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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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• • A—HUMAN NECESSITIES
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  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/31—Combination therapy

Definitions

• the invention ates to stem cell compositions and methods.
• 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.
• MSCs Mesenchymal stem cells
• 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,
• adult stern progenitor cells from abundant autologous origin have the additional advantages over embryonic stem ⁇ '3 ⁇ 4S") cell types given their great availability, low tutuorgenieity and allowing for the avoidance of controversial ethical issues associated with ES cells.
• donor stern progenitor cells should possess desirable therapeutic properties, for example, minima! 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 ve been, em lo ed hi numerous preelimc-al studies m rodents, rabbits and baboon monkeys among others, for bone marrow, s.idn, heart, and corneal transplantation, grail yststts host disease, -hepatic and renal f ilure, luag.
• a major c mpoBent ' of morbidity and mortality attributable t cardiovascular disease occurs as a eaccoque:nce of the partial or complete blockage of vessels carrying blood in the coronar and/or pers hersd vasculature.
• a major c mpoBent ' of morbidity and mortality attributable t cardiovascular disease occurs as a eaccoque:nce of the partial or complete blockage of vessels carrying blood in the coronar and/or pers hersd vasculature.
• 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.
• PAD Peripheral artery disease
• blood vessel occlusion
• new vessels are formed (a process ofier3 ⁇ 4 referred to as "ajj i enesis' 5 ) and smal l vessels are enlarged (termed “arieriogenesis") to replace the ftmciicm of the impaired vessels.
• arieriogenesis a process ofier3 ⁇ 4 referred to as "ajj i enesis' 5 ) and smal l vessels are enlarged (termed “arieriogenesis" to replace the ftmciicm of the impaired vessels.
• ther is a great need for optimised thera eu ic MSC compositions .ami methods of • using them, to ameliorate the symptoms of various, diseases in ene al -audi in the treatment of vascular disease in particular.
• 201002 6181 discloses cultivation of pl ripotent stem cells in a medium that is free of ' serum and feeder cells using blebbistatin as survival factor, increased nu bers of cells are generated by eukiva g the cells in sptuaer flasks or bioresetors.
• Published PCX application VVO20120&281 discloses a me hod for controlling binding of cells to a substrate usin bkbbistatin as a promoter for adhesion of cells to which the ceils usually ha e no or limited affinity.
• 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.
• the vascular d sease is s .result of diabetes.
• the vasc lar disease is a resuk of athemsc!erosis.
• she vascular disease is peripheral vascular disease (PVD), lis still another embo iment, the vascular disease is peripheral artery disea e (FAD).
• VD peripheral vascular disease
• FAD peripheral artery disea e
• the vascular sease is associated with m ankle brachial pressure index of about or less than. 0.9.
• the vascular disease is associated with an aitkle brachial pressure index of about or less than 0.7.
• the vascular disease has resulted in critical limb ischemia, in anoth r embodiment the vascular disease .has resulted in skin ulcerations or gangrene.
• the composition results its or enhances anglogenesis, la iixll another embodiment, the composition results in or enhances angiogenesis by at least about 20 percent compared to control.
• a portion of the population of cells comprises ph ripotent cells.
• the nhsripotect cells are induced phu potent stem cells.
• a portion of the population of cells comprises mnitipotent cells, in still, another embodiment, the rmdtlpotent cells are moltipote stromal cells or meset hyrnal st m cells.
• the ma!tipo eot cells are derived horn kdueed pluripotent stem cells.
• the composition comprises a plmnnaceotically acceptable carrier.
• the .limb function, in the patient improves by about or at least 2-3 grades compared to the same pattern not receiving the composition
• the limb blood flow in the patient irrrpnoves to bout or at least 65-85% of norrrral or entreated limb blood flow
• 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.
• the ischemic- damag ' in the patient improves at a faster rate (or aeeciarsied rate) compared to the same patient not tecelviug the composition.
• the eornposiliori is ad Mstered intravenously.
• the composition is administered intramuscularly, i another embodiment, the composition is administered intramuscularly at or in proximity t
• the non-muscle myosin. 11 antagonist knocks down non-masde m osin ⁇ gene and/or protein expressi n. in yet a fUrther em odiment the n ⁇ i*-n3 ⁇ 4useie myosin II antagonist is an si A. targeted to kaoek dowa non-anisek myosin II gene and/or protein expression.
• the non-nmsc-le myos n II antagonist is an. DMA vector encoding a « siRNA, miRNA or anti-sense RNA targeted to knock down non- muscle myosin 11 gene 3 ⁇ 4nd ⁇ 1 ⁇ 2 protein expression.
