluripoient stem celKNai Commun. 2010 Sep 7:1 ;71.

Daigh, Christine; Fuhrken. Peter; Saivagiotto, Giorgia; Method and composition for the differentiation of stem cells, US20100216181 Al, US Patent application 2010

Schenk, Judith; van den Bos, Christian; Roseobanm, Claudia; Nie, Ying: Method for eontrolUhg binding of cells to a substrate, 2012 WO201.2062819, Patent application

Cai L, Johnstone BH, Cook TG, Liang Z, Tnktuev D, Cornetia K Ingram DA, Rosen ED, March KL., Suppression of hepatocyte growth factor production impairs the ability of adipose-derived stem cells to promote ischemic tissue revascularization. Stem Cells. 2007 Dec;25(12):3234-43, Epub 2007 Sep 27

Goto T. , Fukuyama N., Aku A., Kanabuchi K., Kimura K, , Taira H., Tanaka E.„ Wskana N., Mori H., and lnoue H., Search for appropriate experimental methods to create stable hind-limb ischemia in mouse, Tokai 1 Exp Clin Med,, Vol. 31, No. 3. pp. 128-132, 2006

OBCD principles of Good Laboratory Practice ENV/MC/CREM (98)17.

Stabile E., Bumett M.S., Waikins C., Kinnaird T, Bschis A., la Sala A., Miller J.M., Shou M., Epstein S.E., Fuehs S. linpaired asteriogesle response to acute hiodiimb ischemia In, CP4- kaockout mice. Circulation. 2003;15 ;108(2):205-210

EXAMPLES

Disclosed herein is the testing of BM MSCs in a mouse model for hind limb peripheral ischemia. Peripheral artery disease (PAD) is a form of peripheral vescular diseases (PVD) in which there are partial or total blockage of blood supply to a limb, usually the leg, leading to impaired blood flow and hypoxia in the tissue. When PAD advances it reaches the stage of critical limb ischemia (CLl) with skin ulcerations, gangrene and unavoidable amputations. One of the most promising innovative treatments for diabetic vascular complications is stem cell-based products that enhance angiogenesis (TReft-3) Hind limb ischemia animal models have been used to evaluate various therapeutic approaches addressing stem cell transplantation.

Animal handling was according to the National Instiute of Health (NIH) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animals were housed in polysufone (PSU) cages (7-8/eage) measuring 42.5 x 265,6 x 18.3 cm, with stainless steel top grill having tacilitis for pelleted food and drinking water in glass-clear polycarbonate bottle; bedding: steam sterilized clean paddy husk (Harlan, Sani-chip, Cat#: 106S8216) was used and bedding material was changed along with the cage at least twice a week. Animals were fed. ad libitum a commercial rodent diet (Teklad Certified Global 18% Protein Diet cat #: 106S8216). Animals have free access to autoclaved and acidified drinking water (pH between. 2.5 and 3.5) obtained from the municipality supply. Animals were housed under standard laboratory conditions, air conditioned and filtered (HEPA F6/6) with adequate fresh air supply (Minimum 15 air ehanges/hour). Animals were kept in a climate controlled environment. Temperatures range was 20-24ºC and RH range was 30-70% with 12 hours light and 1 2 hours dark cycle. Species/Strain: Mouse/Achymic Nude; Gender / Number/Age:

Male/ 152/9- 10 weeks; Source: Harlan Laboratories, Israel; Body weight The average body weight was 28.4 g at study initiation (day 1). The minimum and maximum weights of the group were within a range οf =20 % of group mean weight; Acclimation period: 5- 10 days; Identification: Three position ear notching and cage cards. No animals was found in a moribund condition or showed severe pain and enduring signs of severe distress. Surgery Day was defined as "DA Y 0" in this study. On the day of surgery anesthesia was induced by 1 .5 to 3.0% isoflurane, 1.5% N2O and 0,5% O2.

Test items: Human Bone Marrow Mesenchymal Stem Cells (Lonza Cologne GmbH). Vehicles: DMEM + 10 % FCS, Optional: 10 μΜ Blebbistatin (BB),

Under anesthesia, the mouse was placed with ventral side up. A 0.5 - 1.0 em incision was made in the skin in the inguinal area. The femoral artery was ligsted twice with 6-0 silk thread and transected between the ligatures. The wound was closed with 4-0' silk thread and the mouse was allowed to recover.

