WO2014013029A1 - Use of preparations comprising exosomes derived from mesenchymal stem cells (mscs) in the prevention and therapy of inflammatory conditions - Google Patents
Use of preparations comprising exosomes derived from mesenchymal stem cells (mscs) in the prevention and therapy of inflammatory conditions Download PDFInfo
- Publication number
- WO2014013029A1 WO2014013029A1 PCT/EP2013/065219 EP2013065219W WO2014013029A1 WO 2014013029 A1 WO2014013029 A1 WO 2014013029A1 EP 2013065219 W EP2013065219 W EP 2013065219W WO 2014013029 A1 WO2014013029 A1 WO 2014013029A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- exosomes
- mscs
- cells
- pharmaceutical preparation
- exosome
- Prior art date
Links
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 133
- 238000002360 preparation method Methods 0.000 title claims abstract description 41
- 230000002265 prevention Effects 0.000 title claims abstract description 13
- 210000001808 exosome Anatomy 0.000 title claims description 182
- 238000002560 therapeutic procedure Methods 0.000 title abstract description 22
- 230000004968 inflammatory condition Effects 0.000 title abstract description 5
- 208000009329 Graft vs Host Disease Diseases 0.000 claims abstract description 45
- 208000024908 graft versus host disease Diseases 0.000 claims abstract description 45
- 230000006378 damage Effects 0.000 claims abstract description 9
- 210000004027 cell Anatomy 0.000 claims description 71
- 230000000694 effects Effects 0.000 claims description 37
- 230000002519 immonomodulatory effect Effects 0.000 claims description 36
- 238000011282 treatment Methods 0.000 claims description 35
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 33
- 210000001519 tissue Anatomy 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 29
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 27
- 102000004127 Cytokines Human genes 0.000 claims description 26
- 108090000695 Cytokines Proteins 0.000 claims description 26
- 108010074328 Interferon-gamma Proteins 0.000 claims description 25
- 201000010099 disease Diseases 0.000 claims description 25
- 102100040247 Tumor necrosis factor Human genes 0.000 claims description 24
- 238000000338 in vitro Methods 0.000 claims description 23
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 21
- 239000012636 effector Substances 0.000 claims description 20
- 230000004044 response Effects 0.000 claims description 20
- 239000002245 particle Substances 0.000 claims description 19
- 230000003110 anti-inflammatory effect Effects 0.000 claims description 18
- -1 T-cell proliferation Proteins 0.000 claims description 17
- 208000006011 Stroke Diseases 0.000 claims description 15
- 230000004054 inflammatory process Effects 0.000 claims description 15
- 206010061218 Inflammation Diseases 0.000 claims description 14
- 102100037850 Interferon gamma Human genes 0.000 claims description 13
- 230000006028 immune-suppresssive effect Effects 0.000 claims description 13
- 239000006166 lysate Substances 0.000 claims description 13
- 238000001990 intravenous administration Methods 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 208000028867 ischemia Diseases 0.000 claims description 11
- 229920001223 polyethylene glycol Polymers 0.000 claims description 11
- 238000001556 precipitation Methods 0.000 claims description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 101000958041 Homo sapiens Musculin Proteins 0.000 claims description 10
- 102000046949 human MSC Human genes 0.000 claims description 10
- 238000001802 infusion Methods 0.000 claims description 10
- 206010052779 Transplant rejections Diseases 0.000 claims description 9
- 239000006143 cell culture medium Substances 0.000 claims description 9
- 210000004700 fetal blood Anatomy 0.000 claims description 9
- 102000003814 Interleukin-10 Human genes 0.000 claims description 8
- 108090000174 Interleukin-10 Proteins 0.000 claims description 8
- 208000029028 brain injury Diseases 0.000 claims description 8
- 210000000056 organ Anatomy 0.000 claims description 8
- 239000002202 Polyethylene glycol Substances 0.000 claims description 7
- 230000001537 neural effect Effects 0.000 claims description 7
- 102100028967 HLA class I histocompatibility antigen, alpha chain G Human genes 0.000 claims description 6
- 108010024164 HLA-G Antigens Proteins 0.000 claims description 6
- 206010021143 Hypoxia Diseases 0.000 claims description 6
- 230000006931 brain damage Effects 0.000 claims description 6
- 231100000874 brain damage Toxicity 0.000 claims description 6
- 230000007954 hypoxia Effects 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 108090001007 Interleukin-8 Proteins 0.000 claims description 5
- 210000000577 adipose tissue Anatomy 0.000 claims description 5
- 238000010322 bone marrow transplantation Methods 0.000 claims description 5
- 239000003937 drug carrier Substances 0.000 claims description 5
- 208000027866 inflammatory disease Diseases 0.000 claims description 5
- 210000000653 nervous system Anatomy 0.000 claims description 5
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 5
- 210000005059 placental tissue Anatomy 0.000 claims description 5
- 230000036470 plasma concentration Effects 0.000 claims description 5
- 210000003954 umbilical cord Anatomy 0.000 claims description 5
- 230000006052 T cell proliferation Effects 0.000 claims description 4
- 210000000988 bone and bone Anatomy 0.000 claims description 4
- 230000001413 cellular effect Effects 0.000 claims description 4
- 229940124531 pharmaceutical excipient Drugs 0.000 claims description 3
- 239000003181 biological factor Substances 0.000 claims description 2
- 108020004707 nucleic acids Proteins 0.000 claims description 2
- 102000039446 nucleic acids Human genes 0.000 claims description 2
- 150000007523 nucleic acids Chemical class 0.000 claims description 2
- 102000004890 Interleukin-8 Human genes 0.000 claims 1
- 108700011259 MicroRNAs Proteins 0.000 claims 1
- 210000004556 brain Anatomy 0.000 abstract description 4
- 230000003961 neuronal insult Effects 0.000 abstract description 3
- 238000011476 stem cell transplantation Methods 0.000 abstract description 3
- 241001465754 Metazoa Species 0.000 description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 17
- 210000001744 T-lymphocyte Anatomy 0.000 description 17
- 238000012360 testing method Methods 0.000 description 16
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 15
- 238000002474 experimental method Methods 0.000 description 12
- 230000000770 proinflammatory effect Effects 0.000 description 12
- 210000000130 stem cell Anatomy 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 10
- 210000001772 blood platelet Anatomy 0.000 description 10
- 239000003636 conditioned culture medium Substances 0.000 description 10
- 108010047620 Phytohemagglutinins Proteins 0.000 description 9
- 230000035755 proliferation Effects 0.000 description 9
- 208000024891 symptom Diseases 0.000 description 9
- 238000002054 transplantation Methods 0.000 description 9
- 210000001185 bone marrow Anatomy 0.000 description 8
- 210000002865 immune cell Anatomy 0.000 description 8
- 230000001885 phytohemagglutinin Effects 0.000 description 8
- 235000019624 protein content Nutrition 0.000 description 8
- 230000001225 therapeutic effect Effects 0.000 description 8
- 230000006907 apoptotic process Effects 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 6
- 238000011510 Elispot assay Methods 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 239000002158 endotoxin Substances 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 230000000735 allogeneic effect Effects 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 230000004069 differentiation Effects 0.000 description 5
- 230000001506 immunosuppresive effect Effects 0.000 description 5
- 230000001939 inductive effect Effects 0.000 description 5
- 229920006008 lipopolysaccharide Polymers 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 210000000822 natural killer cell Anatomy 0.000 description 5
- 210000002536 stromal cell Anatomy 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 206010012735 Diarrhoea Diseases 0.000 description 4
- 102100028970 HLA class I histocompatibility antigen, alpha chain E Human genes 0.000 description 4
- 101000986085 Homo sapiens HLA class I histocompatibility antigen, alpha chain E Proteins 0.000 description 4
- 101000635799 Homo sapiens Run domain Beclin-1-interacting and cysteine-rich domain-containing protein Proteins 0.000 description 4
- 241000700159 Rattus Species 0.000 description 4
- 102100030852 Run domain Beclin-1-interacting and cysteine-rich domain-containing protein Human genes 0.000 description 4
- 230000001154 acute effect Effects 0.000 description 4
- 210000003719 b-lymphocyte Anatomy 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 230000001172 regenerating effect Effects 0.000 description 4
- 150000003431 steroids Chemical class 0.000 description 4
- 108010004141 HLA-B35 Antigen Proteins 0.000 description 3
- 206010061216 Infarction Diseases 0.000 description 3
- 108090000176 Interleukin-13 Proteins 0.000 description 3
- 102000003816 Interleukin-13 Human genes 0.000 description 3
- 108010002350 Interleukin-2 Proteins 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 230000002490 cerebral effect Effects 0.000 description 3
- 238000000432 density-gradient centrifugation Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000007574 infarction Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 230000035992 intercellular communication Effects 0.000 description 3
- 210000005087 mononuclear cell Anatomy 0.000 description 3
- RTGDFNSFWBGLEC-SYZQJQIISA-N mycophenolate mofetil Chemical compound COC1=C(C)C=2COC(=O)C=2C(O)=C1C\C=C(/C)CCC(=O)OCCN1CCOCC1 RTGDFNSFWBGLEC-SYZQJQIISA-N 0.000 description 3
- 229960004866 mycophenolate mofetil Drugs 0.000 description 3
- 208000010125 myocardial infarction Diseases 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 238000011046 pyrogen test Methods 0.000 description 3
- 210000003289 regulatory T cell Anatomy 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 230000001018 virulence Effects 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 2
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 2
- 102100027221 CD81 antigen Human genes 0.000 description 2
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 229920001917 Ficoll Polymers 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- 102000053171 Glial Fibrillary Acidic Human genes 0.000 description 2
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 2
- 108010061486 HLA-B27 Antigen Proteins 0.000 description 2
- 102000012153 HLA-B27 Antigen Human genes 0.000 description 2
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 description 2
- 101001092197 Homo sapiens RNA binding protein fox-1 homolog 3 Proteins 0.000 description 2
- 206010062016 Immunosuppression Diseases 0.000 description 2
- 108090000978 Interleukin-4 Proteins 0.000 description 2
- 102000004388 Interleukin-4 Human genes 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- 101150093388 MSC3 gene Proteins 0.000 description 2
- 201000003793 Myelodysplastic syndrome Diseases 0.000 description 2
- 239000008118 PEG 6000 Substances 0.000 description 2
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 2
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 2
- 102100035530 RNA binding protein fox-1 homolog 3 Human genes 0.000 description 2
- 101100078001 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) MSC2 gene Proteins 0.000 description 2
- QJJXYPPXXYFBGM-LFZNUXCKSA-N Tacrolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1\C=C(/C)[C@@H]1[C@H](C)[C@@H](O)CC(=O)[C@H](CC=C)/C=C(C)/C[C@H](C)C[C@H](OC)[C@H]([C@H](C[C@H]2C)OC)O[C@@]2(O)C(=O)C(=O)N2CCCC[C@H]2C(=O)O1 QJJXYPPXXYFBGM-LFZNUXCKSA-N 0.000 description 2
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 2
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 2
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 description 2
- 206010047700 Vomiting Diseases 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 230000002293 adipogenic effect Effects 0.000 description 2
- 210000004504 adult stem cell Anatomy 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 230000033115 angiogenesis Effects 0.000 description 2
- 230000003376 axonal effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000003750 conditioning effect Effects 0.000 description 2
- 229960004397 cyclophosphamide Drugs 0.000 description 2
- 230000006735 deficit Effects 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 230000003511 endothelial effect Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 2
- 239000003018 immunosuppressive agent Substances 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 230000000302 ischemic effect Effects 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 239000006249 magnetic particle Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 108091070501 miRNA Proteins 0.000 description 2
- 238000012009 microbiological test Methods 0.000 description 2
- 239000011859 microparticle Substances 0.000 description 2
- 238000007799 mixed lymphocyte reaction assay Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 101150117600 msc1 gene Proteins 0.000 description 2
- 208000031225 myocardial ischemia Diseases 0.000 description 2
- 238000001543 one-way ANOVA Methods 0.000 description 2
- 230000002188 osteogenic effect Effects 0.000 description 2
- 230000003076 paracrine Effects 0.000 description 2
- 230000007414 peripheral immune response Effects 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 230000036573 scar formation Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 229960001967 tacrolimus Drugs 0.000 description 2
- QJJXYPPXXYFBGM-SHYZHZOCSA-N tacrolimus Natural products CO[C@H]1C[C@H](CC[C@@H]1O)C=C(C)[C@H]2OC(=O)[C@H]3CCCCN3C(=O)C(=O)[C@@]4(O)O[C@@H]([C@H](C[C@H]4C)OC)[C@@H](C[C@H](C)CC(=C[C@@H](CC=C)C(=O)C[C@H](O)[C@H]2C)C)OC QJJXYPPXXYFBGM-SHYZHZOCSA-N 0.000 description 2