• the- soa-muscie . myosin il antago ist s aa Angiotensi II receptor antagonist or aagio ensin receptor bl ckea
• the non ⁇ rnascle myosin II antagonist is an angiotenshvoonverting- en&y.i «e (ACE) inhibitor.
• the non-muscle myosin II antagonist is 2,3 ⁇ bi3 ⁇ 4 ⁇ anediona ⁇ 2-monoxime ( .DM),
• th non-mnscle myosin It antagonist is biebbistutin or analog, derivative or variant mereof; in still a .further embodimen the noo-masek myosin II antagonist is a pyrrohdiaorie derivative.
• the non-muscle myosin II antagonist is a molecule ' torn. the. class of pyrroHdmones,
• kits comprising: a population of mul ipotent celts; a non-muscle myosin .11 antagonist: a pharmaceutically aeceplable carrier; and optionally, instructions for administering these to a patient diagnosed with disease which is amenable, to treatmen us ng stem cell therea y.
• the disease is a cardiovascular disease.
• the disease is peripheral artery disease, la one embodiment, the cells are- MSCs.
• the non-muscle myosin ! antagonist is bi.ebbistatin.
• the cardiovascular disease is peripheral artery disease.
• the cells are MSCs r the nommuscis myosin il antagonist is bkbbsstatia and the cardiovascular disease is. peripheral artery disease.
• the kit farmer comprises a cardiovascular drag- in a f rther embodiment the kit further comprises a stent, Jn still another embodiment, me- kit further comprises a dnsg-ehiting stent, in one embodiment, the items comprising the kit are In separate containers.
• the population of ultipotent ceils: the non- muscle myosin II antagonist; and the pharmaceutically acceptable carrier are in the s me container, e,g., a syringe or automated adndnistration device, Tb artery disease.
• the kit may hav cells that a e MSCs, The .non-mnsole etye&n II atttagoaist is Ideally olebbistatin. and the d sease is a peripheral artery disease.
• the kit may forther comp ises! a cardiovascular drug, aad or a stent &ot a dnig-eittiing stem.
• the stems may be in separate containers, in the same container or, in a syringe.
• Another aspect of the invention el tes to a method of reducing the rate of amputation in a peripheral artery disease patient treated with a composition comprising sViSCs, comprising contacting the MSCs with s sion-niuscle myosin II antagonist prior to the treatment, in one embodiment, the no -muscle myosin II antagonist is blehhistatim
• the rate is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, ' 80%, or 90% o more.
• Another aspect of the invention relates to a stem ceil composition for treating a disease amenable to treatment with Stem eel! therap comprising a population of rnahipotem cells; 3 ⁇ 4nd a non-muscle myosin, it antagonist, in.
• the cells are MSCs.
• the non-muscte myos sif nta onist is biebbistatin .
• Another aspect of the in vention relates to a method of making a stem cell composition for treating a disease amenable to treat ent with stem cell therapy comprising a population of muitipotertt cells: and s non-mcseJs myosin ⁇ . ⁇ 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 fttrther aspect of the invention s directed to method of .accelerating the healing of a disease amenable to treatment with stem ceil therapy comprising a treating a patient with a- composition comprising a population of mu!tipotem cells; and a non-mu$ek 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.
• the cells are MSCs, in other embodiments * the NM II antagonist is biebbistatin, In yet further embodiments, the- disease are a cardiovascular disease.
• Figures 1 A-B show Blood Flow ⁇ %) over Time; The median lis the group was computed across all valid animals.
• Biebbisiathi (BB) accelerates .and enhances blood How regeneration by mesenchymal stem ceil implantation, Stem ceils ⁇ administered in the presence of hkhhisla n (I A, Group 4, di mond) showed aster and enhanced regeneration in. blood flo in animals compared to stem ceils that .have been pre-tre&ted with biebbistatin but were I platHed in die absence of biebbistatin (I A, Group 5, solid black square).
• Figure 2A-B sho Limb Function. (%> over Timet The median, in the group was c mputed across all valid animals. The data show mat biebbistatin (BB) presence with MSCs supports las; improvement in limb function. Despite the blood perfusion improvement in other treated groups, Utah functional deterioration and limb necn;?sls Iricrease as 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.
• BB mat biebbistatin
• Figure 3A-8 show Ischemic Severity Score (%): The median i tire group was computed across aU valid, animals, MSCs applied in conjunction, with blebbiststin impede severity in Ischemic Score, Analysis of the ischemia severity score over time sho consistently better resuUs for Group 4 . a «d 5 when applied intramuscularly; although media controls ranged within the scatter. ' ot® thai 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 :m.
• 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 endpohtt over time.
• These plots provide a convenient met od r presenting many groups on a single graph.
• the evatnahoo of blood ow ( ' %) is depicted as slopes boxplots by group in order to point out t e low variation (indica ed by the small box sizes) within controls and ' five best performing group in the study ' M ' SC in.
