Patent Application: US-201715490778-A

Abstract:
disclosed are compositions of matter , protocols , and methods of treatment of neurological manifestations using stem cells , stem cell stimulators , and combination treatments . in one embodiment , a patient suffering neurological manifestations of a viral infection is administered a therapeutically active dose of mesenchymal stem cells at a frequency and concentration sufficient to induce amelioration , remission or cure of neurological manifestations .

Description:
the current invention discloses compositions of matters , protocols , and cells useful for the treatment of neurological manifestations of zika virus . in one embodiment the invention provides means of utilizing mesenchymal stem cells for treatment of neurological manifestations of zika virus . in one embodiment , msc are generated according to protocols previously utilized for treatment of patients utilizing bone marrow derived msc . specifically , bone marrow is aspirated ( 10 - 30 ml ) under local anesthesia ( with or without sedation ) from the posterior iliac crest , collected into sodium heparin containing tubes and transferred to a good manufacturing practices ( gmp ) clean room . bone marrow cells are washed with a washing solution such as dulbecco &# 39 ; s phosphate - buffered saline ( dpbs ), rpmi , or pbs supplemented with autologous patient plasma and layered on to 25 ml of percoll ( 1 . 073 g / ml ) at a concentration of approximately 1 - 2 ′ 10 7 cells / ml . subsequently the cells are centrifuged at 900 g for approximately 30 min or a time period sufficient to achieve separation of mononuclear cells from debris and erythrocytes . said cells are then washed with pbs and plated at a density of approximately 1 ′ 10 6 cells per ml in 175 cm 2 tissue culture flasks in dmem with 10 % fcs with flasks subsequently being loaded with a minimum of 30 million bone marrow mononuclear cells . the mscs are allowed to adhere for 72 h followed by media changes every 3 - 4 days . adherent cells are removed with 0 . 05 % trypsin - edta and replated at a density of 1 ′ 10 6 per 175 cm 2 . said bone marrow msc may be administered intravenously , or in a preferred embodiment , intrathecally in a patient suffering from zika virus neurological manifestations . although doses may be determined by one of skill in the art , and are dependent on various patient characteristics , intravenous administration may be performed at concentrations ranging from 1 - 10 million msc per kilogram , with a preferred dose of approximately 2 - 5 million cells per kilogram . in some embodiments of the invention msc are transferred to possess enhanced neuromodulatory and neuroprotective properties . said transfection may be accomplished by use of lentiviral vectors , said means to perform lentiviral mediated transfection are well - known in the art and discussed in the following references [ 1 - 7 ]. some specific examples of lentiviral based transfection of genes into msc include transfection of sdf - 1 to promote stem cell homing , particularly hematopoietic stem cells [ 8 ], fgf - 18 to promote osteogenic differentiation [ 9 ], gdnf to treat parkinson &# 39 ; s in an animal model [ 10 ], hgf to accelerate remyelination in a brain injury model [ 11 ], akt to protect against pathological cardiac remodeling and cardiomyocyte death [ 12 ], trail to induce apoptosis of tumor cells [ 13 - 16 ], pge - 1 synthase for cardioprotection [ 17 ], nur77 to enhance migration [ 18 ], bdnf to reduce ocular nerve damage in response to hypertension [ 19 ], hif - 1 alpha to stimulate osteogenesis [ 20 ], dominant negative ccl2 to reduce lung fibrosis [ 21 ], interferon beta to reduce tumor progression [ 22 ], hla - g to enhance immune suppressive activity [ 23 ], htert to induce differentiation along the hepatocyte lineage [ 24 ], cytosine deaminase [ 25 ], oct - 4 to reduce senescence [ 26 , 27 ], bambi to reduce tgf expression and protumor effects [ 28 ], ho - 1 for cardioprotection [ 29 ], light to induce antitumor activity [ 30 ], mir - 126 to enhance angiogenesis [ 31 , 32 ], bcl - 2 to induce generation of nucleus pulposus cells [ 33 ], telomerase and myocardin to induce cardiogenesis [ 34 ], cxcr4 to accelerate hematopoietic recovery [ 35 ] and reduce renal allograft rejection [ 36 ], wnt11to promote chondrogenesis [ 37 ], islet - 1 to promote pancreatic differentiation [ 38 ], il - 27 to reduce autoimmune disease [ 39 ], ace - 2 to reduce sepsis [ 40 ], cxcr4 to reduce liver failure [ 41 ], and lung injury [ 42 ], and the hgf antagonist nk4 to reduce cancer [ 43 ]. cell cultures are tested for sterility weekly , endotoxin by limulus amebocyte lysate test , and mycoplasma by dna - fluorochrome stain . in order to determine the quality of msc cultures , flow cytometry is performed on all cultures for surface expression of sh - 2 , sh - 3 , sh - 4 msc markers and lack of contaminating cd14 - and cd - 45 positive cells . cells were detached with 0 . 05 % trypsin - edta , washed with dpbs + 2 % bovine albumin , fixed in 1 % paraformaldehyde , blocked in 10 % serum , incubated separately with primary sh - 2 , sh - 3 and sh - 4 antibodies followed by pe - conjugated anti - mouse igg ( h + l ) antibody . confluent msc in 175 cm 2 flasks are washed with tyrode &# 39 ; s salt solution , incubated with medium 199 ( m199 ) for 60 min , and detached with 0 . 