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 2
- 238000005199 ultracentrifugation Methods 0.000 description 2
- 230000004584 weight gain Effects 0.000 description 2
- 235000019786 weight gain Nutrition 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000012438 2',3'-Cyclic-Nucleotide Phosphodiesterases Human genes 0.000 description 1
- 108010022794 2',3'-Cyclic-Nucleotide Phosphodiesterases Proteins 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 102100022464 5'-nucleotidase Human genes 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- WOVKYSAHUYNSMH-UHFFFAOYSA-N BROMODEOXYURIDINE Natural products C1C(O)C(CO)OC1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-UHFFFAOYSA-N 0.000 description 1
- COVZYZSDYWQREU-UHFFFAOYSA-N Busulfan Chemical compound CS(=O)(=O)OCCCCOS(C)(=O)=O COVZYZSDYWQREU-UHFFFAOYSA-N 0.000 description 1
- 102100032912 CD44 antigen Human genes 0.000 description 1
- 102100025222 CD63 antigen Human genes 0.000 description 1
- 102100027217 CD82 antigen Human genes 0.000 description 1
- 241001459819 Carassius gibelio Species 0.000 description 1
- 102000003952 Caspase 3 Human genes 0.000 description 1
- 108090000397 Caspase 3 Proteins 0.000 description 1
- 206010008089 Cerebral artery occlusion Diseases 0.000 description 1
- 206010068051 Chimerism Diseases 0.000 description 1
- VWFCHDSQECPREK-LURJTMIESA-N Cidofovir Chemical compound NC=1C=CN(C[C@@H](CO)OCP(O)(O)=O)C(=O)N=1 VWFCHDSQECPREK-LURJTMIESA-N 0.000 description 1
- 102100033215 DNA nucleotidylexotransferase Human genes 0.000 description 1
- 101150061941 Dcx gene Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 206010015150 Erythema Diseases 0.000 description 1
- 108010039471 Fas Ligand Protein Proteins 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 1
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 1
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 description 1
- 101000914469 Homo sapiens CD82 antigen Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 1
- 241000701027 Human herpesvirus 6 Species 0.000 description 1
- 208000032382 Ischaemic stroke Diseases 0.000 description 1
- 206010025538 Malignant ascites Diseases 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 208000007201 Myocardial reperfusion injury Diseases 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000036110 Neuroinflammatory disease Diseases 0.000 description 1
- 101150092610 PKH2 gene Proteins 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 206010038270 Refractory anaemia with an excess of blasts Diseases 0.000 description 1
- 208000033501 Refractory anemia with excess blasts Diseases 0.000 description 1
- 208000032411 Refractory with Excess of Blasts Anemia Diseases 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- 241000725643 Respiratory syncytial virus Species 0.000 description 1
- 208000031709 Skin Manifestations Diseases 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 108050002568 Tumor necrosis factor ligand superfamily member 6 Proteins 0.000 description 1
- 101800000859 Tumor necrosis factor ligand superfamily member 6, soluble form Proteins 0.000 description 1
- 102400000084 Tumor necrosis factor ligand superfamily member 6, soluble form Human genes 0.000 description 1
- 102100032807 Tumor necrosis factor-inducible gene 6 protein Human genes 0.000 description 1
- 101710169430 Tumor necrosis factor-inducible gene 6 protein Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- ZHAFUINZIZIXFC-UHFFFAOYSA-N [9-(dimethylamino)-10-methylbenzo[a]phenoxazin-5-ylidene]azanium;chloride Chemical compound [Cl-].O1C2=CC(=[NH2+])C3=CC=CC=C3C2=NC2=C1C=C(N(C)C)C(C)=C2 ZHAFUINZIZIXFC-UHFFFAOYSA-N 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 208000024340 acute graft versus host disease Diseases 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- 206010000891 acute myocardial infarction Diseases 0.000 description 1
- 238000011316 allogeneic transplantation Methods 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 239000002269 analeptic agent Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 230000001494 anti-thymocyte effect Effects 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 230000003140 astrocytic effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000012867 bioactive agent Substances 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 208000002352 blister Diseases 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 229950004398 broxuridine Drugs 0.000 description 1
- 229960002092 busulfan Drugs 0.000 description 1
- 230000005961 cardioprotection Effects 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- CZPLANDPABRVHX-UHFFFAOYSA-N cascade blue Chemical compound C=1C2=CC=CC=C2C(NCC)=CC=1C(C=1C=CC(=CC=1)N(CC)CC)=C1C=CC(=[N+](CC)CC)C=C1 CZPLANDPABRVHX-UHFFFAOYSA-N 0.000 description 1
- 230000023402 cell communication Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 210000002583 cell-derived microparticle Anatomy 0.000 description 1
- 230000008614 cellular interaction Effects 0.000 description 1
- 210000003710 cerebral cortex Anatomy 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000002648 chondrogenic effect Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 229960000724 cidofovir Drugs 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 102000003675 cytokine receptors Human genes 0.000 description 1
- 108010057085 cytokine receptors Proteins 0.000 description 1
- 238000003568 cytokine secretion assay Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000012137 double-staining Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000001378 electrochemiluminescence detection Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 238000001839 endoscopy Methods 0.000 description 1
- 231100000321 erythema Toxicity 0.000 description 1
- 230000028023 exocytosis Effects 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 102000010660 flotillin Human genes 0.000 description 1
- 108060000864 flotillin Proteins 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 210000004744 fore-foot Anatomy 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 230000003779 hair growth Effects 0.000 description 1
- 208000014951 hematologic disease Diseases 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 238000010562 histological examination Methods 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 230000008102 immune modulation Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000037189 immune system physiology Effects 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 229940125721 immunosuppressive agent Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 229960000598 infliximab Drugs 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 208000020442 loss of weight Diseases 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000000207 lymphocyte subset Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 description 1
- 229960001924 melphalan Drugs 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 210000000274 microglia Anatomy 0.000 description 1
- 201000007309 middle cerebral artery infarction Diseases 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000000337 motor cortex Anatomy 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 210000002487 multivesicular body Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 208000016586 myelodysplastic syndrome with excess blasts Diseases 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 230000031990 negative regulation of inflammatory response Effects 0.000 description 1
- 210000001577 neostriatum Anatomy 0.000 description 1
- 230000004766 neurogenesis Effects 0.000 description 1
- 230000007171 neuropathology Effects 0.000 description 1
- 230000004112 neuroprotection Effects 0.000 description 1
- 230000035771 neuroregeneration Effects 0.000 description 1
- 230000001928 neurorestorative effect Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000009984 peri-natal effect Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 238000011176 pooling Methods 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000009101 premedication Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 210000002804 pyramidal tract Anatomy 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008263 repair mechanism Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000010825 rotarod performance test Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- YEENEYXBHNNNGV-XEHWZWQGSA-M sodium;3-acetamido-5-[acetyl(methyl)amino]-2,4,6-triiodobenzoate;(2r,3r,4s,5s,6r)-2-[(2r,3s,4s,5r)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound [Na+].CC(=O)N(C)C1=C(I)C(NC(C)=O)=C(I)C(C([O-])=O)=C1I.O[C@H]1[C@H](O)[C@@H](CO)O[C@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 YEENEYXBHNNNGV-XEHWZWQGSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012385 systemic delivery Methods 0.000 description 1
- 208000016595 therapy related acute myeloid leukemia and myelodysplastic syndrome Diseases 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 238000012384 transportation and delivery Methods 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Classifications
-
- 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
- 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
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1841—Transforming growth factor [TGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/2053—IL-8
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/2066—IL-10
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
- A61K38/217—IFN-gamma
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- 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
Definitions
- the present invention relates to the use of exosome-preparations derived from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) for the prevention or for therapy of inflammatory conditions, such as, for example, pre- and postnatally acquired damages of the brain (i.e. neuronal damages) or in complications following stem cell transplantation ("graft vs host-disease", GvHD).
- MSCs tissue-derived mesenchymal stem-cells
- HSC Hematopoietic stem cells
- EPC endothelial progenitor cells
- MSC mesenchymal stem cells
- Treg regulatory T cells exerting immune modulatory and pro- regenerative activities by direct cellular action or secretion of pro-regenerative, anti-apoptotic, immunomodulatory factors.
- the suppressive activity of Tregs from umbilical cord blood (UCB) has been attributed to the observed reduced severity of graft versus host disease (GvHD) in UCB transplantation (Tolar J, Hippen KL, Blazar BR. Immune regulatory cells in umbilical cord blood: T regulatory cells and mesenchymal stromal cells. Br J Haematol 2009 October; 147(2):200-6).
- MSCs Mesenchymal stem/stromal cells
- MSCs were successfully applied to treat steroid-refractory GvHD (Le Blanc K, Rasmusson I, Sundberg B, Goth- erstrom C, Hassan M, Uzunel M et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004 May 1 ; 363(9419): 1439- Since then, a number of different studies have addressed the impact of MSCs on GvHD with different outcomes. In most of these studies, the infusion of MSCs appeared to be safe. However, a positive impact on GvHD is still discussed controversially (Baron and Storb, 2011).
- MSCs can be raised from virtually every tissue within the human body, they are a very rare population comprising about 0.001 to 5 % of cells in different tissues (Bieback K. Basic Biology of Mesenchymal Stem Cells. Transfus Med Hemother 2008; 35(3): 151-2).
- Exosomes are naturally occurring small membrane vesicles (80-160 nm) released by a huge variety of cell species. Containing a combination of lipids and proteins as well as RNAs, exosomes participate in intercellular communication processes. They can be isolated from all body fluids including blood plasma, urine, saliva, breast milk, bronchial lavage fluid, cerebral spinal fluid, amniotic fluid and malignant ascites (Ludwig AK, Giebel B. Exosomes: Small vesicles participating in intercellular communication. Int J Biochem Cell Biol 2011 October 19).
- exosomes were discovered in 1983 as small vesicles that are released by exocytosis upon fusion of so called multivesicular bodies with the plasma membrane.
- exosomes were isolated from B lymphocytes, and were demonstrated to exhibit antigen-presenting characteristics. Since then, their role in immune-biological settings has been investigated in an increasing number of studies. It turned out that exosomes derived from mature dendritic cells for example exert immune-stimulatory functions, while tumor-derived exosomes often mediate immune-suppressive functions (Ludwig AK, Giebel B. Exosomes: Small vesicles participating in intercellular communication. Int J Biochem Cell Biol 2011 October 19).
- Timmers et al. (in Timmers L, Lim SK, Arslan F, Armstrong JS, Hoefer IE, Doevendans PA, Piek JJ, El Oakley RM, Choo A, Lee CN, Pasterkamp G, de Kleijn DP. Reduction of myocardial infarct size by human mesenchymal stem cell conditioned medium. Stem Cell Res. 2007 Nov; l(2): 129-37. Epub 2008 Mar 8) describe the use of secretions of human MSCs as a therapeutic option in acute myocardial infarct. These MSCs are raised from human Embryonic Stem Cells (hESCs).
- hESCs human Embryonic Stem Cells
- MVs microvesicles
- exosomes as emerging new biomarkers and potential regulators of inflammation and immunological processes.
- the particles contain proteins and genetic information including DNA and mi- croRNAs that may be of importance for cell/cell communication.
- MV modulation may emerge as a therapeutic approach in organ rejection therapy.
- Horstman et al. (in: Horstman LL, Jy W, Minagar A, Bidot CJ, Jimenez JJ, Alexander JS, Ahn YS. Cell-derived microparticles (MPs) and exosomes in neuro inflammatory disorders.