• Figure 5 shows bieboistatin impedes severe complications during the healing process.
• a major problem in pre-chnica! 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 araptttations 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, Da a dearly.
• Inventive MSC compositions have great regenerative potential in a validated animal model
• bkhbistatin dramatically and unexpectedly accelerate an enhances the therapeutic potentitsl of MSCs and redoees severe complications in healing.
• the term "about” is used to indicate that a value, includes the inherent variation of error for the device, the .method being emplo ed, 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%, U 14%, 15%, 16%, 1 7%, 18%, 19% or 20% variability depending on the situation.
• compositions of the invention cm be used to achieve methods of the invention.
• t eat or “treatment” is .referred to herein as administration of a substance to a subject with the purpose to c re, alleviate, relieve, remedy, p e ent, or ameliorate a disorder, symptoms of the disorder, a disease state ec ndar to he disorder, or predisposition toward the disorder.
• An. "effective amount” Is an amount of the substance dial is capable of if ⁇ 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. in ication of or feels an effect).
• 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,0 neurological disorders and cardiovascular medicine; as well as Acute lymphoblastic leukemia (ALL), Acute myeloid, leukemia. (AML), Burkitt's lymphoma.
• ALL Acute lymphoblastic leukemia
• AML Acute myeloid, leukemia.
• Burkitt's lymphoma Burkitt's lymphoma.
• Chronic myeloid .leukemia CML
• Juvenile myelomonoeytic lenken a jMMI.
• Non-Hodgfcin's ! n-usbom Hodgkitvs lymphoma Lymphomatold granulomatosis
• MBS ysiodysplastie syndrome
• CMML Chronic sn elomonocyhc leukemia
• Bone Marrow Failure Syndromes Ams-gakaryocyiic5 thrombocytopenia.
• Autoimmune neutropenia severe.
• Congenital dyserydiarpoietic anemia Cyclic neutropenia, Diaroond-Slackfan anemia, Evan ' s syn r me, Faneoni anemia, Glaoxmam s disease.
• Juvenile derraaiornyositis Kost ansvs syndrome, Red cell aplasia, Schwachman syndrome.
• Severe aplastic anemrs Congenital sideroblastic anemia, Thrombocytopenia with absent radius (TAR. syndrome).
• IKK gamma deficiency Imnaune dysregnlatlou po!yendocririeopaihy, X inked Mucolipidosis, Type 11, My3 ⁇ 4lokathexis Xdioked immunodeficiency. Severe c m ined immunodeficiency. Adenos ne deaminas defic en y, Wiskott-Aidneh syndrome, X-iiaked agammaglobulinemia, X-imked jympbomvliferatiye disease, Omeno's syndrome, Reticular, dysplasia, Thymic dysplasia. Leukocyte adhesion deficiency.
• Osteopetrosis Laogerhans cell histiocytosis, Hemophagocyoc lymphohistiocytosfe, Acute ⁇ 3 ⁇ 4 Chronic Kidney Disease, Alzheimer's disease, Anti-Aging, Arthritis, Asthma, Cardiac Stem Cell Therapy, Cerebral Marcii ' on (Stroke), Cerebral Palsy (Stoke), Chronic Obstructive Pulmonary Disease (COPD), Congestive Heart Failure, Diabetes eilnus (Type 1 II).
• Fibromyalgia Immu e Deficiencies, ischemic Heart Disease, Lupus, Multiple Sclerosis, Myocardial Infarction, Osteoarthritis, Osteoporosis, Parkinson's Disease, Peripheral Arterial Disease, Rheumatoid Arthritis, Stem Cell Therap in Plastic Surgery, Traumatic Brain injury and Neurological Diseases,
• Vascular or cardiovascular disease as referred to herein is characterised by clinical events inckKlmg clinical, symptoms and clinical signs.
• Clinical symptoms are those experiences reported by a patient that indicate to the clinician the presenc of pathology.
• Clinical signs are those objective findings on physical or laboratory examinations that indicate to the clinician the presence of athol gy, Cardiovascular disease” includes both “coronary artery disease” and ''peripheral vascular disease/' both terras being defined below.
• Clinical s mpt ms in cardiovascular disease include chest pain, shortness of breath, weakness, fai ting spells, alterations .in consciousness, extremity pairs, paroxysmal nocturnal dyspnea, transient ischemic attacks and other such phenomena experienced b the patient
• Clinical signs in cardiovascular disease include such findings as E&G abnormaliti s, altered per
• a cardiovascular diseas .r s l(i»g from a fragile plaque disorder can be termed a "fragile plaque disease/"
• Clinical event associated with fragile plaque disease include those signs and symptoms where the rapture of a fragile plaque with subsequent acute thrombosis or with distal em oiixadorj are hallmarks. Examples of fragile plaque disease include certain strokes and myocardial infarctions.