05 % trypsin - edta ( gibco ). cells from 10 flasks were detached at a time and mscs were resuspended in 40 ml of m199 + 1 % human serum albumin ( hsa ; american red cross , washington d . c ., usa ). mscs harvested from each 10 - flask set were stored for up to 4 h at 4 ° c . and combined at the end of the harvest . a total of 2 - 10 ′ 10 6 msc / kg were resuspended in m199 + 1 % hsa and centrifuged at 460 g for 10 min at 20 ° c . cell pellets were resuspended in fresh m199 + 1 % hsa media and centrifuged at 460 g for 10 min at 20 ° c . for three additional times . total harvest time was 2 - 4 h based on msc yield per flask and the target dose . harvested msc were cryopreserved in cryocyte ( baxter , deerfield , ill ., usa ) freezing bags using a rate controlled freezer at a final concentration of 10 % dmso ( research industries , salt lake city , utah , usa ) and 5 % hsa . on the day of infusion cryopreserved units were thawed at the bedside in a 37 ° c . water bath and transferred into 60 ml syringes within 5 min and infused intravenously into patients over 10 - 15 min . patients are premedicated with 325 - 650 mg acetaminophen and 12 . 5 - 25 mg of diphenhydramine orally . blood pressure , pulse , respiratory rate , temperature and oxygen saturation are monitored at the time of infusion and every 15 min thereafter for 3 h followed by every 2 h for 6 h . in one embodiment of the invention msc are transfected with anti - apoptotic proteins to enhance in vivo longevity . the present invention includes a method of using msc that have been cultured under conditions to express increased amounts of at least one anti - apoptotic protein as a therapy to inhibit or prevent apoptosis . in one embodiment , the msc which are used as a therapy to inhibit or prevent apoptosis have been contacted with an apoptotic cell . the invention is based on the discovery that msc that have been contacted with an apoptotic cell express high levels of anti - apoptotic molecules . in some instances , the msc that have been contacted with an apoptotic cell secrete high levels of at least one anti - apoptotic protein , including but not limited to , stc - 1 , bcl - 2 , xiap , survivin , and bcl - 2xl . methods of transfecting antiapoptotic genes into msc have been previously described which can be applied to the current invention , said antiapoptotic genes that can be utilized for practice of the invention , in a nonlimiting way , include gata - 4 [ 44 ], fgf - 2 [ 45 ], bcl - 2 [ 33 , 46 ], and ho - 1 [ 47 ]. based upon the disclosure provided herein , msc can be obtained from any source . the msc may be autologous with respect to the recipient ( obtained from the same host ) or allogeneic with respect to the recipient . in addition , the msc may be xenogeneic to the recipient ( obtained from an animal of a different species ). in one embodiment of the invention msc are pretreated with agents to induce expression of antiapoptotic genes , one example is pretreatment with exendin - 4 as previously described [ 48 ]. in a further non - limiting embodiment , msc used in the present invention can be isolated , from the bone marrow of any species of mammal , including but not limited to , human , mouse , rat , ape , gibbon , bovine . in a non - limiting embodiment , the msc are isolated from a human , a mouse , or a rat . in another non - limiting embodiment , the msc are isolated from a human . based upon the present disclosure , msc can be isolated and expanded in culture in vitro to obtain sufficient numbers of cells for use in the methods described herein provided that the msc are cultured in a manner that promotes contact with a tumor endothelial cell . for example , msc can be isolated from human bone marrow and cultured in complete medium ( dmem low glucose containing 4 mm l - glutamine , 10 % fbs , and 1 % penicillin / streptomycin ) in hanging drops or on non - adherent dishes . the invention , however , should in no way be construed to be limited to any one method of isolating and / or to any culturing medium . rather , any method of isolating and any culturing medium should be construed to be included in the present invention provided that the msc are cultured in a manner that provides msc to express increased amounts of at least one anti - apoptotic protein . culture conditions for growth of clinical grade msc have been described in the literature and are incorporated by reference [ 49 - 82 ]. any medium capable of supporting msc in vitro may be used to culture the msc . media formulations that can support the growth of msc include , but are not limited to , dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ), alpha modified minimal essential medium (. alpha . mem ), and roswell park memorial institute media 1640 ( rpmi media 1640 ) and the like . said media and conditions for culture of msc - and by virtue of the invention msc are known in the art . typically , up to 20 % fetal bovine serum ( fbs ) or 1 - 20 % horse serum is added to the above medium in order to support the growth of msc . a defined medium , however , also can be used if the growth factors , cytokines , and hormones necessary for culturing msc are provided at appropriate concentrations in the medium . media useful in the methods of the invention may contain one or more compounds of interest , including , but not limited to , antibiotics , mitogenic or differentiation compounds useful for the culturing of msc . the cells may be grown at temperatures between 27 . degree . c . to 40 . degree . c ., preferably 31 . degree . c . to 37 . degree . c ., and more preferably in a humidified incubator . the carbon dioxide content may be maintained between 2 % to 10 % and the oxygen content may be maintained between 1 % and 22 %. the invention , however , should in no way be construed to be limited to any one method of isolating and culturing msc . rather , any method of isolating and culturing msc should be construed to be included in the present invention . antibiotics which can be added into the medium include , but are not limited to , penicillin and streptomycin . the concentration of penicillin in the culture medium , in a non - limiting embodiment , is about 10 to about 200 units per ml . the concentration of streptomycin in the culture medium is , in a non - limiting embodiment , about 10 to about 200 . mu . g / ml . msc which express increased amounts of at least one anti - apoptotic protein may be administered to an animal in an amount