- MPs Cell-derived microparticles
- Tables include a listing of bioactive agents known to be carried on MP, many of which were heretofore considered strictly soluble, and some of which can be transferred from cell to cell via MP vectors, for example certain cytokine receptors.
- WO 2009/105044 describes a particle secreted by a mesenchymal stem cell and comprising at least one biological property of a mesenchymal stem cell.
- the biological property may comprise a biological activity of a mesenchymal stem cell conditioned medium (MSC-CM) such as cardioprotection or reduction of infarct size.
- the particle may comprise a vesicle or an exo- some.
- the claims encompass exosomes obtainable from human embryonic stem cells derived from human embryos.
- WO 2012/020308 is directed at compositions and methods for the treatment of tissue damage (e.g., acute or chronic) and related diseases, disorders or conditions based on the use of pathfinder cells, extracellular secretomes thereof, and/or pathfinder cell-associated micro R As (miRNAs).
- tissue damage e.g., acute or chronic
- pathfinder cells e.g., extracellular secretomes thereof
- pathfinder cell-associated micro R As miRNAs
- the present invention provides a method for treating inflammation comprising a step of administering a population of cells, or extracellular secretomes thereof, to an individual suffering from a disease, disorder or condition characterized by inflammation of one or more tissues, wherein the cells are originated from an adult tissue and wherein the cells induce an anti-inflammatory response.
- WO 2012/053976 discloses the use of exosomes that are secreted by mesenchymal stem cells in order to promote hair growth and wound healing. These effects are disclosed in connection with pro -inflammatory cytokines and proteases. WO 2012/053976 speculates about an immune-mo dulatory effect of the exosome-preparation.
- WO 2012/020307 discloses the therapeutic use of micro vesicles including exosomes in the treatment of inflammation and lesions of cerebral trauma.
- WO 2012/087241 describes the use of exosomes for the treatment of the diseases as mentioned.
- MSCs Mesenchymal stromal cells
- GvHD severe graft-versus-host disease
- Multicenter randomized studies are ongoing to more precisely assess the impact of MSC infusion on GvHD prevention/treatment, whereas further research is performed in vitro and in animal models with the aims of determining the best way to expand MSCs ex vivo as well as the most efficient dose and schedule of MSCs administration (see Baron and Storb, 2011).
- MSCs can be raised from virtually every tissue within the human body, they are a very rare population comprising about 0.001 to 5 % of cells in different tissues.
- UCB contains MSC at low frequency.
- MSCs derived from human embryonic ESC meet ethical concerns.
- a pharmaceutical preparation comprising exosomes, obtainable by a method comprising the following steps:
- exosomes as a novel, very potent tool in regenerative and in immune-mo dulatory therapies, the inventors have set up new purification strategies and technologies to enrich for and to analyze exosomes. Amongst other indications, the inventors evaluated their applicability for use in immune-mo dulatory (inflammatory) therapies, such as GvHD therapy, and already treated an otherwise treatment-resistant grade IV acute GvHD patient with MSC- derived exosomes. The GvHD symptoms of the patient decreased dramatically with the specific exosome therapy and the patient remained stable for five months.
- immune-mo dulatory (inflammatory) therapies such as GvHD therapy
- a pharmaceutical preparation wherein said neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) comprising exosomes are selected from human MSCs, and preferably from human MSCs derived from umbilical cord blood, umbilical cord tissue, placenta, bone or adipose tissue.
- the stem cells of the present invention are strictly non-embryonic derived stem cells.
- the MSCs that have been cultured in the presence of platelet lysate, and the preparation can contain said lysate and/or fractions thereof.
- the pharmaceutical preparation according to the present invention is specifically enriched for exosomes.
- any suitable method for purifying and/or enriching can be used, such as methods comprising magnetic particles, filtration, dialysis, ultracentrifugation, ExoQuickTM (Systems Biosciences, CA, USA), and/or chromatography.
- preferred is a method that comprises polyethylene glycol precipitation and/or chromatographical- ly enrichment using the monolithic technology (e.g. CIM®, BIA separations, Austria) as stationary phases instead of columns packed with porous particles.
- Monoliths are continuous stationary phases that are cast as a homogeneous column in a single piece and prepared in various dimensions with agglomeration-type or fibrous microstructures.
- fractions being enriched with exosomes are tested for their in vitro immunomodulatory effect, in particular an anti-inflammatory effect and/or immune suppressive effect, and can further be analyzed, in microbiological, in virulence and in pyrogen tests to, for example, excluded possible contaminations.
- these fractions can be studied with regard to protein content, and particle size.
- fractions being enriched with exosomes were particularly useful in the methods according to the present invention, if they exhibited strong in vitro immunomodulatory effects in activity tests, where, following the addition of said exosome fraction, a reduced IL- ⁇ , TNF-a and/or IFN- ⁇ cytokine response of effector cells of a donor could be found.
- ELISpot assays showed that the IL- ⁇ , TNF-a and/or IFN- ⁇ cytokine response of effector cells are impaired towards allogeneic cells in the presence of exosome containing fractions.
- Other methods that could be used to test for in vitro immunomodulatory effects include, for example, Luminex, ELISA, and/or flow cytometry.
- the present invention is thus based on the novel concept for an improved prevention and treatment of diseases, in particular in patients suffering from a having a risk of an inflammatory disease, neuronal disease, GvHD, stroke, and ischemia and associated complications, for example, for avoiding inflammatory reactions prior or during surgery, and the prevention of inflammatory conditions and reactions of patients that are connected to a life support machine.
- the diseases can be selected from pre- or postnatal damages of the nervous system, such as for example, brain damages related to hypoxia, inflammation, and/or ischemia.
- the diseases can be selected from graft-versus-host disease, or transplant rejections following organ transplantations, respectively.
- the exosomes-enriched fractions derived from adult mesenchymal stem cells (MSCs) that were enriched using a polyethylene glycol precipitation protocol are prophylactically and/or therapeutically transfused into patients, in particular neonates and/or patients receiving transplants and/or patients undergoing surgery.
- the exosomes-enriched fractions are derived from MSCs that are selected from human adult MSCs, and preferably from human MSCs derived from umbilical cord blood, umbilical cord tissue, placenta, bone or adipose tissue, and MSCs that have been cultured in platelet lysate.
- the pharmaceutical preparation according to the present invention preferably is enriched for exosomes that comprise biological factors, such as, for example, proteins, such as antiinflammatory cytokines, IL-10, TGF- ⁇ , and HLA-G, and/or nucleic acids, such as, for example, miR As.
- biological factors such as, for example, proteins, such as antiinflammatory cytokines, IL-10, TGF- ⁇ , and HLA-G
- nucleic acids such as, for example, miR As.
- the present invention constitutes a multimodal intervention, and thus not only a specific factor is used (and only a part of the cascade (or of the underlying clinical phenotype) would be intervened with), but biologically complex and endogenous mediators and modulators are used. These components are found in every human, and therefore no significant adverse side- effects are expected.
- a pharmaceutical preparation according to the present invention wherein said levels of TGF- ⁇ as measured in the activity tests are at least ten times higher (at 60.5 x 10 8 particles/mL) in the preparation, preferably at least 20 times higher, than in plasma levels of healthy controls.
- a pharmaceutical preparation according to the present invention wherein the numbers of IL- ⁇ , TNF-a and/or IFN- ⁇ producing PBMC were found to be reduced in said patient more than fifty percent after the last application (p ⁇ 0.0001, One-way ANOVA), compared to the cytokines' responses before MSC-exosome administration.
- a pharmaceutical preparation according to the present invention wherein said exosomes have a size of between about 70 to 200 nm, preferably between about 70 to 140 nm, or more preferably between about 70 to 120 nm.
- “About” shall mean a +/- 10% deviation.
- the exosomes are positive for cellular exosome markers, and even further preferred the protein content of the pharmaceutical preparation is higher than 1 mg/ml.
- the pharmaceutical preparation according to the present invention is suitable for i.v. administration, such as for example, intravenous administration or infusion into a patient in need thereof.
- Another aspect of the present invention then relates to a method for producing a pharmaceutical preparation according to the present invention, comprising the following steps: a) providing a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs), optionally from MSCs that have been cultured in platelet lysate, comprising exosomes, b) enriching said exosomes, optionally comprising polyethylene glycol precipitation, c) determining an in vitro immunomodulatory effect, in particular an antiinflammatory effect and/or immune suppressive effect, of said enriched exosomes by, for example, a reduced IL- ⁇ , TNF-a, T-cell proliferation, and/or IFN- ⁇ cytokine response of effector cells of a donor, d) selecting those enriched exosomes that exhibit
- a method for producing a pharmaceutical preparation wherein said neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) comprising exosomes are selected from human MSCs, and preferably from human MSCs derived from umbilical cord blood, umbilical cord tissue, placenta, bone or adipose tissue.
- the stem cells of the present invention are strictly non-embryonic derived stem cells.
- the MSCs that have been cultured in the presence of platelet lysate, and the preparation can contain said lysate and/or fractions thereof.
- the method for producing a pharmaceutical preparation according to the present invention comprises the step of specifically enriching for exosomes.
- any suitable method for purifying and/or enriching can be used, such as methods comprising magnetic particles, filtration, dialysis, ultracentrifugation, ExoQuickTM (Systems Biosciences, CA, USA), and/or chromatography.
- preferred is a method that comprises polyethylene glycol precipitation and/or a monolithic method (see above), since using these methods, surprisingly active fractions containing exosomes could be obtained.
- fractions that were enriched for exosomes are further analyzed in microbiological tests, virulence tests, protein content, pyrogen tests, and particle size, in order to identify the most suitable fraction according to the invention. It could be found that fractions that were enriched for exosomes were particularly useful in the methods according to the present invention, if they exhibited strong in vitro immunomodulatory effects in activity tests, where upon the addition of said exosome fraction, a reduced IL- ⁇ , TNF-a and/or IFN- ⁇ cytokine response of effector cells of a donor could be found.
- a method according to the present invention wherein said levels of TGF- ⁇ as measured in the activity tests are at least ten (or at least twenty) times higher (at 60.5 x 10 8 particles/mL) in the preparation than in plasma levels of healthy controls.
- said exosomes have a size of between about 70 to 200 nm, preferably between about 70 to 140 nm, or more preferably between about 70 to 120 nm.
- “About” shall mean a +/- 10% deviation.
- the exosomes are positive for cellular exosome markers, and even further preferred the protein content of the pharmaceutical preparation is higher than 1 mg/ml.
- the method for producing a pharmaceutical preparation according to the present invention comprises d) admixing said enriched exosomes of step c) with at least one suitable pharmaceutical excipient and/or carrier.
- suitable pharmaceutically acceptable excipient and/or carrier can be used.
- said pharmaceutically acceptable excipient and/or carrier renders said preparation suitable for i.v. administration, such as for example, intravenous administration or infusion.
- Yet another aspect of the present invention relates to a pharmaceutical preparation comprising exosomes derived from a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs), preferably according to the present invention as described herein for use in the prevention and/or treatment of diseases.
- MSCs tissue-derived mesenchymal stem-cells
- the diseases that are prevented and/or treated using the pharmaceutical preparation comprising exosomes derived from a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) according to the present invention are selected from the group consisting of inflammatory diseases, neuronal diseases, transplant rejections, GvHD, stroke, and ischemia.
- the diseases that are prevented and/or treated using the pharmaceutical preparation comprising exosomes derived from a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) according to the present invention are selected from pre- or postnatal damages of the nervous system, such as for example, brain damages related to hypoxia, inflammation, and/or ischemia.
- MSCs tissue-derived mesenchymal stem-cells
- the diseases that are prevented and/or treated using the pharmaceutical preparation comprising exosomes derived from a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) according to the present invention are selected from graft-versus-host disease, and transplant rejections following organ or bone marrow transplantation.
- Yet another aspect of the present invention relates to a method for preventing and/or the treatment of a disease selected from the group consisting of inflammatory diseases, neuronal diseases, transplant rejections, stroke, and ischemia in a patient, comprising administering to said patient an effective amount of a pharmaceutical preparation according to the present invention.
- a disease selected from the group consisting of inflammatory diseases, neuronal diseases, transplant rejections, stroke, and ischemia in a patient, comprising administering to said patient an effective amount of a pharmaceutical preparation according to the present invention.