• a cardiovascular disease resulting from -an oc lusive 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 target tissue.
• occlusive disease includes claudication, pain while resting, angina, and gangrene, as well as physical and laborator findings indicative of vessel stenosis arid decreased distal -perfusion.
• a cardiovascular disease resulting from restenosis can be termed m in-sient stenosis disease, in-s ent stenosis disease includes the signs and symptoms resulting from the progressive blockage of an.
• arterial stent that ha been positioned as part of a procedure like a percutaneous transluminal angioplasty, where the presence o 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 "c ronary artery disease " ( ⁇ "GAD" ⁇ refers to a vascular disorder relating to the blockage of arteries serving the heart. Blockage can oc u suddenly, by mechanisms such as plaque rapture or embolization. Blockage can occur progressively, with narrowing of the artery via myointi al hyperplasia, and plaque Ibnnation. Those clinical s: from the blockage of sterns serving the Mart, are mmiiestations ' of coronary rtery disease. Manifestations of coronary artery disease include angina, ischemia, myocardial .infarction, cardiomyopathy, congestive heart failure, arrhythmias and aneu sm formation.
• Blockage can occur suddenly, fey mechanisms such as pla ue .rupture or emboliz t on,- 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.
• -peripheral vascular diseases include, inter alia, cl udication, 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.
• the ankle brachial r ssure index ( ⁇ 1/ ⁇ ) may be determined.
• 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 ma be suspected,.
• An A.B1 ratio of about or less than 0,5 or 0,4 may he used as -a threshol for diagnosis.
• Peripheral artery disease is a form or peripheral vascular diseases (PVD) in which there are partial or total, blockage of blood supply to a limb, us ally the leg, leading to impaired blood flow and hypoxia in tire t ssue.
• PVD peripheral vascular diseases
• CLi critical limb ischemia
• 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, treatme t of diabetes, treatment of high blood pressure, A €E-inhibitors, exercise, and eilosiazoL Additional treatment options can involve stents, e.g.. drag-ekning stents. Such, as paciiiaxel-eluting stents.
• stents e.g.. drag-ekning stents.
• Such, as paciiiaxel-eluting stents are all envisaged to be provided with such treatment options. See for example, Burns et af , BMJ, 2003 March 15; 326(7389): 584-588 ⁇
• Peripheral artery disease is commonly divided in the Fontaine stages, introduced by Rene Fontaine in 1 5 tor ischemia: I ) Mild pain when walking (claudication ⁇ , incomplete blood vessel obstruction: 2 ⁇ Severe pain when walkin relatively short distances (inlermittenl claudication), pain, triggered, by walking "after a distance of > 150 ra in stage li-a nd alter 150 in stage ⁇ -b"; 3 ⁇ Fain while resting (rest pain), mostl in the feet, increasing when the limb is raised; 4) Biological tissue loss (gangrene) and difficulty walking.
• the compositions sad methods .
• compositions and methods disclosed herein are envisaged to result m the improvement of peripheral artery disease by improving the Rutherf r Stage of a patient by about or at least 1 , 2, 3. , 5 or 6 Stages over a period of at 10 least or about 1, 2, 3. 4, 5, b, 7, 8, 9. 10, 11 , 12, 13, .1 4, 15, 16, 17, 18, 19, 20. 31 , 2 2, 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 com ared to control
• composition, claimed herein results or enhances i S angiogenesis.
• composition results in or enhances angiogenesis fey at least 20 percent compa ed to control,.
• the composition claimed herein comprises a pharmaceutically acceptable carrier,
• 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, I I . 12.
• hebemt damage can he expressed as a morphological grade.
• the mo:nphologieal. grades are; K 0 W for substantial, absence of necrosis, s for m ⁇ .rosis substantially limiting to toes (toes .loss ⁇ , .
• necrosis x nd n for necrosis x nd n to a dorsum pedis (foot loss), "3" tor necrosis extending to a crus (force loss), and "4" for necrosis extending to- a thigh (total h»d-lmib loss ⁇ .
• the eomposihons and meth s disclosed herein, are envisaged to result m the reduction of isensmio damage in a patient by about or at least !
• compositions ami methods disclosed herein arc envisaged so result in the improvement, in limb blood Bow of a patient to about or at least 2, 3, 4, 5, 6, 7, 8 radiation 9, 1.0, 11 , 12, 1 3.
