Patent Application: US-201515116805-A

Abstract:
the invention disclosed provides populations and compositions of mesenchymal stem and progenitor cells derived from adult tissues that are subjected to a process or processes designed to enhance a biological mechanism prior to implantation of the cells for therapeutic use . the processes involve preconditioning of said mesenchymal stem cells with a physiological stressor in vitro , so as to enhance therapeutically significant biological properties . specific embodiments of the invention include stimulation of angiogenic and immune modulatory properties by preconditioning with interferon - gamma , as well as other physiologically - relevant stressors such as activation of toll like receptors . immunologically - relevant properties of preconditioned cells including the treatment of cytokine storm associated with sepsis , or viral infection . in the angiogenic embodiment of the invention , cells are used for the treatment of peripheral artery disease , its advanced form , critical limb ischemia , or ischemic heart failure .

Description:
the invention provides means of augmenting immune modulatory and angiogenic properties of msc through preconditioning with agents that induce a stress response in msc . previous clinical utilization of msc involves administration of cultured cells . while culture under defined conditions allows for expansion of cells , which in many times are administered without differentiation . while artificially generated msc are usually administered after in vitro culture , their migratory route from administration site to target site for therapeutic activity involves various biological stimuli , as well as locations of anatomical sequestration such as the spleen , liver , or lung . accordingly , the invention provides means to “ prime ” msc in vitro before administration , for augmentation of homing , immune modulatory , and therapeutic activity . “ treat ” or “ treatment ” means improving the symptoms and ameliorating autoimmune , septic , or pulmonary disease . additionally , “ treat ” means improving ischemic conditions . methods for measuring the rate of “ treatment ” efficacy are known in the art and include , for example , assessment of inflammatory cytokines . “ angiogenesis ” means any alteration of an existing vascular bed or the formation of new vasculature which benefits tissue perfusion . this includes the formation of new vessels by sprouting of endothelial cells from existing blood vessels or the remodeling of existing vessels to alter size , maturity , direction or flow properties to improve blood perfusion of tissues . as used herein the terms , “ angiogenesis ,” “ revascularization ,” “ increased collateral circulation ,” and “ regeneration of blood vessels ” are considered as synonymous . “ chronic wound ” means a wound that has not completely closed in twelve weeks since the occurrence of the wound in a patient having a condition , disease or therapy associated with defective healing . conditions , diseases or therapies associated with defective healing include , for example , diabetes , arterial insufficiency , venous insufficiency , chronic steroid use , cancer chemotherapy , radiotherapy , radiation exposure , and malnutrition . a chronic wound includes defects resulting in inflammatory excess ( e . g ., excessive production of interleukin - 6 ( il - 6 ), tumor necrosis factor - alpha ( tnf -. alpha . ), and mmps ), a deficiency of important growth factors needed for proper healing , bacterial overgrowth and senescence of fibroblasts . a chronic wound has an epithelial layer that fails to cover the entire surface of the wound and is subject to bacterial colonization . “ therapeutically effective amount ” means the amount of cells , conditioned media or exosomes that , when administered to a mammal for treating a chronic wound , or angiogenic insufficiency is sufficient to effect such treatment . the “ therapeutically effective amount ” may vary depending on the size of the wound , and the age , weight , physical condition and responsiveness of the mammal to be treated . “ therapeutic agent ” means to have “ therapeutic efficacy ” in modulating angiogenesis and / or wound healing and an amount of the therapeutic is said to be a “ angiogenic modulatory amount ”, if administration of that amount of the therapeutic is sufficient to cause a significant modulation ( i . e ., increase or decrease ) in angiogenic activity when