- the disease that is prevented and/or treated is selected from pre- or postnatal damages of the nervous system, such as for example, brain damages related to hypoxia, inflammation, ischemia, graft-versus-host disease, and transplant rejections following organ transplantation.
- said preventing and/or the treatment according to the present invention comprises an administration through i.v. administration, such as for example, intravenous administration or infusion.
- More preferred is a method for preventing and/or the treatment of a disease according to the present invention, wherein said patient is a newborn.
- the invention thus also relates to the application of exosome-enriched fractions for the treatment of acquired neonatal as well as adult neural defects, for example the treatment of brain damages in neonates and grown-ups after hypoxia, inflammation, ischemia, etc. Furthermore, the treatment of ischemic strokes can be treated, and the adverse effect of the stroke can be reduced and/or reversed.
- the invention thus also relates to the application of exosome-enriched fractions for the treatment of transplantation-related complications.
- Treatments can be both prophylactic and/or therapeutic.
- mesenchymal stem cells became one of the most intensively studied adult stem cell entities within the last 15 years.
- MSCs mesenchymal stem cells
- MSCs might improve clinical outcomes by paracrine effects rather than by their previously proposed engraftment into damaged host tissues (Lee et al, 2009).
- the group of Lim earlier showed that the supernatants of in vitro expanded embryonic stem cell derived MSCs contain small extracellular vesicles, so called exosomes, whose infusion is sufficient to mediate a reduction of myocardial infarction sizes (see Lim et al, 2010).
- Figure 1 shows the effect of exosome preparations on newborn rats that were partially confronted with LPS.
- Figure 2 shows an example for the analyses of exosomes-containing fractions during the purification thereof using a monolithic column.
- Figure 3 shows the transplantation-relevant effects of exosomes on a patient in a treatment scheme according to the examples.
- the patient As symptom of GvHD the patient has diarrhea of more than 3 liters per day. After administration of MSC-derived exosome-enriched fractions, the symptoms were drastically reduced, without that other side-effects could be observed.
- Figure 4 also shows the transplantation-relevant effects of exosomes on a patient in a treatment scheme according to the examples.
- the patient developed inflammations of the skin.
- the symptoms were drastically reduced, without that other side-effects could be observed.
- FIG. 5 shows that MSC-fractions, such as MSCl Exo reduce the number of IL- ⁇ , TNF-a and IFN- ⁇ secreting PBMCs in almost all settings, in particular in vitro. Immune suppressive effects on the cytokine responses of the other exosome preparations (MSC2 Exo, MSC3 Exo and MSC4 Exo) are slightly less prominent.
- FIG. 6 shows that depending on the exosome fraction, MSC-enriched exosomes suppress, do not modulate or increase the proliferation rates of treated T cells, respectively (Example III).
- Figure 7 shows that certain exosome fractions such as Exo MSC A (fraction 3 in Fig. 2) and MSC Exo F (fraction 7 in Fig. 2) suppress proliferation of PHA stimulated T cells and IL- lbeta and TNF alpha release of stimulated T cells (see Example III).
- Figure 8 shows that the effects of the in vitro studies (A) correlate with the effect in vivo (B).
- Distinct MSC exosome preparations contain different cytokine compositions
- exosome-enriched fractions derived from the four MSC preparations were analyzed for their content of anti- and pro -inflammatory as well as apoptosis-inducing molecules (Table 1).
- the exosome preparations contained high quantities of the anti-inflammatory molecules IL-10, TGF- ⁇ and HLA-G.
- pro-inflammatory cytokines IL- ⁇ , IL-2, IL-6, IL-17a, IL-21 and TNF-a nor the apoptosis-inducing molecule sFasL were detected in any of the exosome preparations (Table 1).
- MSCl Exo contained elevated TGF- ⁇ levels, which surpassed TGF- ⁇ plasma levels of healthy donors by more than the factor 10.
- MSCl Exo differed from the others preparations with regard to the anti-inflammatory cytokine IL-10 and the pro -inflammatory cytokine IFN- ⁇ .
- the ratio of IL-10 to IFN- ⁇ varied between the four exosome preparations for almost two orders of magnitude (Table 1); the highest ratio was found for MSC1 Exo (1.02).
- Distinct MSC exosome preparations reveal different capabilities to suppress inflammatory responses in vitro
- Immune modulatory impacts of the MSC exosome preparations were tested in a mixed lymphocyte reaction using the HLA class I negative K562 as target cells.
- HLA-E*01 :03 or HLA-B27-transfected K562 variants and cells of the HLA class I positive, respiratory syncytial virus infected (RSV) B-cell line were additionally used as allogeneic target cells.
- RSV respiratory syncytial virus infected
- MSC1 Exo Compared to the co-cultures without exosomes, MSC1 Exo reduced the number of IL- ⁇ ⁇ , TNF-a and IFN- ⁇ secreting PBMCs in almost all settings (Figure 5), in particular in vitro. Immune suppressive effects on the cytokine responses of the other exosome preparations (MSC2 Exo, MSC3 Exo and MSC4 Exo) were less prominent (Figure 5).
- Primary human MSCs were raised from mononuclear cells obtained by Ficoll density gradient centrifugation of bone marrow and raised mainly as described before (Hemeda et al, 2010). Instead of 10% fetal bovine serum (FBS) the inventors supplement the basal media with 5% human pooled platelet lysates. To exclude variations caused by the usage of different platelet lysate preparations, the inventors performed all experiments with the same batch of platelet lysates that the inventors produce by pooling 3040 individual human platelet samples.
- FBS fetal bovine serum
- MSCs were characterized by flow cytometry as CD90 + /CD73 + /CD105 + /CD44 + cells lacking the expression of CD45 and the endothelial marker CD31. MSC differentiation capability along the osteogenic, cartilage and adi- pogenic lineage was evaluated.
- conditioned media were harvested every 48 h and passed through a 0.22 ⁇ filter membrane to remove cell debris and larger vesicles. CM were collected and stored at -20°C. After thawing, CM were unified and processed by PEG precipitation. Briefly, PEG 6000 was added to CM. Following incubation for 8-12 h at 4°C exosomes were precipitated by centrifugation for 30 min at 1500 g. Obtained pellets were resolved in 1 mL 0.9% NaCl, washed in a total volume of 45 mL 0.9% NaCl for 12 h at 4°C and precipitated by ul- tracentrifugation for 2 h at 100,000 g. Again pellets were resolved in 1 mL of 0.9% NaCl, diluted with 0.9% NaCl to required concentrations and stored as 1 mL aliquots at -80°C until use.
- Protein concentrations of obtained aliquots were determined. Of each lot, 10 ⁇ of exosome containing fraction was used to perform Western blots with antibodies recognizing exosome specific marker proteins, such as TsglOl, CD63, CD81 or CD82. Additionally, the inventors measured the concentration and size of the exosome by the NTA method as described previously (Sokolova et al, 2011). The concentration of pro- and anti-inflammatory cytokines (e.g., IL- ⁇ , IL-6, IL-8; TNF-a, IFN- ⁇ , IL-4, IL-10, IL-13, TGF- ⁇ , TSG-6) and of the Fas- ligand within the exosome fractions were measured using the Luminex method. Other methods that could be used include, for example, ELIspot, ELISA, and/or flow cytometry.
- cytokines e.g., IL- ⁇ , IL-6, IL-8; TNF-a, IFN- ⁇ , IL-4,
- the inventors established the T-cell/MNC proliferation assay within the group.
- MNCs or magnetic cell separation enriched CD3 + cells were stained either with CFSE or PKH2 and raised in the presence of either a T cell/MNC proliferation stimulating agents (e.g. LPS, PMA, PHA, anti-CD3 beads) or of cells of the leukemic cell line K562.
- a T cell/MNC proliferation stimulating agents e.g. LPS, PMA, PHA, anti-CD3 beads
- the inventors established a protocol allowing them to document T-cell/MNC proliferation by flow cytometry.
- the inventors searched for a stimulant whose impact on the T cellslMNCs could effectively be modulated by MSC exosomes. Finally, the inventors found a stimulant, which triggered proliferation of different leukocytes within MNC fractions. Using gating strategies for B, T and NK cells the inventors analyzed impacts of MSC exosomes on these cells separately. To estimate effects of MSC-derived exosomes on the cytokine secretion of B, T and NK cells the inventors used the ELISpot assay.
- the inventors adapted a flow cytometry based cytokine secretion assay (Miltenyi, Bergisch-Gladbach), allowing them to study the effect of MSC exosomes on the release of pro -inflammatory cytokines (specifically IFN-a, TNF-a and IFN- ⁇ ) by stimulated immune cells.
- the inventors compared the immunomodulatory activities of 15-20 independently obtained MSC exosome fractions. The inventors expected that some of these fractions would show strong immunomodulatory activity, whereas others would have no effect on the immune response of stimulated immune cells. The three samples with the highest and the lowest immunomodulatory activities were compared in more detail. The inventors also comprehensively compared the expression profiles of the MSC propagations, from which corresponding exosome fractions were obtained. The inventors identified sets of candidate proteins that predicted immunomodulatory effects. The inventors validated these markers by evaluating their expression in additional MSC and exosome samples.
- MSC-derived exosomes using PEG preparations with high and low immunomodulatory capabilities in vitro were used for the in vivo approach.
- C57M6 mice were submitted to 30 minutes of intraluminal middle cerebral artery occlusion. This model is well characterized and has been extensively used in the inventors' lab before, resulting in localized infarcts of the striatum and most lateral cerebral cortex (Doeppner et al, 20096). Dissolved in a volume of 250 ⁇ , 2 x 10 6 cell equivalents of exosomes were injected over 10 min via tail vein on days 1, 3, and 5 post-stroke.
- Post-ischemic motor and coordination deficits were analyzed using the rota rod, the tight rope and the corner turn test as previously described (Zhang et al, 2002; Doeppner et al, 201 lb). All animals were trained 1-2 days before induction of stroke to ensure sufficient test performance. Actual tests are performed on days 7, 14, 28, 56 and 84. Using the rota rod test, animals were put on a treadmill with an accelerating speed of 4-40 rpm. The maximum speed was achieved after 260 s, and maximum testing time was 300 s. The time until animals drop was registered and statistically analyzed. For the tight rope test, animals were placed on a 60 cm long rope grasping the string with their forepaws.
- mice received daily intraperitoneal injections of bromodeoxyuridine (BrdU) on days 8-18, to indirectly assess post-ischemic cell proliferation.
- PrdU bromodeoxyuridine
- immunohistochemical protocols using the marker CD31 were employed (Wang et al, 2005). Tissue samples were collected for studies other than immunohistochemistry, such as realtime PCR, which allowed to evaluate post- ischemic inflammation, neuronal viability and astroglial scar formation. These assays included real-time PCR protocols according to the manufacturers' protocols.
- GvHD a condition, in which the new immune system recognizes the patient's tissue as 'foreign' and attacks it.
- transplanted patients receive immunosuppressant treatments. Patients refractory to such treatments have a poor prognosis, while transplanted MSCs might improve the clinical response (Gotherstrom et al, 2004; Le Blanc et al, 2004).
- the immunomodulatory activities of MSCs result from secreted soluble factors rather than direct cellular interactions between engrafted MSCs and the patient's immune cells (Baron and Storb, 2012).
- the inventors hypothesized that the effectors are MSC-derived exosomes.
- the patient was stable for more than 5 months. Even though the inventors cannot conclude at this state that the MSC exosomes led to the suppression of the GvHD symptoms, the patient's immune cells surprisingly showed a similar immune modulation as cells of healthy donors cells which were treated with exosomes in vitro.
- BM Human bone marrows
- Primary human MSCs were generated from mononu- clear cells obtained by Ficoll (Biocoll Separating Solution, Biochrom AG, Berlin, Germany) density gradient centrifugation of bone marrow.
- the mononuclear cells were cultured in tissue culture 6 well plates at a density of 2 x 10 6 cells per well in MSC basal media (PAN) supplemented with 5% thrombocyte lysate, 1% glutamine, and 1% penicillin-streptomycin (PAA) for 24 hours. After 24 hours of incubation non-adherent cells were removed by medium exchange.