• 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 Broad infection or necrosis
• MSCs peripheral artery disease
• the treatment of patients with a com osition which, includes a combination of MSCs and an II antagonists reduces the rates of such amputation by about or at least 2, 3, 4, 5, 0,, 7, 8, 9, TO, 1 1 , 12, 13, 1.4, 15, 16, .17, 18, 19, 20, 21,, 22, 23, 24, 25, 26, .27, 28, 29, 30, 31, 32, 33. 34, 35, 36.
• Cardiovascular Disease Drugs'- as defined herein refers to medicaments oxeinl for the mana mtsnt of diabetes, hypertension (e.g., ACE-inhibitors), cholesterol (e.g., statins), and antiplatelet drugs (e.g though aspirin, clopidogrei), anti -eoagnlasm (e.g., warfarin), diuretics and beta-blockers. Additionally, oiiostaml or pentoxifylline are cardiovascular diabetes drugs.
• the term '3 ⁇ 4agiogenesis' ' 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 psx ⁇ oess in growth and development, as well as wonnd healing. If is also a iuadamentai step in the transition of tumors trom a dormant state to a malignant state. Angiogenesis is said to be taking place when there (s about or at least 2, 3.
• the speed of healing increases by about or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 1 , 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, : 6, 47, 48, 49, 50, 51 , 52, 53, 5 , 55, 56, 57, 38 ⁇ 59.
• Patient as used he e n refers to a aramaliao subject diagnosed with or suspected of a i g or developin cardiovascular disease.
• Exemplary atients m be humans, apes, do s, pigs, cattle, cats, horses, goats, sheep, rodents sod other mammalians that can benefit from stem ceil therapies.
• composition, administration e,g,, injec ion
• composition, administration may he performed by tntrgvenon ( ⁇ , ⁇ .) mjeetiotj, sub-cutaneous (s.c.) injection, intradermal (i.d-l injection, intraperitoneal (i.p,) injecti n, or intramuscular (Lot.) injection,
• s.c. subcutaneous injection
• i.d-l injection intradermal
• i.p intraperitoneal injecti.p,
• injecti n intramuscular
• Parenteral administration can be, for example, by bolus injection er by gradual erist over iims.
• 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 ! , 2, 3, 4, 5, 6, 7.
• the a of the ischemic cardiovascular disease lesion e.g,, vascular stenosis/bbekage, .necrotic tissue or site of gangrenous infection.
• a minist ation ma be mtravenoys, intramuscular, ai or .near a site of a disease-associated lesion and ' or intrtiumseukir at or in proximity to he site of ischemic damage.
• a patient in need thereof is referred to herein as a patient diagnosed ish or suspected of having cardiovascular disease.
• the patient has or is likely to develop peripheral artery disease.
• i- Tofipotenc ! is referred to hereht 3 ⁇ 4S he ability of s ng e ceil to divkle and/produce all the differentiated ceils m organism, including nclude po nd zygotes. In some organisms, cells can dedifferentiate arsd regain t tlpoteaey,
• Pluripotency' is referred- to herein ss the potential to di fereniiaie into my of the three germ layers; endoderm. (intend* stomach Imfe , gastrointestinal tract, the !uags), mesoderm (muscle, bose, blood, urogenital), or ectoderm (epidermal tissues and nervous system),
• Induced pluripotem stem ceils or (“IPS ceils *' ⁇ re similar to natural pluripotent stem cells, such as embtyomc stem (ES) cells, in many aspects, such as the expression of certain, stem cell enes and/orteios, chromatin, meth lallon patterns, doubling time, emhryoid body formation, teratoma formation, viable chimera .formation, and potency and differentiability.
• Induced plurrpotenl cells may be derived fro for example, adult stomach, liver, skin, ceils and blood ceils.
• IPS ceils may be derived by transaction, of certain stem celh-assoeiated genes into noi*- piunpoterrt cells, such as adult fibroblasts.
• transteetion may be achieved through viral vectors, such as retroviruses, for example,
• Transiected genes can include, but arc not limited to, master traBscriptiontd regulators Oet-3/ (PooSfl) and Sox2 Oct-4, anog and Lm2$ transgenes.
• Sub-populations of transfeeted ceils 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, as tnuitipotent progenitor cells which have the potential to give rise to multiple ceil types, but a number ofiiaeages mors limited than a pluripotent stem ceil, fo 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 ⁇ " esench al stem ceils or Multipoint.
• Stromal Ceil (both referred to as “MSCs”) are referred m herein, as h ing nmlt?ps3 ⁇ 4e3 ⁇ 4t stromal cells t ai can differentiate into & variety of cell types, including but not limited to: osteoblast ' s (bone cells), chondrocytes (cartilage cells), and adipocytes (fateells).