effective to provide a therapeutic effect . the animal may be a mammal , including but not limited to , human and non - human primates . the msc can be suspended in an appropriate diluent . suitable excipients for injection solutions are those that are biologically and physiologically compatible with the msc and with the recipient , such as buffered saline solution or other suitable excipients . the composition for administration can be formulated , produced , and stored according to standard methods complying with proper sterility and stability . the msc may have one or more genes modified or be treated such that the modification has the ability to cause the msc to self - destruct or “ commit suicide ” because of such modification , or upon presentation of a second drug ( eg ., a prodrug ) or signaling compound to initiate such destruction of the msc . in one embodiment of the invention , msc are isolated from the subepithelial layer ( sl ) of the umbilical cord . isolated cells from the sl can have a variety of characteristic markers that distinguish them from cell previously isolated from umbilical cord samples . it should be noted that these isolated cells are not derived from the wharton &# 39 ; s jelly , but rather from the sl . various cellular markers that are either present or absent can be utilized in the identification of these sl - derived cells , and as such , can be used to show the novelty of the isolated cells . for example , in one aspect , the isolated cell expresses at least three cell markers selected from cd29 , cd73 , cd90 , cd146 , cd166 , ssea4 , cd9 , cd44 , cd146 , or cd105 , and the isolated cell does not express at least three cell markers selected from cd45 , cd34 , cd14 , cd79 , cd106 , cd86 , cd80 , cd19 , cd117 , stro - 1 , or hla - dr . in another aspect , the isolated cell expresses at least five cell markers selected from cd29 , cd73 , cd90 , cd146 , cd166 , ssea4 , cd9 , cd44 , cd146 , or cd105 . in another aspect , the isolated cell expresses at least eight cell markers selected from cd29 , cd73 , cd90 , cd146 , cd166 , ssea4 , cd9 , cd44 , cd146 , or cd105 . in a yet another aspect , the isolated cell expresses at least cd29 , cd73 , cd90 , cd166 , ssea4 , cd9 , cd44 , cd146 , and cd105 . in another aspect , the isolated cell does not express at least five cell markers selected from cd45 , cd34 , cd14 , cd79 , cd106 , cd86 , cd80 , cd19 , cd117 , stro - 1 , or hla - dr . in another aspect , the isolated cell does not express at least eight cell markers selected from cd45 , cd34 , cd14 , cd79 , cd106 , cd86 , cd80 , cd19 , cd117 , stro - 1 , or hla - dr . in yet another aspect , the isolated cell does not express at least cd45 , cd34 , cd14 , cd79 , cd106 , cd86 , cd80 , cd19 , cd117 , stro - 1 , and hla - dr . additionally , in some aspects , the isolated cell can be positive for sox2 , oct4 , or both sox2 and oct4 . in a further aspect , the isolated cell can produce exosomes expressing cd63 , cd9 , or both cd63 and cd9 . a variety of techniques can be utilized to extract the isolated cells of the present disclosure from the sl , and any such technique that allows such extraction without significant damage to the cells is considered to be within the present scope . in one aspect , for example , a method of culturing stem cells from the sl of a mammalian umbilical cord can include dissecting the subepithelial layer from the umbilical cord . in one aspect , for example , umbilical cord tissue can be collected and washed to remove blood , wharton &# 39 ; s jelly , and any other material associated with the sl . for example , in one non - limiting aspect the cord tissue can be washed multiple times in a solution of phosphate - buffered saline ( pbs ) such as dulbecco &# 39 ; s phosphate - buffered saline ( dpbs ). in some aspects the pbs can include a platelet lysate ( i . e . 10 % prp lysate of platelet lysate ). any remaining wharton &# 39 ; s jelly or gelatinous portion of the umbilical cord can then be removed and discarded . the remaining umbilical cord tissue ( the sl ) can then be placed interior side down on a substrate such that an interior side of the sl is in contact with the substrate . an entire dissected umbilical cord with the wharton &# 39 ; s jelly removed can be placed directly onto the substrate , or the dissected umbilical cord can be cut into smaller sections ( e . g . 1 - 3 mm ) and these sections can be placed directly onto the substrate . a variety of substrates are contemplated upon which the sl can be placed . in one aspect , for example , the substrate can be a solid polymeric material . one example of a solid polymeric material can include a cell culture dish . the cell culture dish can be made of a cell culture treated plastic as is known in the art . in one specific aspect , the sl can be placed upon the substrate of the cell culture dish without any additional pretreatment to the cell culture treated plastic . in another aspect , the substrate can be a semi - solid cell culture substrate . such a substrate can include , for example , a semi - solid culture medium including an agar or other gelatinous base material . following placement of the sl on the substrate , the sl is cultured in a suitable medium . in some aspects it is preferable to utilized culture media that is free of animal and human components or contaminants . as one example , fig2 shows the culturing of cells from the sl . the culture can then be cultured under either normoxic or hypoxic culture conditions for a period of time sufficient to establish primary cell cultures . ( e . g . 3 - 7 days in some cases ). after primary cell cultures have been established , the sl tissue is removed and discarded . cells or stem cells are further cultured and expanded in larger culture flasks in either a normoxic or hypoxic culture conditions . while a variety of suitable cell culture media are contemplated , in one non - limiting example the media can be dulbecco &# 39 ; s modified eagle medium ( dmem ) glucose ( 500 - 6000 mg / ml ) without phenol red , 1 . times . glutamine , 1 . times . neaa , and 0 . 