administered to a subject ( e . g ., an animal model or human patient ) needing modulation of angiogenesis . “ growth factor ” can be a naturally occurring , endogenous or exogenous protein , or recombinant protein , capable of stimulating cellular proliferation and / or cellular differentiation and cellular migration . “ about ” or “ approximately ” means within an acceptable range for the particular value as determined by one of ordinary skill in the art , which will depend in part on how the value is measured or determined , e . g ., the limitations of the measurement system . for example , “ about ” can mean a range of up to 20 %, preferably up to 10 %, more preferably up to 5 %, and more preferably still up to 1 % of a given value . alternatively , particularly with respect to biological systems or processes , the term can mean within an order of magnitude , preferably within 5 - fold , and more preferably within 2 - fold , of a value . unless otherwise stated , the term ‘ about ’ means within an acceptable error range for the particular value . “ pharmaceutically acceptable ” refers to a natural or synthetic substance means that the substance has an acceptable toxic effect in view of its much greater beneficial effect , while the related the term , “ physiologically acceptable ,” means the substance has relatively low toxicity . “ endothelial cell mitogen ” means any protein , polypeptide , variant or portion thereof that is capable of , directly or indirectly , inducing endothelial cell growth . such proteins include , for example , acidic and basic fibroblast growth factors ( afgf ) ( genbank accession no . np . sub .- 149127 ) and bfgf ( genbank accession no . aaa52448 ), vascular endothelial growth factor ( vegf ) ( genbank accession no . aaa35789 or np . sub .- 001020539 ), epidermal growth factor ( egf ) ( genbank accession no . np . sub .- 001954 ), transforming growth factor . alpha . ( tgf -. alpha .) ( genbank accession no . np . sub .- 003227 ) and transforming growth factor . beta . ( tfg -. beta .) ( genbank accession no . 1109243a ), platelet - derived endothelial cell growth factor ( pd - ecgf ) ( genbank accession no . np . sub .- 001944 ), platelet - derived growth factor ( pdgf ) ( genbank accession no . 1109245a ), tumor necrosis factor . alpha . ( tnf -. alpha .) ( genbank accession no . caa26669 ), hepatocyte growth factor ( hgf ) ( genbank accession no . baa14348 ), insulin like growth factor ( igf ) ( genbank accession no . p08833 ), erythropoietin ( genbank accession no . p01588 ), colony stimulating factor ( csf ), macrophage - csf ( m - csf ) ( genbank accession no . aab59527 ), granulocyte / macrophage csf ( gm - csf ) ( genbank accession no . np . sub .- 000749 ), monocyte chemotactic protein - 1 ( genbank accession no . p13500 ) and nitric oxide synthase ( nos ) ( genbank accession no . aaa36365 ). see , klagsbrun , et al ., annu . rev . physiol ., 53 : 217 - 239 ( 1991 ); folkman , et al ., j . biol . chem ., 267 : 10931 - 10934 ( 1992 ) and symes , et al ., current opinion in lipidology , 5 : 305 - 312 ( 1994 ). variants or fragments of a mitogen may be used as long as they induce or promote endothelial cell or endothelial progenitor cell growth . preferably , the endothelial cell mitogen contains a secretory signal sequence that facilitates secretion of the protein . proteins having native signal sequences , e . g ., vegf , are preferred . proteins that do not have native signal sequences , e . g ., bfgf , can be modified to contain such sequences using routine genetic manipulation techniques . see , nabel et al ., nature , 362 : 844 ( 1993 ). “ mesenchymal stem cell ” or “ msc ” refers to cells that are ( 1 ) adherent to plastic , ( 2 ) express cd73 , cd90 , and cd105 antigens , while being cd14 , cd34 , cd45 , and hla - dr negative , and ( 3 ) possess ability to differentiate to osteogenic , chondrogenic and adipogenic lineage . as used herein , “ mesenchymal stromal cell ” or “ msc ” can be derived from any tissue including , but not limited to , bone marrow , adipose tissue , amniotic fluid , endometrium , trophoblast - derived tissues , cord blood , wharton jelly , placenta , amniotic tissue , derived from pluripotent stem cells , and tooth . as used herein , “ mesenchymal stromal cell ” or “ msc ” includes cells that are cd34 positive upon initial isolation from tissue but are similar to cells described about phenotypically and functionally . as used herein , “ msc ” includes