• mesenchymal stem, cells are btained fr m bone marrow, hi another one embodiment mesenchymal $ten3 ⁇ 4 ceils are obtained from developing tooth bud of the man ibular t ird mit ' kc in another embodiment, MSCs are obtained from amniotic Ikdd, In another embodiment MSCs may he obtained irom the umbilical cord, tissue, e.g., from Whartxu s jelly and the umbilical cord blood. In a further embodiment MSCs re isolated from adipose tissue. Alternatively, MSCs a e isolated from Wharton's jelly.
• MSCs are derived from IPS cells as, for example, described in Guillanl et at, "Human, mesenchymal stem cells derived from induced pluripotsnt stem cells d .waregukte ' NK. cell cytolytic machinery Blood, July 2% 201 ! ..
• MSCs preferably have a small cell body with a le cell processes ihat-are long and thin.
• the cell body may contain, s large;, round nucleus with a pr minen nucleolus, which is surrounded by finely dispersed chromatin particles, giving the nuclais a clear, appearance.
• the remainder of the cell bod contains a small amount of go!gi. apparatus, rough, endoplasmic reticulum, mitochondria, and polyribosomes, MSCs which are long and this, are widel dispersed and the adjacent extracellular matrix is populated ' by a few reticular fibrils but is devoid of the oilier types of collagen fibr ils.
• Cell attachment is not a homogeneous process but rather occurs through ⁇ particular 20n.es, so-called focal, adhesion zones.
• cells exert tension by means of interna! motor proteins.
• the eytoskeletoo may become or nised and functions m a variety of processes Including transport scaffolding and cell survival Upon de-attaching ceils, ihese may quickly constrict as a function of the internal tension build up by .motor proteins and the previously organised iatemal structures (eytoskeletal and other) can be disturbed. If not allowed to reattach to surfaces, such cells may initiate cell death programs which have been described as at gehment-dependeot apepotosis/anoibls.
• Myosins include a family of Al - ependent motor roteins and are gene all kno n tor their role in muscle contraction and their involvement in. a wide range of other eukaryotis motility processes. They are generally re&nonsibi ⁇ for actin-based motility. Generally, m os n ii (also known as; conventional myosin) is the myosin type responsible for roducin muscle contraction in muscle cells,.
• the major eyioskeletsl -motor prole (n es onsible for generating ceil tens o is non-rnuscie myosin II (referred to as myosin ii), "Non-Muscle Myosin IP of i; NM IF ' is m acti -bindin protein thai has act?** cross-linking and contractile ro erties and is regulated m part through the phosphorylation of its light and heavy chains.
• HM 1:1 molecules arc comprised of three pairs of peptides: two heavy chains of 230 kOa :t two 20 IcD regulator light c ains (RLCs) that regu te 11 activity and two 1 3 ⁇ 4 essential light chains ELCs) that stabilise the heavy chairs structure,
• RLCs right-chain regulator light c ains
• ELCs essential light chains
• three mamm lia MM ii isoforms have both overiapning and uniq e properties.
• the two globular head domains of NM II contain a bindin site for hoth ATP and actm and Ibc are ibliowed. by cock re ions, each of which binds the two functionally different light chains,
• the neck domain acts as a lever arm to amplify head rotation - hile the chemical energy of ATP is converted into d e mechanical ' .m vement of the myosin head..
• the rod domains of KM H self-associate to form bipolar filaments (anti- parallel arrays of myosin molecules), which are smaller ih those found m cardiac and skeletal, m.oscle,
• NM H acts to integrate processes that drive ceil migration and adhesion, It is also an important end point on which many signaling pathways converge, largely through Rho GTFases.
• NM II itself is ti htly regulated at different levels, including at the love
• the .regulation of Mg ' ⁇ -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 a « ⁇ 3 the heavy chains.
• the function of the BLC pair is to stabilize the HC and there is. no evkleace that they undergo reversible phosphorylation. See also Vieerite-Man ⁇ anares et al, Nat Rev MM Cell Biol 2009 November: K 1 I): 778-790,
• Non-Muscle ysosin II antagonist refers to an agent thai directly or indirectl inhibits, blocks or re verses the activity of Myosin II as it occurs is nature or in fire 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, mR A, iR A, miRNA, siRNA (see e.g dislike Ma et al, Mo!. Biol Ceil November 15, 2010 vol, 2 i no. 22 3952-3962 or Kim el al J Biol Cheer.
• nucleic acid including r&NA, mR A, iR A, miRNA, siRNA
• NM II antagonist can. be an siRNA targeted o .knock down the expression of the NM 0. encoding mRNA. or the mRNA of a gene associated 3 ⁇ 4» the ⁇ -re ul ion of NM II; or a vector encoding such an siRNA.