1 - 20 % prp lysate or platelet lysate . another example of suitable media can include a base medium of dmem low glucose without phenol red , 1 . times . glutamine , 1 . times . neaa , 1000 units of heparin and 20 % prp lysate or platelet lysate . in another example , cells can be cultured directly onto a semi - solid substrate of dmem low glucose without phenol red , 1 . times . glutamine , 1 . times . neaa , and 20 % prp lysate or platelet lysate . in a further example , culture media can include a low glucose medium ( 500 - 1000 mg / ml ) containing 1 . times . glutamine , 1 . times . neaa , 1000 units of heparin . in some aspects , the glucose can be 1000 - 4000 mg / ml , and in other aspects the glucose can be high glucose at 4000 - 6000 mg / ml . these media can also include 0 . 1 %- 20 % prp lysate or platelet lysate . in yet a further example , the culture medium can be a semi - solid with the substitution of acid - citrate - dextrose acd in place of heparin , and containing low glucose medium ( 500 - 1000 mg / ml ), intermediate glucose medium ( 1000 - 4000 mg / ml ) or high glucose medium ( 4000 - 6000 mg / ml ), and further containing 1 . times . glutamine , 1 . times . neaa , and 0 . 1 %- 20 % prp lysate or platelet lysate . in some aspects , the cells can be derived , subcultured , and / or passaged using tryple . in another aspect , the cells can be derived , subcultured , and / or passaged without the use of tryple or any other enzyme . in one aspect , sl cells can be cultured from a mammalian umbilical cord in a semi - solid prp lysate or platelet lysate substrate . such cells can be cultured directly onto a plastic coated tissue culture flask as has been described elsewhere herein . after a sufficient time in either normoxic or hypoxic culture environments the media is changed and freshly made semi - solid prp lysate or platelet lysate media is added to the culture flask . the flask is continued to be cultured in either a normoxic or hypoxic culture environment . the following day the media becomes a semi - solid prp - lysate or platelet lysate matrix . the cells can be continued to be cultured in this matrix being until further use . in one specific aspect , ingredients for a semi solid culture can include growth factors for expanded cell culture of differentiation . non - limiting examples can include fgf , vegf , fndc5 , 5 - azacytidine , tgf - beta1 , tgf beta2 , insulin , its , igf , and the like , including combinations thereof . in some cases , allogenic confirmation of sl cells , either differentiated or undifferentiated , can be highly beneficial , particularly for therapeutic uses of the cells . in such cases , mixed lymphocyte reactions can be performed on the cells to confirm the allogenic properties of the cells . in certain aspects , a cell derived as described herein does not cause a mixed lymphocyte response or t - cell proliferation . in certain aspects , a cell derived as described herein can be recombinantly modified to express one or more genes and or proteins . in one technique , a gene or genes can be incorporated into an expression vector . approaches to deliver a gene into the cell can include without limitation , viral vectors , including recombinant retroviruses , adenoviruses , adeno - associated virus , lentivirus , poxivirus , alphavirus , herpes simplex virus - 1 , recombinant bacterial , eukaryotic plasmids , and the like , including combinations thereof . plasmid dna may be delivered naked or with the help of exosomes , cationic liposomes or derivatized ( antibody conjugated ) polylysine conjugates , gramicidin s , artificial viral envelopes , other intracellular carriers , as well as direct injection of the genes . in some aspects , non - viral gene delivery methods can be used , such as for example , scaffold / matrix attached region ( s / mar )- based vector . furthermore , in some aspects , isolated sl cells can be used to produce an exosome population . these exosome populations can be utilized for a variety of research and therapeutic uses . in one aspect , for example , cells are cultured as described in either a normoxic or hypoxic culture environment and supernatants are collected at each media change . exosomes can then be purified from the supernatants using an appropriate purification protocol . one not - limiting example of such a protocol is the exoquick isolation system by systembio . purified exosomes can be utilized for further manipulation , targeting , and therapeutic use . the exosomes specific to the sl cells are positive for cd63 expression . the dosage of the msc varies within wide limits and may be adjusted to the individual requirements in each particular case . the number of cells used depends on the age , weight , sex , and condition of the recipient , the number and / or frequency of administrations , the disease or disorder being treated , and the extent or severity thereof , and other variables known to those of skill in the art . the amniotic fluid - derived stem cells described in this invention are capable of self - renewal in tissue culture , maintain euploidy for & gt ; 1 year in culture , share markers with human es cells , and are capable of differentiating into all three germ layers of the developing embryo , endoderm , mesoderm and ectoderm . in a preferred embodiment the regenerative amniotic fluid cells are found in the amnion harvested during the second trimester of human pregnancies . it is known that amniotic fluid contains multiple morphologically - distinguishable cell types , the majority of the cells are prone to senescence and are lost from cultures . in one embodiment , fibronectin coated plates and culture conditions described in u . s . pat . no . 