cells that are isolated from tissues using cell surface markers selected from the list comprised of ngf - r , pdgf - r , egf - r , igf - r , cd29 , cd49a , cd56 , cd63 , cd73 , cd105 , cd106 , cd140b , cd146 , cd271 , msca - 1 , ssea4 , stro - 1 and stro - 3 or any combination thereof , and satisfy the isct criteria either before or after expansion . as used herein , “ mesenchymal stromal cell ” or “ msc ” includes cells described in the literature as bone marrow stromal stem cells ( bmssc ), marrow - isolated adult multipotent inducible cells ( miami ) cells , multipotent adult progenitor cells ( mapc ), mesenchymal adult stem cells ( mascs ), multistem ®, prochymal ®, remestemcel - l , mesenchymal precursor cells ( mpcs ), dental pulp stem cells ( dpscs ), plx cells , plx - pad , allostem ®, astrostem ®, ixmyelocel - t , msc - ntf , nurown ™, stemedyne ™- msc , stempeucel ®, stempeucelcli , stempeuceloa , hiqcell , hearticellgram - ami , revascor ®, cardiorel ®, cartistem ®, pneumostem ®, promostem ®, homeo - gh , ac607 , pda001 , sb623 , cx601 , ac607 , endometrial regenerative cells ( erc ), adipose - derived stem and regenerative cells ( adrcs ). the preferred animal subject of the present invention is a mammal . by the term “ mammal ” is meant an individual belonging to the class mammalia . the invention is particularly useful in the treatment of human subjects . the present invention provides for methods of treatment of acute respiratory distress syndrome and cytokine storm , which methods comprise administering to a subject in need of such treatment a therapeutically effective amount of msc that have been subjected to therapeutic preconditioning , combinations of msc with monocytes , and conditioned media from msc or msc combined with monocytes . in one particular embodiment exosomes are purified from msc or msc combined with monocytes . while it is possible to use a composition provided by the present invention for therapy as is , it may be preferable to administer it in a pharmaceutical formulation , e . g ., in admixture with a suitable pharmaceutical excipient , diluent , or carrier selected with regard to the intended route of administration and standard pharmaceutical practice . accordingly , in one aspect , the present invention provides a pharmaceutical composition or formulation comprising at least one active composition , or a pharmaceutically acceptable derivative thereof , in association with a pharmaceutically acceptable excipient , diluent , and / or carrier . the excipient , diluent and / or carrier must be “ acceptable ” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof . in one embodiment , msc are generated from bone marrow mononuclear cells ( bm mnc ). alternatively , other tissue sources can be used including adipose tissue , peripheral blood , amniotic membrane , umbilical cord blood or whartons jelly . the culture method for generating begins with the enrichment of bm mnc from the starting material ( e . g ., tissue ) by removing red blood cells and some of the polynucleated cells using a conventional cell fractionation method . for example , cells are fractionated by using a ficoll .®. density gradient separation . the volume of starting material needed for culture is typically small , for example , 40 to 50 ml , to provide a sufficient quantity of cells to initiate culture . however , any volume of starting material may be used . nucleated cell concentration is then assessed using an automated cell counter , and the enriched fraction of the starting material is inoculated into a biochamber ( cell culture container ). the number of cells inoculated into the biochamber depends on its volume . prior to inoculation , a biochamber is primed with culture medium . illustratively , the medium used in accordance with the invention comprises three basic components . the first component is a media component comprised of imdm , mem , dmem , rpmi 1640 , alpha medium or mccoy &# 39 ; s medium , or an equivalent known culture medium component . the second is a serum component which comprises at least horse serum or human serum and may optionally further comprise fetal calf serum , newborn calf serum , and / or calf serum . optionally , serum free culture mediums known in the art may be used . the third component is a corticosteroid , such as hydrocortisone , cortisone , dexamethasone , solumedrol , or a combination of these , preferably hydrocortisone . the cells and media are then passed through the biochamber at a controlled ramped perfusion schedule during culture process . the cells are cultures for 2 , 4 , 6 , 8 , 10 , 12 , 14 , 16 or more days . preferably , the cells are cultured for 12 days . these cultures are typically carried out at a ph which is roughly physiologic , i . e . 