• die NM II antagonists include Angiotensin II Type 1 Receptor Antagonists, (eg., the nou-pepiide AT ! receptor antagonist F -739 sec Fuji et al, Hypertension, i 999;33 ⁇ 973- 980) as well as Angiotensin II receptor antagonists, also known as angiotensin: receptor ' h ' foetes (ARBs), ATI. -receptor antagonists or sartans, are a group of phnmiace3 ⁇ 4meais ⁇ which modulaie the T «sm-angsoteussn-a!dosterone system.
• Angiotensin II Type 1 Receptor Antagonists eg., the nou-pepiide AT ! receptor antagonist F -739 sec Fuji et al, Hypertension, i 999;33 ⁇ 973- 980
• Angiotensin II receptor antagonists also known as angiotensin: receptor ' h
• Losartan, srbssarian, olmes sian, eaodesar an and valsarian include (he tetrazo!e group (a ring w h .tour nitrogen and one carbon); Losaslan, irbesartan, obnesartan, candesartaa, aad iebnisartau include one or mo imidazole groups.
• N&n-Mosck Mysosin 0. antagonists also include ACE inhibitors.
• An ACE inhibitor or angioiei3 ⁇ 4ir ⁇ eoirverting"er5»:j3 ⁇ 4ne inhibitor
• An ACE inhibitor is a medication pharmacentie l drug used primarily for the treatment of high blood pressure (hypeoeosion) and weak heart oiuscie (congestive besrf failure).
• Exemplary ACE inhibitors include cspiopril, sofenoprii enalapril ramiprih quinapril, perindoprsi, iisinopril, benazepril, ImidapriL zoknoprii, trandolapril, fosinopriL easofcimns and laetokinin% a as hydrogen lactoiripeptides Va!-Pro-Pm and ilc- ⁇ ro-Fm produced by the probiotie Lactobacillus hdvetkus or derived fkms .casein have been shown to have ACE-ii-lnhitiog asd antihypertensive functions,
• Non-Muscle ysosin II antagonists also inekide 2.3-b «tai) ⁇ i o «C"2-mO ⁇ >oxh Ve ( ⁇ 0 ) aod inhibitors of myosin II adenosine triphosphatase (ATPase) activity.
• bkbbistatm (a i ⁇ pb.eayk i -2-pyo3 ⁇ 4lidiaooe derivative) which is specific pharmacologic inhibitor of skeletal muscle and nonmuscle myosin U adenosine triphosphatase (ATPase) activity.
• Blebbistatin is highly active stnaii mokeu!e that inhibits ceil h bhing. This compound is eell-permsam, benign, and, importantly, readily reversible. It has been described as a survival factor in stem cell e3 ⁇ 4lmres.
• the ern:: v 3 ⁇ 4febbstathr ! includes a racemie mixture.
• the temt biebhistaiin refers to . -blshbistatm.
• the term biebhistaiin refers to S-biebbistatm.
• Non-Muscle Mysosin II antagonist is present in single dose at a concentration between abo t 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 Of 7-8 ⁇ .
• the therapeutic compositions dis losed herein are administered in a5 dose about or at least or more han 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 i, ' 20, 21, 22, 3 or 24 iroes per day, week or month over a period of about or ai east or more than I, 2, 3, 4, 6, 2, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 days, eeks or months.
• kits comprising cells and an.
• NM II antagonist Certai:o kit embodiments comprise (a) s container that contains at least one NM II antagonist; and (b) a container havmg siera cells.
• the NM I! may be in. solution, or in lyophilmed- form.
• the kit may optionally mciodo snstmations for (?) use of the solution or (a) rc onstitudotj arid/or ose of ibe lyophilized. fomsutekm; or (ill.) nsing the contents to treat cardiovascular disease.
• the kit may bather comprise one or more of (ill) a bufler, (iv) a diluent, fv) a Slier, (vi) a needle, (v) a syringe, or ( ⁇ ) .an m mi&tsd medical, device,
• the container Is selected from the group eoasistiag oil a bottle, a vial, a syringe, a test cube, Or a mnhi-ase 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, 10,11, 12, 13, .14, 15, 16, 17, 18,19, 20,21,22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33 , 34, 35, 36, 37, 38, 39, 40, 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, 7!., 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83.84, 85, 86, 87, 88, 89, 90, b 92, 93, 94, 95, 96, 97, 8, 99 or 100 ' each times ' 1, 10, 10 10910*, 10 s , ⁇ ,W ⁇ W ⁇ W
• dose eomalos about, at least or m re man 1-20, 2-19, 3-18, 4-17, 5- 16, 6- 15, 7-14, 8 - 13, 9-12 or 10-11 each times 10 6 stem cells.
• die addition of the NM U antagonist in the inventive compositions allow (l e cran 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 st m cells lacking the NM II antagonist in certain embodiments, the addition of the NM II 5 antagonist in the inventive compositioos allow the clinician to provide a.