- PAN MSC basal media
- PAA penicillin-streptomycin
- Adherent cells After confluence had been reached, usually within 14 days, cells were continuously passaged after treatment with 0.25% trypsin. Adherent cells showed fibroblast-like morphology, and were able to differentiate along the adipogenic, chondrogenic, and osteogenic pathways.
- Exosomes were isolated from cell culture supematants of MSCs derived from bone marrow. To remove cells, conditioned media were centrifuged for 5 min at 900 x g, and to remove remaining debris, another centrifugation step was performed for 1 h at 10,000 x g. To remove all particles bigger than 200 nm, the supematants were filtered through 0.2 um pore filters. To concentrate the exosomes, the filtrate was centrifuged for 2 h, 110,000 x g, at 4°C. The obtained precipitates were resuspended in 150 ⁇ PBS and used for further analyses.
- MSC supematants were collected and stored at -20°C. After thawing, the supematants were unified and 3 units/mL of heparin and approx. 3 ⁇ g/mL ActilyseTM (Boehringer Ingelheim, Germany) were added. Supematants were incubated for 3 h at 37°C, and then filtered through 0.22 ⁇ bottle top filters.
- the 45 ml suspensions were split and transferred in half into two ultracentrifugation- vials. 0.9% NaCl was added to a total volume of 38 ml.
- the preparations were centrifuged for 2 h at 110.000 g.
- the supematants were removed from the sediments as obtained; the supematants were resuspended in 1 ml 0.9%> NaCl solution, and pooled. Then, the sample was filled up to 10 ml using 0.9% NaCl solution, and frozen in 1 ml aliquots in 15 ml reaction vials at -80°C.
- the supematants were also kept, and stored frozen at -20°C. Then, the aliquots were diluted 1 :5 (0.9%) NaCl solution), and used in the following assays:
- Exosome specific proteins were identified by Western blot analysis. For this, purified exo- somes were treated with sample buffer (DTT, 0.1 % SDS, 0.1 M Tris HCl, pH 7.0) and spoiled for 5 min at 95°C. Proteins were separated on 12% SDS-PAGE gel and transferred to PVDF membranes. Membranes were blotted with antibodies to TsglOl (Sigma Aldrich), Flotilin 1 (BD Pharmingen), and CD81 (BD Pharmingen), then incubated with appropriate HRP- conjugated secondary antibodies (Dianova) and visualized by enhanced chemiluminescence (Pierce). Nano particle tracking analysis (NTA)
- NTA For particle size determination, NTA was performed with a Nano Sight LM10 instrument equipped with the NTA 2.0 analytical software. All experiments were carried out at 1 :2000 dilutions, resulting in particle concentrations of approx. 3 x 10 8 per mL. Each experiment was carried out in triplicate. The 50% median value (D50) is given in all cases, and the standard deviation was calculated for all data.
- PBMC peripheral blood mononuclear cells
- soluble HLA-G sHLA-G
- cytokines cytokines
- apoptosis-inducing molecules in MSC- exosome preparations were analyzed by ELISA.
- exosomes (approx. 60.5 x 10 8 particles) were solubilized in 1 ml PBS-Tween (1%) and sonicated for 60 sec.
- sHLA-G concentrations were determined as described before (Schutt et al, 2010) with minor modifications: The monoclonal antibody (mAb) G233 (Exbio, Praha, Czech Republic) was used as capture reagent and bound sHLA-G was detected by the bioti- nylated mAb w6/32 (Leinco Technologies, St. Louis, Missouri, USA) followed by AMDEXTM Streptavidin HRP (Amersham, Freiburg, Germany). The detection limit of sHLA- G ELISA was 0.1 ng/ml.
- Concentrations of pro -inflammatory IFN- ⁇ , TNF-a, IL-lb, IL-2, IL- 6, IL-8, IL-17a, IL-21
- anti-inflammatory IL-l RA, IL-4, IL-10, IL-13, TGF- ⁇
- apoptosis-inducing molecules soluble FasL
- PBMC and NK cells isolated by negative selection out of these PBMC (Invitrogen, Düsseldorf, Germany), were adjusted to a fixed concentration of lxlO 6 cells/mL and used as effector cells.
- lxlO 6 effector cells were mixed with approx. 60.5 x 10 8 MSC-exosomes. In this case effector cell suspensions with exosomes and without exosomes were used in cytokine stimulation experiments.
- K562 cell line served as stimulator cells and were adjusted to a concentration of O.lxlO 6 cells/mL. All ELISpot assays were performed in an effector/stimulator ratio of 6: 1.
- 12.5 x 10 4 effector cells and 0.25 x 10 4 stimulator cells and for the IFN- ⁇ ELISpot assay 75 x 10 4 effector cells and 1.5 x 10 4 stimulator cells were seeded with 200 ⁇ culture medium containing 200U/mL IL-2 and 10% fetal calf serum onto MultiScreen-HA ELISpot plates (MAIPS4510, Millipore, Bedford, MA, USA) pre-coated with the respective capture antibody of the ELISpot Kit (eBioscience, Frankfurt, Germany).
- Protein content of the sample 1.1 mg/ml
- Particle size 199.5 +/- 70 nm
- Endotoxin content 0.588 I.E./ml (+/- 50 %) corresponding to 2.94 I.E./ml of the concentrated solution.
- Exosomes derived from four different MSC preparations were analyzed for the content of anti-, pro-, and apoptosis-inducing molecules: Besides the two pro -inflammatory cytokines IFN- ⁇ and IL-8, MSC-exosomes contained preferentially high quantities of the antiinflammatory and/or immune suppressive molecules IL-10, TGF- ⁇ and HLA-G. Especially, TGF- ⁇ levels were found to be ten times higher in the MSC-exosome solution of 60.5 x 10 particles/mL than in plasma levels of healthy controls.
- the influence of the MSC-exosomes on the cytokine response of PBMC or NK cells of PB donors on K562 leukemic cells was proven by IL- ⁇ , TNF-a and IFN- ⁇ ELISpot using.
- the donors effector cells were co-cultured with HLA class I negative K562 or genetically engineered K562 cells expressing HLA-E*01 :03 or HLA-B35. It was found that the IL- ⁇ , TNF- ⁇ and IFN- ⁇ cytokine responses of the donor's effector cells were reduced towards the different allogenic cells in the presence of MSC-exosomes compared to responses in the absence of MSC-exosomes.
- MSC-exosomes are able to impair in vitro the capability of PBMC to produce pro -inflammatory cytokines known to be operative in GvHD.
- AlloSCT allogeneic stem cell transplantation
- MDS RAEB-T myelodysplasia syndrome
- PBSC peripheral blood stem cells
- infliximab (10 mg/kg BW once per week over a four week period) was initiated and 17 sessions of extracorporeal photopheresis were performed over four months. Throughout this time the basic immunosuppression with steroids, mostly in combination with either tacrolimus or MMF, was continued.
- the MSC-exosomes were administered between August, 1 st to August, 15 th 2011 in increasing doses.
- the patient received pre-medication with steroids and anti-histamins prior to each MSC- exosome application and was closely monitored in the intensive care unit. The therapy was tolerated very well, and no side effects were detected. After each administration, blood samples were taken and analyzed for their capability to produce IL- ⁇ , TNF-a, and IFN- ⁇ in response towards K562, and K562 transfected with HLA-E*01 :03 or HLA-B27 (see above).
- the PBMC of the patient started to lessen their cytokine reaction towards the stimulator cells.
- the numbers of IL- ⁇ , TNF-a and IFN- ⁇ producing PBMC were found to be reduced more than fifty percent after the last application (p ⁇ 0.0001, One-way ANOVA).
- p ⁇ 0.0001, One-way ANOVA the MSC-exosomes are able to impair in-vivo the capability of PBMC to produce pro -inflammatory cytokines known to be operative in GvHD.
- PB-MNCs of 10 different peripheral blood donors were pooled. Ali- quots of 400.000 cells were cultured in triplicates each in a total volume of 200 ⁇ , either in the presence or absence of 0.02 U of the same exosome fractions presented in Figure 6.
- Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti- inflammatory protein TSG-6.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Virology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Marine Sciences & Fisheries (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Dermatology (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Transplantation (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to the use of exosome-preparations derived from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) for the prevention or for therapy of inflammatory conditions, such as, for example, pre- and postnatally acquired damages of the brain (i.e. neuronal damages) or in complications following stem cell transplantation ("graft vs host-disease", GvHD).
Description
Use of preparations comprising exosomes derived from mesenchymal stem cells (MSCs) in the prevention and therapy of inflammatory conditions
The present invention relates to the use of exosome-preparations derived from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) for the prevention or for therapy of inflammatory conditions, such as, for example, pre- and postnatally acquired damages of the brain (i.e. neuronal damages) or in complications following stem cell transplantation ("graft vs host-disease", GvHD).
Background of the invention
Different cell populations appear as candidates for cell-based therapies, in particular in neonates: Hematopoietic stem cells (HSC), endothelial progenitor cells (EPC), mesenchymal stem cells (MSC) as well as regulatory T cells (Treg) exerting immune modulatory and pro- regenerative activities by direct cellular action or secretion of pro-regenerative, anti-apoptotic, immunomodulatory factors. The suppressive activity of Tregs from umbilical cord blood (UCB) has been attributed to the observed reduced severity of graft versus host disease (GvHD) in UCB transplantation (Tolar J, Hippen KL, Blazar BR. Immune regulatory cells in umbilical cord blood: T regulatory cells and mesenchymal stromal cells. Br J Haematol 2009 October; 147(2):200-6).
Mesenchymal stem/stromal cells (MSCs) have emerged as one of the most intensely studied adult stem cell population within the last 15 years (Bieback K, Brinkmann I. Mesenchymal stromal cells from human perinatal tissues: From biology to cell therapy. World J Stem Cells 2010 August 26; 2(4):81-92). The therapeutic interest initially was based on their multilineage differentiation potential. However, it recently turned out that only few cells engraft and differentiate after either local or systemic delivery of MSCs in a number of different injury models. Subsequent to the detection of profound immune modulatory activities, MSCs were successfully applied to treat steroid-refractory GvHD (Le Blanc K, Rasmusson I, Sundberg B, Goth- erstrom C, Hassan M, Uzunel M et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004 May 1 ; 363(9419): 1439-
Since then, a number of different studies have addressed the impact of MSCs on GvHD with different outcomes. In most of these studies, the infusion of MSCs appeared to be safe. However, a positive impact on GvHD is still discussed controversially (Baron and Storb, 2011). Although MSCs can be raised from virtually every tissue within the human body, they are a very rare population comprising about 0.001 to 5 % of cells in different tissues (Bieback K. Basic Biology of Mesenchymal Stem Cells. Transfus Med Hemother 2008; 35(3): 151-2).
Exosomes are naturally occurring small membrane vesicles (80-160 nm) released by a huge variety of cell species. Containing a combination of lipids and proteins as well as RNAs, exosomes participate in intercellular communication processes. They can be isolated from all body fluids including blood plasma, urine, saliva, breast milk, bronchial lavage fluid, cerebral spinal fluid, amniotic fluid and malignant ascites (Ludwig AK, Giebel B. Exosomes: Small vesicles participating in intercellular communication. Int J Biochem Cell Biol 2011 October 19).
Initially, exosomes were discovered in 1983 as small vesicles that are released by exocytosis upon fusion of so called multivesicular bodies with the plasma membrane. In 1996, exosomes were isolated from B lymphocytes, and were demonstrated to exhibit antigen-presenting characteristics. Since then, their role in immune-biological settings has been investigated in an increasing number of studies. It turned out that exosomes derived from mature dendritic cells for example exert immune-stimulatory functions, while tumor-derived exosomes often mediate immune-suppressive functions (Ludwig AK, Giebel B. Exosomes: Small vesicles participating in intercellular communication. Int J Biochem Cell Biol 2011 October 19).
In 2010, Lai and co-workers provided evidence that exosomes released from MSCs mediate at least a proportion of the regenerative effects of clinically applied MSCs (Lai RC, Arslan F, Lee MM, Sze NS, Choo A, Chen TS et al. Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Res 2010 May; 4(3):214-22). Since intravenously administered MSCs get rapidly trapped into the lung and are hardly found within infarcted hearts (Lee et al, 2009), the group of Lim searched for paracrine MSC effectors and showed in a first set of experiments that the supematants of in vitro expanded MSCs are sufficient to mediate beneficial effects. Next, they purified exosomes from MSC supematants and infused
them in a murine myocardial ischemia model. Again, they observed a reduction of the infarct size (Lai et al, 2010).