7 , 569 , 385 are used to grow cells from amniotic fluid harvests from normal 16 - 18 week pregnancies . the cells of the invention are of fetal origin , and have a normal diploid karyotype . growth of the amniotic fluid stem cells as described in the invention for use in neurological ischemic conditions results in cells that are multipotent , as several main cell types have been derived from them . as used herein , the term “ multipotent ” refers to the ability of amniotic fluid regenerative cells to differentiate into several main cell types . the mafsc cells may also be propagated under specific conditions to become “ pluripotent .” the term “ pluripotent stem cells ” describes stem cells that are capable of differentiating into any type of body cell , when cultured under conditions that give rise to the particular cell type . the amniotic fluid regenerative cells are preferably isolated from humans . however , the amniotic fluid regenerative cells may be isolated in a similar manner from other species . examples of species that may be used to derive the amniotic fluid regenerative cells include but are not limited to mammals , humans , primates , dogs , cats , goats , elephants , endangered species , cattle , horses , pigs , mice , rabbits , and the like . the amniotic fluid - derived cells and mafsc can be recognized by their specific cell surface proteins or by the presence of specific cellular proteins . typically , specific cell types have specific cell surface proteins . these surface proteins can be used as “ markers ” to determine or confirm specific cell types . typically , these surface markers can be visualized using antibody - based technology or other detection methods . the surface markers of the isolated mafsc cells derived from independently - harvested amniotic fluid samples were tested for a range of cell surface and other markers , using monoclonal antibodies and facs analysis . these cells can be characterized by the following cell surface markers : ssea3 , ssea4 , tra - 1 - 60 , tra - 1 - 81 , tra - 2 - 54 . the mafsc cells can be distinguished from mouse es cells in that the mafsc cells do not express the cell surface marker ssea1 . additionally , mafsc express the stem cell transcription factor oct - 4 . the mafsc cells can be recognized by the presence of at least one , or at least two , or at least three , or at least four , or at least five , or at least six , or all of the following cellular markers ssea3 , ssea4 , tra - 1 - 60 , tra - 1 - 81 , tra - 2 - 54 and oct - 4 . the mafsc cultures express very little or no ssea - 1 marker . in addition to the embryo stem cell markers ssea3 , ssea4 , tra1 - 60 , tra1 - 81 , tra2 - 54 , oct - 4 the amniotic fluid regenerative cells also expressed high levels of the cell surface antigens that are normally found on human mesenchymal stem cells , but not normally on human embryo stem cells . this set of markers includes cd13 ( 99 . 6 %) aminopeptidase n , cd44 ( 99 . 7 %) hyaluronic acid - binding receptor , cd49b ( 99 . 8 %) collagen / laminin - binding integrin alpha2 , and cd105 ( 97 %) endoglin . the presence of both the embryonic stem cell markers and the hmsc markers on the mafsc cell cultures indicates that amniotic fluid - derived mafsc cells , grown and propagated as described here , represent a novel class of human stem cells that combined the characteristics of hes cells and of hmsc cells . in some embodiments of the invention , at least about 90 %, 94 %, 97 %, 99 %, or 100 % of the cells in the culture express cd13 . in additional embodiments , at least about 90 %, 94 %, 97 %, 99 %, or 100 % of the cells in the culture express cd44 . in some embodiments of the invention , a range from at least about 90 %, 94 %, 97 %, 99 %, 99 . 5 %, or 100 % of the cells in the culture express cd49b . in further embodiments of the invention , a range from at least about 90 %, 94 %, 97 %, 99 %, 99 . 5 %, or 100 % of the cells in the culture express cd105 . in one particular embodiment of the invention , the amniotic fluid regenerative cells are human stem cells that can be propagated for an indefinite period of time in continuous culture in an undifferentiated state . the term “ undifferentiated ” refers to cells that have not become specialized cell types . a “ nutrient medium ” is a medium for culturing cells containing nutrients that promote proliferation . the nutrient medium may contain any of the following in an appropriate combination : isotonic saline , buffer , amino acids , antibiotics , serum or serum replacement , and exogenously added factors . in one embodiment , mesenchymal stem cells used are amniotic fluid stem cells . said amniotic fluid stem cells , or “ regenerative cells ” may be grown in an undifferentiated state for as long as desired ( and optionally stored as described above ), and can then be cultured under certain conditions to allow progression to a differentiated state . by “ differentiation ” is meant the process whereby an unspecialized cell acquires the features of a specialized cell such as a heart , liver , muscle , pancreas or other organ or tissue cell . the amniotic fluid regenerative cells , when cultured under certain conditions , have the ability to differentiate in a regulated manner into three or more subphenotypes . once sufficient cellular mass is achieved , cells can be differentiated into endodermal , mesodermal and ectodermal derived tissues in vitro and in vivo . this planned , specialized differentiation from undifferentiated cells towards a specific cell type or tissue type is termed “ directed differentiation .” exemplary cell types that may be prepared from amniotic fluid regenerative cells using directed differentiation include but are not limited to fat cells , cardiac muscle cells , epithelial cells , liver cells , brain cells , blood cells , neurons , glial cells , pancreatic cells , and the like . general methods relating to stem cell differentiation techniques that may be useful for differentiating the amniotic fluid regenerative cells of this invention can be found in general texts such as : teratocarcinomas and embryonic stem cells : a practical approach ( e . j . robertson , ed ., irl press ltd . 