On DAY 1 , 24 hours post-surgery. each treated animal was injected intramuscular at two sites (the proximal and the distal side of the surgical wound) or Intravenous into the tail vein. For intramuscular injection the animals were injected 50 μl at each site, total 100 μl per animal. For intravenous Injection the animals were injected by 200 μl per animal See Table 1 for Groups Allocation.


Body weight was measured on study day - 1 prior to the surgery and once weekly thereafter. Blood flow measurement: Blood flow in. legs from both sides was measured with a non-costacl Laser Doppler before surgery and on days: 1. 3, 7, 14, 21, 28 and 35 post operation, Blood flow measurements were expressed as the ratio of the flow in the ischemic limb to that in the normal limb (Goto et al. Tokai J Exp Clin Med., Vol. 31, No. 3, pp. 128-132, 2006). Consistently best regeneration was shown in Group 4, animals treated with MSC+BB (Fig.1). Macroscopic evalustion of ischemic severity: Macroscopic evaluation of the ischemic limb was performed once a week post operation by using morphological grades for necrotic area (Goto et al.): See Table 2.


in vivo assessment of limb funotion and Ischemic damage. Semi -qaantitative assessment of impaired use of the ischemic Iimb was .performed once a week post-surgery using the following scale (Stabile et al., Circulation , 2003; 15; 108(2):205-210). See Table 3.


Limb function is graded as "Not applicable" in case of partial or full limb amputation In such case blood flow measurements was not included in the statistical analysis,

is vivo assessment of limb function. Semi-quantitative assessment of impaired use of the ischemic limb was performed on days 7 up to 35 by vising graded fimcdomd scales. An improvement in limb fonction starting from day 14 was found in animals from group 4 treated with cells versus control animal groups 1 and 2 (Fig 2A). That improvement was found in animals groups 3 and 5 on day 35 after ischemia. In animals administered test article intravenously, improvement in limb function starting from, day 28 was found in group 9 compare to control groups 6 and 7 (Fig 28).

EXAMPLE 2 - Statiscal Analysis

The purpose of this study was to test the efficacy of the tested stem cells as a possible treatment to alleviate HLl symptoms in an animal nude mouse model.

Study and points were: Clinical score; Body weight measurements; Blood flow measurements Ranking analysis: Each group was given a ranking from 1 to 10 on each of the first 3 eodpoints measured (% Blood Flow, Limb Frmction and Ischemic Severity Score). Rank=1 is highest and Rank= 10 is lowest When two groups are tied, both receive the same rank "in the middle"; e.g. if two groups were tied in Rank = 4, both received the score 4.5 (twice of which is the sum of ranks 4 md 5). For each endpoints, the median was computed of each group (the median was used to ensure that extreme value will not affect results). On each endpoint. groups were sorted by median and each given its respective rank. The average rank was computed for each group over all endpoints

Statistical testing was done using Mixed Model in SAS®. Main comparisons presented are between the two most effective treatments and all the others. Full statistical tenststing results are provided herein. P-values provided both in the report and In the appendixes are not adjusted for multiplicity. To determine significance, use P'< 0.0056. There are many ways to adjust for multiple testing bore based on the interpretation of tests that are meaningful and those that are not. Since this is an exploratory trial, it was decided that adjustment would be considered within each group separately. Thus, for example, Group 4 is compared to 9 other groups so that we determine that a significant result is P < 0.05/9 = 0.0056.

Slope: The slope computed is of the linear trend from surgery to Day 35. This single number for each animal represents its change on an eudpoints over time. Distribution of slopes is provided for each group on each of the endpoints (excluding Weight), using Box Plots, These plots provide a convenient method for presenting many groups on a single graph. They should be read as follows: 25% of data points (in our case, slopes) are between the lower hinge and the bottom of the box-— percentiles 0 to 25; 50% of data points a

and top of the box percentiles- 25 to 75. This is also terned the "inter-quartile range" (IQR).

25% of data points are located between top of the box and upper hinge- percentiles 75 to 100. However, upper and lower fences are defined by a distance of 1 .5*IQR from bottom and top of the box. While they are not actually shown in the plot, values outside these fences are considered outiers. So if there are data points above the upper fence, or below the lower fence, they are indicated separately on the boxplot; in other words, extreme points are not included between lower and upper hinges of the boxplot


In further analyses the imputation of missing dais is done in the following way: Blood Flow (%): Value of the first measurement after the surgery (assumption is that treatment failed so that there is no iasprovement beyond what was observed immediately after surgery); Ischemic Severity Score; 4=''total Hind-Limb Loss" (the worst score possible); Lirnb Funtion: 3 ="Dragging of foot" (the worst score possible); Weight (g): The animals lowest weight at any of the .points measurred up to and including the day of amputation.