6 . 9 to 7 . 6 . the medium is kept at an oxygen concentration that corresponds to an oxygen - containing atmosphere which contains from 1 to 20 vol . percent oxygen , preferably 3 to 12 vol . percent oxygen . the preferred range of 0 . sub . 2 concentration refers to the concentration of 0 . sub . 2 near the cells , not necessarily at the point of 0 . sub . 2 introduction which may be at the medium surface or through a membrane . standard culture schedules call for medium and serum to be exchanged weekly , either as a single exchange performed weekly or a one - half medium and serum exchange performed twice weekly . preferably , the nutrient medium of the culture is replaced , preferably perfused , either continuously or periodically , at a rate of about 1 ml per ml of culture per about 24 to about 48 hour period , for cells cultured at a density of from 2 . times . 10 . sup . 6 to 1 . times . 10 . sup . 7 cells per ml . for cell densities of from 1 . times . 10 . sup . 4 to 2 . times . 10 . sup . 6 cells per ml the same medium exchange rate may be used . thus , for cell densities of about 10 . sup . 7 cells per ml , the present medium replacement rate may be expressed as 1 ml of medium per 10 . sup . 7 cells per about 24 to about 48 hour period . for cell densities higher than 10 . sup . 7 cells per ml , the medium exchange rate may be increased proportionality to achieve a constant medium and serum flux per cell per unit time . a method for culturing bone marrow cells is described in lundell , et al ., “ clinical scale expansion of cryopreserved small volume whole bone marrow aspirates produces sufficient cells for clinical use ,” j . hematotherapy ( 1999 ) 8 : 115 - 127 ( which is incorporated herein by reference ). bone marrow ( bm ) aspirates are diluted in isotonic buffered saline ( diluent 2 , stephens scientific , riverdale , n . j . ), and nucleated cells are counted using a coulter zm cell counter ( coulter electronics , hialeah , fla .). erythrocytes ( non - nucleated ) are lysed using a manual lyse ( stephens scientific ), and mononuclear cells ( mnc ) are separated by density gradient centrifugation ( ficoll - paque .®. plus , pharmacia biotech , uppsala , sweden ) ( specific gravity 1 . 077 ) at 300 g for 20 min at 25 . degree . c . bm mnc are washed twice with long - term bm culture medium ( ltbmc ) which is iscove &# 39 ; s modified dulbecco &# 39 ; s medium ( imdm ) supplemented with 4 mm l - glutamine 9gibco brl , grand island , n . y . ), 10 % fetal bovine serum ( fbs ), ( bio - whittaker , walkersville , md . ), 10 % horse serum ( gibco brl ), 20 . mu . g / ml vancomycin ( vancocin .®. hcl , lilly , indianapolis , ind . ), 5 gentamicin ( fujisawa usa , inc ., deerfield , ill . ), and 5 . mu . m hydrocortisone ( solu - cortef .®, upjohn , kalamazoo , mich .) before culture . reduction in endotoxin induced mortality by administration of ifn - gamma treated wharton &# 39 ; s jelly msc human umbilical cords were obtained from full term women immediately after delivery . after thorough washing with phosphate buffered saline containing penicillin and streptomycin , the cord was cut open and the arteries and veins were removed . the tissue was treated with collagenease blend at a net concentration of 500 μg / ml for 20 hr . post - digestion with collagenease the cells were filtered through a 100μ cell strainer to remove tissue debris . cells were then washed with pbs and cultured in ko - dmem containing 10 % fbs , 2 mm l - glutamine and antibiotics . wjscs were grown till confluence and cells were used at passage 8 for experimentation . to stimulate a stress response , msc were cultured for 48 hours in 150 iu / ml of interferon gamma ( ifn - msc ) or in control dmem media ( control - msc ). female balb / c mice were treated with 20 mg / kg of lps diluted in 100 μl 0 . 9 % saline solution , administered by intraperitoneal ( ip ) injection in a single dose . all groups consisted of 10 mice per group . group 1 was control , which received lps alone . group 2 received msc , 500 , 000 cells per mouse , administered intravenously . group 3 received ifn - 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