1987 ); guide to techniques in mouse development ( p . m . wasserman et al . eds ., academic press 1993 ); embryonic stem cell differentiation in vitro ( m . v . wiles , meth . enzymol . 225 : 900 , 1993 ); properties and uses of embryonic stem cells : prospects for application to human biology and gene therapy ( p . d . rathjen et al ., reprod . fertil . dev . 10 : 31 , 1998 ); and in stem cell biology ( l . m . reid , curr . opinion cell biol . 2 : 121 , 1990 ), each of which is incorporated by reference herein in its entirety . in one embodiment of the invention , the mafsc are allowed to adhere for 72 h followed by media changes every 3 - 4 days . adherent cells are removed with 0 . 05 % trypsin - edta and replated at a density of 1 ′ 10 6 per 175 cm 2 . said mafsc may be administered intravenously , or in a preferred embodiment , intrathecally in a patient suffering radiation associated neurodegenerative manifestations . although doses may be determined by one of skill in the art , and are dependent on various patient characteristics , intravenous administration may be performed at concentrations ranging from 1 - 10 million msc per kilogram , with a preferred dose of approximately 2 - 5 million cells per kilogram . in some embodiments of the invention mafsc are transfected to possess enhanced neuromodulatory and neuroprotective properties . said transfection may be accomplished by use of lentiviral vectors , said means to perform lentiviral mediated transfection are well - known in the art and discussed in the following references [ 10 - 16 ]. some specific examples of lentiviral based transfection of genes into msc , which by include transfection of sdf - 1 to promote stem cell homing , particularly hematopoietic stem cells [ 17 ], gdnf to treat parkinson &# 39 ; s in an animal model [ 18 ], hgf to accelerate remyelination in a brain injury model [ 19 ], akt to protect against pathological cardiac remodeling and cardiomyocyte death [ 20 ], trail to induce apoptosis of tumor cells [ 21 - 24 ], pge - 1 synthase for cardioprotection [ 25 ], nur77 to enhance migration [ 26 ], bdnf to reduce ocular nerve damage in response to hypertension [ 27 ], hif - 1 alpha to stimulate osteogenesis [ 28 ], dominant negative ccl2 to reduce lung fibrosis [ 29 ], interferon beta to reduce tumor progression [ 30 ], hla - g to enhance immune suppressive activity [ 31 ], htert to induce differentiation along the hepatocyte lineage [ 32 ], cytosine deaminase [ 33 ], oct - 4 to reduce senescence [ 34 , 35 ], bambi to reduce tgf expression and protumor effects [ 36 ], ho - 1 for radioprotection [ 37 ], light to induce antitumor activity [ 38 ], mir - 126 to enhance angiogenesis [ 39 , 40 ], bcl - 2 to induce generation of nucleus pulposus cells [ 41 ], telomerase to induce neurogenesis [ 42 ], cxcr4 to accelerate hematopoietic recovery [ 43 ] and reduce unwanted immunity [ 44 ], wnt11 to promote regenerative cytokine production [ 45 ], and the hgf antagonist nk4 to reduce cancer [ 46 ]. cell cultures are tested for sterility weekly , endotoxin by limulus amebocyte lysate test , and mycoplasma by dna - fluorochrome stain . in order to determine the quality of mafsc cultures , flow cytometry is performed on all cultures for surface expression of sh - 2 , sh - 3 , sh - 4 msc markers and lack of contaminating cd14 - and cd - 45 positive cells . cells were detached with 0 . 05 % trypsin - edta , washed with dpbs + 2 % bovine albumin , fixed in 1 % paraformaldehyde , blocked in 10 % serum , incubated separately with primary sh - 2 , sh - 3 and sh - 4 antibodies followed by pe - conjugated anti - mouse igg ( h + l ) antibody . confluent msc in 175 cm 2 flasks are washed with tyrode &# 39 ; s salt solution , incubated with medium 199 ( m199 ) for 60 min , and detached with 0 . 05 % trypsin - edta ( gibco ). cells from 10 flasks were detached at a time and mscs were resuspended in 40 ml of m199 + 1 % human serum albumin ( hsa ; american red cross , washington d . c ., usa ). mscs harvested from each 10 - flask set were stored for up to 4 h at 4 ° c . and combined at the end of the harvest . a total of 2 - 10 ′ 10 6 msc / kg were resuspended in m199 + 1 % hsa and centrifuged at 460 g for 10 min at 20 ° c . cell pellets were resuspended in fresh m199 + 1 % hsa media and centrifuged at 460 g for 10 min at 20 ° c . for three additional times . total harvest time was 2 - 4 h based on msc yield per flask and the target dose . harvested msc were cryopreserved in cryocyte ( baxter , deerfield , ill ., usa ) freezing bags using a rate controlled freezer at a final concentration of 10 % dmso ( research industries , salt lake city , utah , usa ) and 5 % hsa . on the day of infusion cryopreserved units were thawed at the bedside in a 37 ° c . water bath and transferred into 60 ml syringes within 5 min and infused intravenously into patients over 10 - 15 min . patients are premedicated with 325 - 650 mg acetaminophen and 12 . 