Timmers et al. (in Timmers L, Lim SK, Arslan F, Armstrong JS, Hoefer IE, Doevendans PA, Piek JJ, El Oakley RM, Choo A, Lee CN, Pasterkamp G, de Kleijn DP. Reduction of myocardial infarct size by human mesenchymal stem cell conditioned medium. Stem Cell Res. 2007 Nov; l(2): 129-37. Epub 2008 Mar 8) describe the use of secretions of human MSCs as a therapeutic option in acute myocardial infarct. These MSCs are raised from human Embryonic Stem Cells (hESCs).
Fleissner et al. (in: Fleissner F, Goerzig Y, Haverich A, Thum T. Microvesicles as novel bi- omarkers and therapeutic targets in transplantation medicine. Am J Transplant. 2012 Feb; 12(2):289-97. Epub 2011 Nov 14) describe microvesicles (MVs) including exosomes as emerging new biomarkers and potential regulators of inflammation and immunological processes. The particles contain proteins and genetic information including DNA and mi- croRNAs that may be of importance for cell/cell communication. However, their role during and after organ transplantation and immunomodulatory effects is described to be only in its beginning of understanding. The authors speculate that MV modulation may emerge as a therapeutic approach in organ rejection therapy.
Horstman et al. (in: Horstman LL, Jy W, Minagar A, Bidot CJ, Jimenez JJ, Alexander JS, Ahn YS. Cell-derived microparticles (MPs) and exosomes in neuro inflammatory disorders. Int Rev Neurobiol. 2007; 79:227-68) describe MPs and their possible roles in the pathophsyi- ology of some neuropathologies, especially those which are ischemic in nature, but probably also inflammatory ones. Tables include a listing of bioactive agents known to be carried on MP, many of which were heretofore considered strictly soluble, and some of which can be transferred from cell to cell via MP vectors, for example certain cytokine receptors.
Baron and Storb (in: Baron F, Storb R. Mensenchymal Stromal Cells: A New Tool against Graft- versus-Host Disease? Biol Blood Marrow Transplant 2011 September 29) propose that immune modulatory activities of MSCs are thought to derive from secreted soluble factors rather than from direct intercellular interactions of engrafted MSCs with the patient's immune cells.
WO 2009/105044 describes a particle secreted by a mesenchymal stem cell and comprising at least one biological property of a mesenchymal stem cell. The biological property may comprise a biological activity of a mesenchymal stem cell conditioned medium (MSC-CM) such as cardioprotection or reduction of infarct size. The particle may comprise a vesicle or an exo- some. The claims encompass exosomes obtainable from human embryonic stem cells derived from human embryos.
WO 2012/020308 is directed at compositions and methods for the treatment of tissue damage (e.g., acute or chronic) and related diseases, disorders or conditions based on the use of pathfinder cells, extracellular secretomes thereof, and/or pathfinder cell-associated micro R As (miRNAs). In some embodiments, the present invention provides a method for treating inflammation comprising a step of administering a population of cells, or extracellular secretomes thereof, to an individual suffering from a disease, disorder or condition characterized by inflammation of one or more tissues, wherein the cells are originated from an adult tissue and wherein the cells induce an anti-inflammatory response.
WO 2012/053976 discloses the use of exosomes that are secreted by mesenchymal stem cells in order to promote hair growth and wound healing. These effects are disclosed in connection with pro -inflammatory cytokines and proteases. WO 2012/053976 speculates about an immune-mo dulatory effect of the exosome-preparation.
WO 2012/020307 discloses the therapeutic use of micro vesicles including exosomes in the treatment of inflammation and lesions of cerebral trauma. Similarly, WO 2012/087241 describes the use of exosomes for the treatment of the diseases as mentioned.
The currently proposed strategies in the prevention and/or therapy based on exosomes suffer from several disadvantages. Most problematic is their unimodal approach. This is in contrast to the biological complexity of the pathophysiology of the indications to be treated. Instead, it would be required to use a therapeutic cocktail that can interact with very different pathophysiological signaling cascades, and thus provide a more complete and effective prevention or treatment.
Mesenchymal stromal cells (MSCs) represent a heterogeneous subset of multipotent cells that can be isolated from several tissues including bone marrow and fat. MSCs exhibit immuno-
modulatory and anti-inflammatory properties that prompted their clinical use as prevention and/or treatment for severe graft-versus-host disease (GvHD). Although a number of phase I- II studies have suggested that MSC infusion was safe and might be effective for preventing or treating acute GvHD, definitive proof of their efficacy remains lacking thus far. Multicenter randomized studies are ongoing to more precisely assess the impact of MSC infusion on GvHD prevention/treatment, whereas further research is performed in vitro and in animal models with the aims of determining the best way to expand MSCs ex vivo as well as the most efficient dose and schedule of MSCs administration (see Baron and Storb, 2011). Although MSCs can be raised from virtually every tissue within the human body, they are a very rare population comprising about 0.001 to 5 % of cells in different tissues. Especially UCB contains MSC at low frequency. Also, MSCs derived from human embryonic ESC meet ethical concerns.
Finally, exosomes that are obtained from MSCs of individual donors were found to be very heterogenic. Thus, it seems reasonable to assume that subtypes of populations of MSCs with quite different potential are present. Currently, this problem is completely unresolved.
It is therefore an object of the present invention to provide a safe and more effective preparation of exosomes that is suitable for the treatment of diseases and conditions that involve inflammatory reactions. Other objects of the present invention will become apparent to the person of skill upon studying the present description of the invention.
In a first aspect thereof, the above object is solved by a pharmaceutical preparation comprising exosomes, obtainable by a method comprising the following steps:
a) providing a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) comprising exosomes,
b) enriching said exosomes, optionally comprising polyethylene glycol precipitation, c) determining an in vitro immunomodulatory effect, in particular an anti-inflammatory effect and/or immune suppressive effect, of said enriched exosomes by, for example, a reduced IL- 1β, TNF-α, T-cell proliferation, and/or IFN-γ cytokine response of effector cells of a donor, and
d) selecting those enriched exosomes that exhibit an immunomodulatory effect, in particular an anti-inflammatory effect and/or immune suppressive effect.
Considering exosomes as a novel, very potent tool in regenerative and in immune-mo dulatory therapies, the inventors have set up new purification strategies and technologies to enrich for and to analyze exosomes. Amongst other indications, the inventors evaluated their applicability for use in immune-mo dulatory (inflammatory) therapies, such as GvHD therapy, and already treated an otherwise treatment-resistant grade IV acute GvHD patient with MSC- derived exosomes. The GvHD symptoms of the patient decreased dramatically with the specific exosome therapy and the patient remained stable for five months.
Furthermore, in order to prove effectiveness of the prevention and therapy of neuronal damages, in particular in neonates, experiments with LPS confronted rats were performed that showed a clear positive reaction following the administration of the inventive pharmaceutical preparation.
Preferred is a pharmaceutical preparation according to the present invention, wherein said neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) comprising exosomes are selected from human MSCs, and preferably from human MSCs derived from umbilical cord blood, umbilical cord tissue, placenta, bone or adipose tissue. The stem cells of the present invention are strictly non-embryonic derived stem cells. Most preferably, the MSCs that have been cultured in the presence of platelet lysate, and the preparation can contain said lysate and/or fractions thereof.
The pharmaceutical preparation according to the present invention is specifically enriched for exosomes. For this, generally any suitable method for purifying and/or enriching can be used, such as methods comprising magnetic particles, filtration, dialysis, ultracentrifugation, ExoQuick™ (Systems Biosciences, CA, USA), and/or chromatography. Nevertheless, preferred is a method that comprises polyethylene glycol precipitation and/or chromatographical- ly enrichment using the monolithic technology (e.g. CIM®, BIA separations, Austria) as stationary phases instead of columns packed with porous particles. Monoliths are continuous stationary phases that are cast as a homogeneous column in a single piece and prepared in various dimensions with agglomeration-type or fibrous microstructures. (see Iberer, G., Hahn, R., Jungbauer, A. LC-GC, 1999, 17, 998). Using these methods, surprisingly active fractions containing exosomes could be obtained.
Then, in order to identify the most suitable fraction according to the invention fractions being enriched with exosomes are tested for their in vitro immunomodulatory effect, in particular an anti-inflammatory effect and/or immune suppressive effect, and can further be analyzed, in microbiological, in virulence and in pyrogen tests to, for example, excluded possible contaminations. In addition, these fractions can be studied with regard to protein content, and particle size.
It could be found that fractions being enriched with exosomes were particularly useful in the methods according to the present invention, if they exhibited strong in vitro immunomodulatory effects in activity tests, where, following the addition of said exosome fraction, a reduced IL-Ιβ, TNF-a and/or IFN-γ cytokine response of effector cells of a donor could be found. ELISpot assays showed that the IL-Ιβ, TNF-a and/or IFN-γ cytokine response of effector cells are impaired towards allogeneic cells in the presence of exosome containing fractions. Other methods that could be used to test for in vitro immunomodulatory effects include, for example, Luminex, ELISA, and/or flow cytometry.
The present invention is thus based on the novel concept for an improved prevention and treatment of diseases, in particular in patients suffering from a having a risk of an inflammatory disease, neuronal disease, GvHD, stroke, and ischemia and associated complications, for example, for avoiding inflammatory reactions prior or during surgery, and the prevention of inflammatory conditions and reactions of patients that are connected to a life support machine. In one embodiment, the diseases can be selected from pre- or postnatal damages of the nervous system, such as for example, brain damages related to hypoxia, inflammation, and/or ischemia. In another embodiment, the diseases can be selected from graft-versus-host disease, or transplant rejections following organ transplantations, respectively.
In a particularly preferred embodiment of the invention, the exosomes-enriched fractions derived from adult mesenchymal stem cells (MSCs) that were enriched using a polyethylene glycol precipitation protocol, are prophylactically and/or therapeutically transfused into patients, in particular neonates and/or patients receiving transplants and/or patients undergoing surgery.
Preferably, the exosomes-enriched fractions are derived from MSCs that are selected from human adult MSCs, and preferably from human MSCs derived from umbilical cord blood,
umbilical cord tissue, placenta, bone or adipose tissue, and MSCs that have been cultured in platelet lysate.
The pharmaceutical preparation according to the present invention preferably is enriched for exosomes that comprise biological factors, such as, for example, proteins, such as antiinflammatory cytokines, IL-10, TGF-βΙ, and HLA-G, and/or nucleic acids, such as, for example, miR As. This leads to the further advantage according to the invention that a) a multimodal (complex) intervention is performed, b) biological physiological ("self) substances are used, and c) unwanted side effects of the preparation are reduced.
The present invention constitutes a multimodal intervention, and thus not only a specific factor is used (and only a part of the cascade (or of the underlying clinical phenotype) would be intervened with), but biologically complex and endogenous mediators and modulators are used. These components are found in every human, and therefore no significant adverse side- effects are expected.
Preferred is a pharmaceutical preparation according to the present invention, wherein said levels of TGF-βΙ as measured in the activity tests are at least ten times higher (at 60.5 x 108 particles/mL) in the preparation, preferably at least 20 times higher, than in plasma levels of healthy controls. Further preferred is a pharmaceutical preparation according to the present invention, wherein the numbers of IL-Ιβ, TNF-a and/or IFN-γ producing PBMC were found to be reduced in said patient more than fifty percent after the last application (p < 0.0001, One-way ANOVA), compared to the cytokines' responses before MSC-exosome administration.
Further preferred is a pharmaceutical preparation according to the present invention, wherein said exosomes have a size of between about 70 to 200 nm, preferably between about 70 to 140 nm, or more preferably between about 70 to 120 nm. "About" shall mean a +/- 10% deviation. Further preferred, the exosomes are positive for cellular exosome markers, and even further preferred the protein content of the pharmaceutical preparation is higher than 1 mg/ml.