While paired comparisons were done, central results are presented in a way in that they are statistically relevant and can be clearly depicted in graphics. Data are consistent across endpoints and are consistent across different analysis. To obtain this picture. we eliminated Weight from consideration. Descriptive statistics and testing indicated that Weight was not a meaningful measure of efficacy in the model. Each group was ranked on remaining 3 endpoints on day 35 and assessed Average Rank of groups - a measure combining results from ah endpoints.

Evaluating all Parameters Concurrently at Day 35. Procedure: For each parameter (Blood Flow (%), Limb Function and Ischemic Severity Score) the median in the group across all animals was computed. All the groups based on their median from 1 to 10, where 1 is best, were ranked on each of the following parameters: Blood Flow (%) -highest is 1 , lowest is 10; Limb Function— highest is 10, lowest is 1 (worst category is 3, best is 0); Ischemic Severity

Score highest is 10, lowest is 1 (worst category is 4, best is 0); Finally, tor each group we computed the average rank over the three parameters.

From the table it can be seen that there are four general magnitudes of rank:

Group 4 (BM.MSC 1 in DMEM + 88 (im)) has the highest rank and is Far ahead of the 2nd most effective group (5): Average Rank = 1.2, difference from next group is 2.

Group 5 is far behind Group 4 (as indicated in the ballet above) but also ahead of the groups behind it. Specifically, it is 1.2 ranks ahead of the 3rd most effective group (9).

The next seven groups (9, 3, 2, 7, 6, 1 and 8) are in the 3rd category with a difference between best and worst - 2,5 (6.8-4-3), which is equivalent smaller than the distance between 1 st and 3rd places (3.1).

Group 10 (BM.MSC in DMEM, pre-BB (iv)) is far behind the "block of seven groups" in the previous bullet - 3 ranks. Note that Group 10 turned out especially ineffective due to a large number of amputations and so, ineffective values imputed for Day 35.

in Table 6 the results of the Significance Testing are depicted. The two: best groups tare compared with all others on all endpoirsis.

impaired angiogenesis is one of the features of ischemic diseases, 'However* clinical trials to alleviate ischemia have been, disappointing, indicating the need tor new mokctiies and

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therapeutic targets to treat ischemic diseases. Stem cell therapy is a promising approach in cardiovascular medicine. In order to assess the therapeutic activity of stem cells in ischemic tissue, the mouse hind limb ischemia model is used.

It is disclosed herein that that MSCs showed regenerative potential an animal mouse model whereas the substance Blebbistatin. alone had no beneficial effect Tins validated the model in these experiments, a single IV or local admimstration to the ischemic limb of the tested cells surprisingly and nnsxpected, restorerd blood perfusion up to 65-84% of its normal values. Bleed flow .restoration was compatible with other results showing improvement in limb ftmcti.cn and decreased ;snd delayed ischemic severity to the moose Mad- limb ischemia model This effect was achieved in the groups treated with tested cells locally (groups 4 mi 5} or intravenously (group 9),

Blebbistatin dramatically accelerated MSC-triggered regeneration of damaged tissues. Application of Blebbistatin with MSCs drastically reduces severe complications in healing as clearly demonstrated by Group 4 (BM.MSC in DMEM +BB (im)) which is more effective than all groups on % Blood Flow and Limb Function, Including group 5 BM.MSC in DMEM, pre-BB (im). In addition, Blebbistatin drastically improves the regenerative potential of MSCs by reducing severe complications in healing,

Group 4 (BM.MSC in DMEM +BB (im) is more effective than all groups on % Blood Flow and Limb Function, including group 5 BM.MSC in DMEM, pre-BB (im).

The results presented herein demonstrate that application of BM.MSC In the presence of Blebbistatin provides greatest efficacy when applied intramuscular. This combined application showed fastest recovery from the lesion resulting in the most effective treatment The relatively small variation in the best group indicates consistency of efficacy across animals. Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of this invention. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those dest

practice this inve&iion, the preferred compositions, methods., kits, sad. rnesujs for communicating information are described herein.

All references cited herein are incorporated herein by reference to the full extern allowed by law. The discussion of those references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the aceumey amd pertinence of any cited reference.