• kits including cells, NM ⁇ antagonists, pharmaceutically acceptable carriers, and - optionally appropriate instructions, ar 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 aon-muscie myosin II antagonist prior to the treatment.
• die nommnse!e m osin II antagonist is b!ehbistatin.
• the methods of treating can generally involve intramuscular injection of a dose of a composition of MSCs treated with an NM 11 antagonist saeli as- blebbisiatln at or near tlte site of the lesions arising from CVD such as PAD.
• Such injections generally: result in an increase in limb f e ion, blood flow, and ischemic symptoms.
• the use of an 25 NM 11 antagonist snch as blebbistatin In the composition dramatically accelerates MSC- triggered regeneration of damaged tissues.
• application of ' b!ehbistatsu with MSC 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 no be drug e!utingj. stents may be loaded with MSCs which ave been contacted with NM I! ant gonists prior so or durin implantation.
• additional cardiovascular drugs or medical devices such as for example stents (which may or may no be drug e!utingj. stents may be loaded with MSCs which ave been contacted with NM I! ant gonists prior so or durin implantation.
• Peripheral artery d sease is a form of peripheral vesicular diseases i ' PVO) in
• Temperatures range was 20 ⁇ 243 ⁇ 4 and RH range was 30-70% with 12 hotsrs0 light and 1 2 hoar dark cycle, Species/Strain; Mouse/Achyinie Mode; Gender ⁇ r1 ⁇ 2mb «r/Age: Male/ 152/9- 10 weeks; Source: Harlan Laboratories, Israel; Body wei h The average body weight vv&s 28.4 g at study initiation (day l)» The minimum and maximum, weights of the roup were withm a range ⁇ ' ⁇ 20 % of group meau weight; Acclimation period: 5- 10 days; Identification: Three position ear notching and cage cards.
• 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.
• intramuscular injection the animals were injected St) pi at each site, total 100 ⁇ per animal,0
• intravenous Injection the animals were injected hy 200 ul per animal See Table or Groups .Allocation,
• Body weight was measure on. study day - 1 pior to the surgery anil once weekly thereafter.
• Blood flo easurement Stood flow in. legs 1mm fcoih sides was measured with a son- costacl Laser Doppier before su*ge ' ry and on ⁇ J «ys: 1. 3, ?, 14, 21, 28 and 35 post operation, Blood flow oeasuremep * ere expressed as fee ratio of the flow ia the schemic limb to thai io the normal limb (Goto et al Tokai J Exp Glim Med., Vol, 31, No. 3, pp, 128-132, 2006).
• Limb function is graded as "No applicable” in case of partial or mil. limb aroputa om la such case blood flow measurements was net inoiu ed in th statistical analysis,
• Slope The slope compute h of ire linear trend from surgery to Day 35. Tins single number tor each animal represents its change on an eudpoini over time. Distribution of slopes is provided for each group, on each, of the eadpoints (excluding Weight), using Box Plots, These plots provide a c nvenient 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 m the bottom of the box-— ercentiles 0 to 25; 50% of data points a? and. to of the ox percentiles- 25 to ?5. This is a s termed t e "imei-quartile range " (IQR),
• Blood Flow 3 ⁇ 4 ⁇ : Value of the first measurement alter the surgery (assumpuon is that rreaiment failed so thai tfeers s «o .iasprovemaii beyond wha as observed iannediately aiier xorgery); ' Iseliemis Severity Score; 4 : "'' Ota! Hin imh Loss" (the worst sc re possible); Li n ' b Facti n; 3 -"Draggin of foof " (the worst score possible); Weight ⁇ g): The animal's lowest weight at any of th . points ineasured up to and including the day of amputation.
• Gro p 4 (BM.MSC 1» DMEMt 88 (im)) has the highest n s»d is Far ahead of the 2nd most eff ctive group (5): Average Rank ⁇ L2, difference from next, group is 2.
• Group 5 s far behind Group 4 (as indicated in the ballet above) hut also ahead of the ou s 1 S behind it. Specifically, it is 1 ,2 rarrks ahead of the 3rd most effective group (9).
• Stem cell therapy is « pn> ising approach in cardiovascular oiedieme.
• the mome hind limb ischemia m del 5s used. It is disclosed herein that thai MSCs showed regenerative potential m m animal moos m del whereas the substance B!ebblstatin. a ac had no beneficial effect Tins validated the model in these e periments, a single IV or local admi stmiion .to the ischemic limb of the tested ceils surprisingl and nnsxpected, restorer! blood perfusion u to 65-14% of its normal values.
• Group 4 (BM.MSC in DMEM -l-BB (im) is more effective than ail groups on % Blood Flow and Limb Function, including group 5 BM.MSC iu DMEM, pre-BB (im).

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