In another aspect of the present invention, the pharmaceutical preparation according to the present invention, is suitable for i.v. administration, such as for example, intravenous administration or infusion into a patient in need thereof.
Another aspect of the present invention then relates to a method for producing a pharmaceutical preparation according to the present invention, comprising the following steps: a) providing a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs), optionally from MSCs that have been cultured in platelet lysate, comprising exosomes, b) enriching said exosomes, optionally comprising polyethylene glycol precipitation, c) determining an in vitro immunomodulatory effect, in particular an antiinflammatory effect and/or immune suppressive effect, of said enriched exosomes by, for example, a reduced IL-Ιβ, TNF-a, T-cell proliferation, and/or IFN-γ cytokine response of effector cells of a donor, d) selecting those enriched exosomes that exhibit an immunomodulatory effect, in particular an anti-inflammatory effect and/or immune suppressive effect, and e) admixing said enriched exosomes of step d) with at least one suitable pharmaceutical excipient and/or carrier.
Preferred is a method for producing a pharmaceutical preparation according to the present invention, wherein said neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) comprising exosomes are selected from human MSCs, and preferably from human MSCs derived from umbilical cord blood, umbilical cord tissue, placenta, bone or adipose tissue. The stem cells of the present invention are strictly non-embryonic derived stem cells. Most preferably, the MSCs that have been cultured in the presence of platelet lysate, and the preparation can contain said lysate and/or fractions thereof.
The method for producing a pharmaceutical preparation according to the present invention comprises the step of specifically enriching for exosomes. For this, generally any suitable method for purifying and/or enriching can be used, such as methods comprising magnetic particles, filtration, dialysis, ultracentrifugation, ExoQuick™ (Systems Biosciences, CA, USA), and/or chromatography. Nevertheless, preferred is a method that comprises polyethylene glycol precipitation and/or a monolithic method (see above), since using these methods, surprisingly active fractions containing exosomes could be obtained.
Preferred is a method for producing a pharmaceutical preparation according to the present invention, wherein fractions that were enriched for exosomes are further analyzed in microbiological tests, virulence tests, protein content, pyrogen tests, and particle size, in order to identify the most suitable fraction according to the invention.
It could be found that fractions that were enriched for exosomes were particularly useful in the methods according to the present invention, if they exhibited strong in vitro immunomodulatory effects in activity tests, where upon the addition of said exosome fraction, a reduced IL-Ιβ, TNF-a and/or IFN-γ cytokine response of effector cells of a donor could be found. Preferred is a method according to the present invention, wherein said levels of TGF-βΙ as measured in the activity tests are at least ten (or at least twenty) times higher (at 60.5 x 108 particles/mL) in the preparation than in plasma levels of healthy controls. Further preferred is a method according to the present invention, wherein said exosomes have a size of between about 70 to 200 nm, preferably between about 70 to 140 nm, or more preferably between about 70 to 120 nm. "About" shall mean a +/- 10% deviation. Further preferred, the exosomes are positive for cellular exosome markers, and even further preferred the protein content of the pharmaceutical preparation is higher than 1 mg/ml.
Furthermore, the method for producing a pharmaceutical preparation according to the present invention comprises d) admixing said enriched exosomes of step c) with at least one suitable pharmaceutical excipient and/or carrier. In general any suitable pharmaceutically acceptable excipient and/or carrier can be used. Preferably, said pharmaceutically acceptable excipient and/or carrier renders said preparation suitable for i.v. administration, such as for example, intravenous administration or infusion.
Yet another aspect of the present invention then relates to a pharmaceutical preparation comprising exosomes derived from a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs), preferably according to the present invention as described herein for use in the prevention and/or treatment of diseases.
Preferably, the diseases that are prevented and/or treated using the pharmaceutical preparation comprising exosomes derived from a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) according to the present invention are selected from the group consisting of inflammatory diseases, neuronal diseases, transplant rejections, GvHD, stroke, and ischemia.
Preferably, the diseases that are prevented and/or treated using the pharmaceutical preparation comprising exosomes derived from a cell culture medium supernatant from neonatal or adult
tissue-derived mesenchymal stem-cells (MSCs) according to the present invention are selected from pre- or postnatal damages of the nervous system, such as for example, brain damages related to hypoxia, inflammation, and/or ischemia.
Most preferably, the diseases that are prevented and/or treated using the pharmaceutical preparation comprising exosomes derived from a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) according to the present invention are selected from graft-versus-host disease, and transplant rejections following organ or bone marrow transplantation.
Yet another aspect of the present invention according to the present invention then relates to a method for preventing and/or the treatment of a disease selected from the group consisting of inflammatory diseases, neuronal diseases, transplant rejections, stroke, and ischemia in a patient, comprising administering to said patient an effective amount of a pharmaceutical preparation according to the present invention. Preferably, the disease that is prevented and/or treated is selected from pre- or postnatal damages of the nervous system, such as for example, brain damages related to hypoxia, inflammation, ischemia, graft-versus-host disease, and transplant rejections following organ transplantation.
Preferably, said preventing and/or the treatment according to the present invention comprises an administration through i.v. administration, such as for example, intravenous administration or infusion.
More preferred is a method for preventing and/or the treatment of a disease according to the present invention, wherein said patient is a newborn.
The invention thus also relates to the application of exosome-enriched fractions for the treatment of acquired neonatal as well as adult neural defects, for example the treatment of brain damages in neonates and grown-ups after hypoxia, inflammation, ischemia, etc. Furthermore, the treatment of ischemic strokes can be treated, and the adverse effect of the stroke can be reduced and/or reversed.
The invention thus also relates to the application of exosome-enriched fractions for the treatment of transplantation-related complications. Here, primarily the treatment of GvHD, and the
rejection of solid organs following transplantation have to be named. Treatments can be both prophylactic and/or therapeutic.
Based on their proposed broad multi-lineage differentiation potential mesenchymal stem cells (MSCs) became one of the most intensively studied adult stem cell entities within the last 15 years. Up to now, more than 300 different NIH-registered studies have addressed potential therapeutic impacts of clinically administered MSCs in a variety of clinical settings including treatment of acute myocardial infarction, stroke and acute kidney failure.
Several studies showed that the vast majority of intravenously administered MSCs get rapidly trapped in the lung and are rarely recovered in other tissues. Therefore, MSCs might improve clinical outcomes by paracrine effects rather than by their previously proposed engraftment into damaged host tissues (Lee et al, 2009). Indeed, the group of Lim earlier showed that the supernatants of in vitro expanded embryonic stem cell derived MSCs contain small extracellular vesicles, so called exosomes, whose infusion is sufficient to mediate a reduction of myocardial infarction sizes (see Lim et al, 2010).
The present invention will now be described further in the following examples, nevertheless, without being limited thereto. For the purpose of the present invention, all references as cited are incorporated by reference in their entireties.
Figure 1 shows the effect of exosome preparations on newborn rats that were partially confronted with LPS.
Figure 2 shows an example for the analyses of exosomes-containing fractions during the purification thereof using a monolithic column.
Figure 3 shows the transplantation-relevant effects of exosomes on a patient in a treatment scheme according to the examples. As symptom of GvHD the patient has diarrhea of more than 3 liters per day. After administration of MSC-derived exosome-enriched fractions, the symptoms were drastically reduced, without that other side-effects could be observed.
Figure 4 also shows the transplantation-relevant effects of exosomes on a patient in a treatment scheme according to the examples. As symptom of the GvHD, the patient developed
inflammations of the skin. After administration of MSC-derived exosome-enriched fractions, the symptoms were drastically reduced, without that other side-effects could be observed.
Figure 5 shows that MSC-fractions, such as MSCl Exo reduce the number of IL-Ιβ, TNF-a and IFN-γ secreting PBMCs in almost all settings, in particular in vitro. Immune suppressive effects on the cytokine responses of the other exosome preparations (MSC2 Exo, MSC3 Exo and MSC4 Exo) are slightly less prominent.
Figure 6 shows that depending on the exosome fraction, MSC-enriched exosomes suppress, do not modulate or increase the proliferation rates of treated T cells, respectively (Example III).
Figure 7 shows that certain exosome fractions such as Exo MSC A (fraction 3 in Fig. 2) and MSC Exo F (fraction 7 in Fig. 2) suppress proliferation of PHA stimulated T cells and IL- lbeta and TNF alpha release of stimulated T cells (see Example III).
Figure 8 shows that the effects of the in vitro studies (A) correlate with the effect in vivo (B).
Examples
Example I
Distinct MSC exosome preparations contain different cytokine compositions
The exosome-enriched fractions derived from the four MSC preparations were analyzed for their content of anti- and pro -inflammatory as well as apoptosis-inducing molecules (Table 1). Apart from the pro-inflammatory cytokines IFN-γ and IL-8, the exosome preparations contained high quantities of the anti-inflammatory molecules IL-10, TGF-βΙ and HLA-G. Neither the pro -inflammatory cytokines IL-Ιβ, IL-2, IL-6, IL-17a, IL-21 and TNF-a, the antiinflammatory cytokines IL-Ιβ Ra and IL13, nor the apoptosis-inducing molecule sFasL were detected in any of the exosome preparations (Table 1).
Especially the MSCl exosome preparation {MSCl Exo) contained elevated TGF-β levels, which surpassed TGF-β plasma levels of healthy donors by more than the factor 10. In addition MSCl Exo differed from the others preparations with regard to the anti-inflammatory cytokine IL-10 and the pro -inflammatory cytokine IFN-γ. The ratio of IL-10 to IFN-γ varied
between the four exosome preparations for almost two orders of magnitude (Table 1); the highest ratio was found for MSC1 Exo (1.02).
Table 1: Cytokine contents of the MSC exosome preparations. ^Levels are given in pg/mL; Levels are given in ng/mL; ^Preparation used for treatment; (4->N=20 for healthy controls
Distinct MSC exosome preparations reveal different capabilities to suppress inflammatory responses in vitro
Immune modulatory impacts of the MSC exosome preparations were tested in a mixed lymphocyte reaction using the HLA class I negative K562 as target cells. In order to reflect an allogeneic transplantation setting stable HLA-B35, HLA-E*01 :03 or HLA-B27-transfected K562 variants and cells of the HLA class I positive, respiratory syncytial virus infected (RSV) B-cell line were additionally used as allogeneic target cells. In three independent experiments target cells were co-cultured with PBMCs of healthy donors, either in the presence or absence
of MSC exosomes. Numbers of IL-Ιβ, TNF-a and IFN-γ secreting PBMCs were documented within the ELISpot assay as mean percentage ± SEM.
Compared to the co-cultures without exosomes, MSC1 Exo reduced the number of IL-Ι β, TNF-a and IFN-γ secreting PBMCs in almost all settings (Figure 5), in particular in vitro. Immune suppressive effects on the cytokine responses of the other exosome preparations (MSC2 Exo, MSC3 Exo and MSC4 Exo) were less prominent (Figure 5). These findings suggest that depending on the expanded MSCs, MSC-derived exosomes exert different immune modulatory functions.
Immunomodulatory effects of exosomes in vitro
Propagation of MSCs
Primary human MSCs were raised from mononuclear cells obtained by Ficoll density gradient centrifugation of bone marrow and raised mainly as described before (Hemeda et al, 2010). Instead of 10% fetal bovine serum (FBS) the inventors supplement the basal media with 5% human pooled platelet lysates. To exclude variations caused by the usage of different platelet lysate preparations, the inventors performed all experiments with the same batch of platelet lysates that the inventors produce by pooling 3040 individual human platelet samples. Growth conditions in the presence of human platelet lysates instead of animal sera such as FBS comply with GMP standards and are applicable for clinical settings (Doucet et al., 2005; Schallmoser et al, 2007 & 2009; Lange et al, 2007). MSCs were characterized by flow cytometry as CD90+/CD73+/CD105+/CD44+ cells lacking the expression of CD45 and the endothelial marker CD31. MSC differentiation capability along the osteogenic, cartilage and adi- pogenic lineage was evaluated.