5 - 25 mg of diphenhydramine orally . blood pressure , pulse , respiratory rate , temperature and oxygen saturation are monitored at the time of infusion and every 15 min thereafter for 3 h followed by every 2 h for 6 h . in one embodiment of the invention mafsc are transfected with anti - apoptotic proteins to enhance in vivo longevity . the present invention includes a method of using mafsc that have been cultured under conditions to express increased amounts of at least one anti - apoptotic protein as a therapy to inhibit or prevent apoptosis . in one embodiment , the mafsc which are used as a therapy to inhibit or prevent apoptosis have been contacted with an apoptotic cell . the invention is based on the discovery that mafsc that have been contacted with an apoptotic cell express high levels of anti - apoptotic molecules . in some instances , the mafsc that have been contacted with an apoptotic cell secrete high levels of at least one anti - apoptotic protein , including but not limited to , stc - 1 , bcl - 2 , xiap , survivin , and bcl - 2xl . methods of transfecting antiapoptotic genes into mafsc have been previously described which can be applied to the current invention , said antiapoptotic genes that can be utilized for practice of the invention , in a nonlimiting way , include gata - 4 [ 47 ], fgf - 2 [ 48 ], bcl - 2 [ 41 , 49 ], and ho - 1 [ 50 ]. based upon the disclosure provided herein , mafsc can be obtained from any source . the mafsc may be autologous with respect to the recipient ( obtained from the same host ) or allogeneic with respect to the recipient . in addition , the mafsc may be xenogeneic to the recipient ( obtained from an animal of a different species ). in one embodiment of the invention mafsc are pretreated with agents to induce expression of antiapoptotic genes , one example is pretreatment with exendin - 4 as previously described [ 51 ]. in a further non - limiting embodiment , mafsc used in the present invention can be isolated , from the bone marrow of any species of mammal , including but not limited to , human , mouse , rat , ape , gibbon , bovine . in a non - limiting embodiment , the mafsc are isolated from a human , a mouse , or a rat . in another non - limiting embodiment , the mafsc are isolated from a human . based upon the present disclosure , mafsc can be isolated and expanded in culture in vitro to obtain sufficient numbers of cells for use in the methods described herein provided that the msc are cultured in a manner that promotes contact with a tumor endothelial cell . for example , msc can be isolated from human bone marrow and cultured in complete medium ( dmem low glucose containing 4 mm l - glutamine , 10 % fbs , and 1 % penicillin / streptomycin ) in hanging drops or on non - adherent dishes . the invention , however , should in no way be construed to be limited to any one method of isolating and / or to any culturing medium . rather , any method of isolating and any culturing medium should be construed to be included in the present invention provided that the mafsc are cultured in a manner that provides mafsc to express increased amounts of at least one anti - apoptotic protein . culture conditions for growth of clinical grade msc , which under the scope of the invention mafsc fall under , have been described in the literature and are incorporated by reference [ 52 - 85 ]. in one embodiment of the invention mafsc are administered subcutaneously or intramuscularly in order to endow radiation production by secretion of soluble factors . in another embodiment soluble factors are isolated from said mafsc and administered to patients in need of therapy . in another embodiment mafsc are stimulated in vitro to produce soluble factors by treatment with hypoxia . this invention calls for the administration of stem cells or neuronal cells to individuals experiencing the effects of cns neuron loss . the cells can be administered a variety of ways : the cells of the present invention are administered and dosed in accordance with good medical practice , taking into account the clinical condition of the individual patient , the site and method of administration , scheduling of administration , patient age , sex , body weight and other factors known to medical practitioners . the pharmaceutically “ effective amount ” for purposes herein is thus determined by such considerations as are known in the art . the amount must be effective to achieve improvement including , but not limited to , improved cognition , survival rate or more rapid recovery , or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art . in the method of the present invention , the cells of the present invention can be administered in various ways as would be appropriate to implant in the central nervous system . the amniotic fluid stem cells can be administered intracisternally , parenchymally , intravenously , intraventricularly or by other convenient means . various aspects of the invention relating to the above are enumerated in the following paragraphs : aspect 1 . a method of treating neurological manifestations of a viral infection comprising the steps of : a ) obtaining a patient suffering from a viral infection neurological manifestation ; b ) administering a therapeutic dose of stem cells ; c ) assessing effect of said stem cell administration ; and d ) performing additional administrations of said stem cells based on neurological response achieved . aspect 2 . the method of claim 1 , wherein said stem cells are selected from a group comprising of : a ) adipose derived stem cells ; b ) embryonic stem cells ; c ) inducible pluripotent stem cells ; d ) hematopoietic stem cells ; and e ) mesenchymal stem cells . aspect 3 . the method of claim 2 , wherein said mesenchymal stem cell is derived from tissue comprising a group selected from : a ) wharton &# 39 ; s jelly ; b ) bone marrow ; c ) peripheral blood ; d ) mobilized peripheral blood ; e ) endometrium ; f ) hair follicle ; g ) deciduous tooth ; h ) testicle ; i ) adipose tissue ; j ) skin ; k ) amniotic fluid ; l ) cord blood ; m ) omentum ; n ) muscle ; o ) amniotic membrane ; o ) periventricular fluid ; and p ) placental tissue . aspect 4 . the method of claim 3 , wherein said mesenchymal stem cells express a marker or plurality of markers selected from a group comprising of : stro - 1 , cd90 , cd73 , cd105 , cd54 , cd106 , hla - i markers , vimentin , asma , collagen - 1 , fibronectin , lfa - 3 , icam - 1 , pecam - 1 , p - selectin , l - selectin , cd49b / cd29 , cd49c / cd29 , cd49d / cd29 , cd61 , cd18 , cd29 , thrombomodulin , telomerase , cd10 , cd13 , stro - 2 , vcam - 1 , cd146 , and thy - 1 . aspect 5 . the method of claim 4 , wherein said mesenchymal stem cells do not express substantial levels of hla - dr , cd117 , and cd45 . aspect 6 . the method of claim 3 , wherein said mesenchymal stem cells are generated from a pluripotent stem cell . aspect 7 . the method of claim 6 , wherein said pluripotent stem cell is selected from a group comprising of : a ) an embryonic stem cell ; b ) an inducible pluripotent stem cell ; c ) a parthenogenic stem cell ; and d ) a somatic cell nuclear transfer derived stem cell . aspect 8 . the method of claim 7 , wherein said embryonic stem cell population expresses genes selected from a group comprising of : stage - specific embryonic antigens ( ssea ) 3 , ssea 4 , tra - 1 - 60 and tra - 1 - 81 , oct - 3 / 4 , cripto , gastrin - releasing peptide ( grp ) receptor , podocalyxin - like protein ( podxl ), rex - 1 , gctm - 2 , nanog , and human telomerase reverse transcriptase ( htert ). aspect 9 . the method of claim 7 , wherein said inducible pluripotent stem cell possesses markers selected from a group comprising of : cd10 , cd13 , cd44 , cd73 , cd90 , pdgfr - alpha , pd - l2 , and hla - a , b , c and possesses ability to undergo at least 40 doublings in culture , while maintaining a normal karyotype upon passaging . aspect 10 . the method of claim 7 , wherein said parthenogenic stem cells wherein said parthenogenically derived stem cells are generated by addition of a calcium flux inducing agent to activate an oocyte followed by enrichment of cells expressing markers selected from a group comprising of ssea - 4 , tra 1 - 60 and tra 1 - 81 . aspect 11 . the method of claim 7 , wherein said somatic cell nuclear transfer derived stem cells possess a phenotype negative for ssea - 1 and positive for ssea - 3 , ssea - 4 , tra - 1 - 60 , tra - 1 - 81 , and alkaline phosphatase . aspect 12 . the method of claim 6 , wherein said mesenchymal stem cells are differentiated from a pluripotent stem cell source through culture in the presence of an inhibitor of the smad - 2 / 3 pathway . aspect 13 . the method of claim 12 , wherein said mesenchymal stem cells are differentiated from a pluripotent stem cell source through culture in the presence of an inhibitor nucleic acid targeting the smad - 2 / 3 pathway . aspect 14 . the method of claim 13 , wherein said nucleic acid inhibitor is selected from a group comprising of : a ) an antisense oligonucleotide ; b ) a hairpin loop short interfering rna ; c ) a chemically synthesized short interfering rna molecule ; and d ) a hammerhead ribozyme . aspect 15 . the method of claim 13 , wherein said inhibitor of the smad - 2 / 3 pathway is a small molecule inhibitor . aspect 16 . the method of claim 15 , wherein said small molecule inhibitor is sb - 431542 . aspect 17 . the method of claim 6 , wherein a selection process is used to enrich for mesenchymal stem cells differentiated from said pluripotent stem cell population . aspect 18 . the method of claim 17 , wherein said enrichment method comprises of positively selecting for cells expressing a marker associated with mesenchymal stem cells . aspect 19 . the method of claim 18 , wherein said marker of mesenchymal stem cells is selected from a group comprising of : stro - 1 , cd90 , cd73 , cd105 , cd54 , cd106 , hla - i markers , vimentin , asma , collagen - 1 , fibronectin , lfa - 3 , icam - 1 , pecam - 1 , p - selectin , l - selectin , cd49b / cd29 , cd49c / cd29 , cd49d / cd29 , cd61 , cd18 , cd29 , thrombomodulin , telomerase , cd10 , cd13 , stro - 2 , vcam - 1 , cd146 , and thy - 1 . aspect 20 . the method of claim 1 , wherein said immune modulatory cells are autologous , allogeneic or xenogeneic to the recipient . aspect 21 . the method of claim 2 wherein one or more cells are co - administered to said recipient based on specific need for immune modulation in said recipient . aspect 22 . the method of claim 2 , wherein an antigen is administered in combination with immune modulatory cells . aspect 23 . the method of claim 1 , wherein said stem cells is a cell obtained from a subepithelial layer of a mammalian umbilical cord tissue capable of self - renewal and culture expansion ; wherein the isolated cell expresses at least three cell markers selected from the group consisting of cd29 , cd73 , cd90 , cd166 , ssea4 , cd9 , cd44 , cd146 , or cd105 ; and wherein the isolated cell does not express at least three cell markers selected from the group consisting of cd45 , cd34 , cd14 , cd79 , cd106 , cd86 , cd80 , cd19 , cd117 , stro - 1 , or hla - dr . aspect 24 . the method of claim 23 , wherein said isolated cell expresses cd29 , cd73 , cd90 , cd166 , ssea4 , cd9 , cd44 , cd146 , and cd105 . aspect 25 . the method of claim 23 , wherein said isolated cell does not express cd45 , cd34 , cd14 , cd79 , cd106 , cd86 , cd80 , cd19 , cd117 , stro - 1 , and hla - dr . aspect 26 . the method of claim 23 , wherein said isolated cell expresses one or more markers selected from a group comprising of : a ) sox2 ; b ) oct - 4 ; c ) nanog ; and d ) klf - 4 . aspect 27 . the method of claim 1 , wherein said stem cells are treated with neurotrophic factors prior to administration . aspect 28 . the method of claim 27 , wherein said neurotrophic factors are selected from a group comprising of : a ) ngf ; b ) hgf - 1 ; c ) bdnf ; and d ) fgf - 5 . aspect 29 . the method of claim 1 , wherein exosomes are derived from said stem cells and used together , or in substitution of said stem cells . aspect 30 . a method of treating neurological manifestations of a viral infection through reducing microglial production of quinolinic acid through administration of mesenchymal stem cells . aspect 31 . the method of claim 1 , wherein said mesenchymal stem cells are placentally derived and express cd34 upon isolation . 1 . zhang , x . y ., et al ., lentiviral vectors for sustained transgene expression in human bone marrow - 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