Collection of MSC conditioned media and purification of exosomes
Starting at passage 3 conditioned media (CM) were harvested every 48 h and passed through a 0.22 μιη filter membrane to remove cell debris and larger vesicles. CM were collected and stored at -20°C. After thawing, CM were unified and processed by PEG precipitation. Briefly, PEG 6000 was added to CM. Following incubation for 8-12 h at 4°C exosomes were precipitated by centrifugation for 30 min at 1500 g. Obtained pellets were resolved in 1 mL 0.9% NaCl, washed in a total volume of 45 mL 0.9% NaCl for 12 h at 4°C and precipitated by ul- tracentrifugation for 2 h at 100,000 g. Again pellets were resolved in 1 mL of 0.9% NaCl,
diluted with 0.9% NaCl to required concentrations and stored as 1 mL aliquots at -80°C until use.
Quality control of exosomes
Protein concentrations of obtained aliquots were determined. Of each lot, 10 μ of exosome containing fraction was used to perform Western blots with antibodies recognizing exosome specific marker proteins, such as TsglOl, CD63, CD81 or CD82. Additionally, the inventors measured the concentration and size of the exosome by the NTA method as described previously (Sokolova et al, 2011). The concentration of pro- and anti-inflammatory cytokines (e.g., IL-Ιβ, IL-6, IL-8; TNF-a, IFN-γ, IL-4, IL-10, IL-13, TGF- βΐ, TSG-6) and of the Fas- ligand within the exosome fractions were measured using the Luminex method. Other methods that could be used include, for example, ELIspot, ELISA, and/or flow cytometry.
Immunomodulatory effects in vitro
As an immunomodulatory read out, the inventors established the T-cell/MNC proliferation assay within the group. In analogy to the inventors' CD34+ cell studies (Beckmann et al, 2007; Giebel et al, 2006) MNCs or magnetic cell separation enriched CD3+ cells were stained either with CFSE or PKH2 and raised in the presence of either a T cell/MNC proliferation stimulating agents (e.g. LPS, PMA, PHA, anti-CD3 beads) or of cells of the leukemic cell line K562. First, the inventors established a protocol allowing them to document T-cell/MNC proliferation by flow cytometry. Next, the inventors searched for a stimulant whose impact on the T cellslMNCs could effectively be modulated by MSC exosomes. Finally, the inventors found a stimulant, which triggered proliferation of different leukocytes within MNC fractions. Using gating strategies for B, T and NK cells the inventors analyzed impacts of MSC exosomes on these cells separately. To estimate effects of MSC-derived exosomes on the cytokine secretion of B, T and NK cells the inventors used the ELISpot assay. Furthermore, the inventors adapted a flow cytometry based cytokine secretion assay (Miltenyi, Bergisch-Gladbach), allowing them to study the effect of MSC exosomes on the release of pro -inflammatory cytokines (specifically IFN-a, TNF-a and IFN-γ) by stimulated immune cells.
Comparison of immunomodulatory activities of different MSC exosome preparations
Using the aforementioned assays the inventors compared the immunomodulatory activities of 15-20 independently obtained MSC exosome fractions. The inventors expected that some of these fractions would show strong immunomodulatory activity, whereas others would have no
effect on the immune response of stimulated immune cells. The three samples with the highest and the lowest immunomodulatory activities were compared in more detail. The inventors also comprehensively compared the expression profiles of the MSC propagations, from which corresponding exosome fractions were obtained. The inventors identified sets of candidate proteins that predicted immunomodulatory effects. The inventors validated these markers by evaluating their expression in additional MSC and exosome samples.
Immunomodulatory and neurorestorative effects of exosomes in vivo
MSC-derived exosomes using PEG preparations with high and low immunomodulatory capabilities in vitro were used for the in vivo approach. C57M6 mice were submitted to 30 minutes of intraluminal middle cerebral artery occlusion. This model is well characterized and has been extensively used in the inventors' lab before, resulting in localized infarcts of the striatum and most lateral cerebral cortex (Doeppner et al, 20096). Dissolved in a volume of 250 μί, 2 x 106 cell equivalents of exosomes were injected over 10 min via tail vein on days 1, 3, and 5 post-stroke. To compare effects of exosomes with those of MSCs, additional animals are examined, which received transplantations of MSCs used for the generation of exosome fractions. In these animals, a total of 1 x 106 MSCs dissolved in 250 normal saline were transplanted on day 1 post-stroke by intravenous injection. The latter animals receive normal saline injections on days 3 and 5. Vehicle-treated control animal were also prepared that receive injections of 250 μΙ_, normal saline only on days 1, 3 and 5 post-stroke. Four survival time points were examined, at which animals were sacrificed (7 d, 28 d, 56 d and 84 d after the stroke). Finally, four survival times were assessed in a total of 12 animals per condition and time point, resulting in a total of 240 animals, considering two exosome sources (immunomodulatory and nonimmunomodulatory) plus their corresponding MSCs.
Analysis of post-ischemic motor and coordination deficits
Post-ischemic motor and coordination deficits were analyzed using the rota rod, the tight rope and the corner turn test as previously described (Zhang et al, 2002; Doeppner et al, 201 lb). All animals were trained 1-2 days before induction of stroke to ensure sufficient test performance. Actual tests are performed on days 7, 14, 28, 56 and 84. Using the rota rod test, animals were put on a treadmill with an accelerating speed of 4-40 rpm. The maximum speed was achieved after 260 s, and maximum testing time was 300 s. The time until animals drop was registered and statistically analyzed. For the tight rope test, animals were placed on a 60 cm long rope grasping the string with their forepaws. Maximum test time was 60 s, and re-
suits were scored from 0 (minimum) to 20 (maximum) according to a validated score. For the corner turn test, two vertical boards were attached at one side with an angle of 30°, and each mouse was tested for the side chosen over 10 trials per test day. Whereas healthy animals left the corner without side preference, mice suffering from stroke preferentially turned to the left, non-impaired body side. Thereafter, the laterality index (LI) was calculated according to the following formula: (number of left turns - number of right turns)/10.
Analysis of post-ischemic structural brain injury, endogenous neurogenesis, axonal plasticity and angiogenesis
Brain injury was analyzed at the time points given using cresyl violet staining, NeuN staining, TdT -mediated dUTP-biotin nick end labeling (TUNEL) staining and analysis of astroglial scar formation using the astrocytic marker glial fibrillary acidic protein (GFAP). Since transplantation of stem cells can support endogenous repair mechanisms, postischemic neuro- regeneration after exosome treatment was analyzed as well. Therefore, mice received daily intraperitoneal injections of bromodeoxyuridine (BrdU) on days 8-18, to indirectly assess post-ischemic cell proliferation. After sacrifice of animals at the time points given, immuno- histochemial double staining against BrdU and various "differentiation" markers (e.g., Dcx, NeuN, CNPase) were performed (Doeppner et al, 2010b). To evaluate axonal plasticity, animals also received ipsi- and contralateral injections of the anterograde tract tracer biotinylated dextran amine (BDA) and cascade blue labeled dextran amine into the motor cortex on day 70 in order to determine exosome-induced pyramidal tract sprouting on day 84 post-stroke (Reitmeir et al, 2011). For analysis of angiogenesis, immunohistochemical protocols using the marker CD31 were employed (Wang et al, 2005). Tissue samples were collected for studies other than immunohistochemistry, such as realtime PCR, which allowed to evaluate post- ischemic inflammation, neuronal viability and astroglial scar formation. These assays included real-time PCR protocols according to the manufacturers' protocols.
Analysis of post-ischemic cerebral immune cell infiltration and peripheral immune response In order to assess quantitative changes of cerebral immune cell infiltration, FACS analysis was performed on day 7 post-stroke. In line with published protocols (Gelderblom et al, 2009), the inventors analyzed the relative frequency of microglia, neutrophils, macrophages, dendritic cells, natural killer cells and lymphocytes in brain tissue. Since stem cell induced neuroprotection also implies modulation of peripheral immune responses (Schwarting et al, 2008), peripheral immune cell subtypes were analyzed in the spleen, lymph nodes and blood.
More specifically, a differentiation analysis of CD l ib and CDl lc antigen-presenting cells and of lymphocyte subpopulations was performed.
Example II
As an undesired side effect of allogeneic bone marrow transplantation, some patients with malignant hematological diseases develop GvHD, i.e. a condition, in which the new immune system recognizes the patient's tissue as 'foreign' and attacks it. Usually, transplanted patients receive immunosuppressant treatments. Patients refractory to such treatments have a poor prognosis, while transplanted MSCs might improve the clinical response (Gotherstrom et al, 2004; Le Blanc et al, 2004). Meanwhile it is assumed that the immunomodulatory activities of MSCs result from secreted soluble factors rather than direct cellular interactions between engrafted MSCs and the patient's immune cells (Baron and Storb, 2012). The inventors hypothesized that the effectors are MSC-derived exosomes.
Given the history of a female patient with myelodysplasia syndrome that had received allogeneic bone marrow transplantation, the patient developed a refractory, severe GvHD. After written consent and approval by local ethics authorities, the inventors transfused MSC- derived exosomes for the very first time ever in man for compassionate use ('individueller Heilversuch'). Before delivery, the inventors purified exosomes from four different MSC lineages and tested their immunomodulatory properties extensively in vitro.
The inventors observed immunosuppressive properties of these exosomes in 3 out of 4 lineages. Next, the inventors chose exosomes with the strongest immunomodulatory effect, tested their interaction with the patient's blood cells in vitro and transfused the exosomes in intervals of 2-3 days in increasing doses into the patient (Fig. 3). The patient tolerated the exosome application very well and remarkably recovered from her GvHD symptoms during and after the therapy (Fig. 3). The patient was stable for more than 5 months. Even though the inventors cannot conclude at this state that the MSC exosomes led to the suppression of the GvHD symptoms, the patient's immune cells surprisingly showed a similar immune modulation as cells of healthy donors cells which were treated with exosomes in vitro.
Isolation of primary human mesenchymal stem cells
Human bone marrows (BM) were obtained from unrelated donors after informed consent according to the Declaration of Helsinki. Primary human MSCs were generated from mononu-
clear cells obtained by Ficoll (Biocoll Separating Solution, Biochrom AG, Berlin, Germany) density gradient centrifugation of bone marrow. The mononuclear cells were cultured in tissue culture 6 well plates at a density of 2 x 106 cells per well in MSC basal media (PAN) supplemented with 5% thrombocyte lysate, 1% glutamine, and 1% penicillin-streptomycin (PAA) for 24 hours. After 24 hours of incubation non-adherent cells were removed by medium exchange. After confluence had been reached, usually within 14 days, cells were continuously passaged after treatment with 0.25% trypsin. Adherent cells showed fibroblast-like morphology, and were able to differentiate along the adipogenic, chondrogenic, and osteogenic pathways.
Exosome isolation and purification (first variant)
Exosomes were isolated from cell culture supematants of MSCs derived from bone marrow. To remove cells, conditioned media were centrifuged for 5 min at 900 x g, and to remove remaining debris, another centrifugation step was performed for 1 h at 10,000 x g. To remove all particles bigger than 200 nm, the supematants were filtered through 0.2 um pore filters. To concentrate the exosomes, the filtrate was centrifuged for 2 h, 110,000 x g, at 4°C. The obtained precipitates were resuspended in 150 μΐ PBS and used for further analyses.
Exosome isolation and purification (second preferred variant)
PEG-precipitation of exosomes from conditioned media of adult tissue, in particular adipose tissue, bone marrow, placental tissue or umbilical cord derived MSCs
Starting at passage three, the inventors performed media exchanges every other day. MSC supematants were collected and stored at -20°C. After thawing, the supematants were unified and 3 units/mL of heparin and approx. 3 μg/mL Actilyse™ (Boehringer Ingelheim, Germany) were added. Supematants were incubated for 3 h at 37°C, and then filtered through 0.22 μιη bottle top filters.
Subsequently, a fraction of the medium (300 ml) was concentrated in 300 MWCO filters (Sar- torius), the supernatant was precipitated either using 10% or 12% PEG 6000 or 10% PEG8000. For the precipitation, supematants were transferred into 50 ml reaction vials (sterile, Greiner, Germany) prefilled with the precipitation reagent (PEG + NaCl). After mixing the vials were incubated over night (8-12 h at 4°C).
On the next day, vials were centrifuged at 1500 g for 30 min at 4°C. The supematants were carefully removed (25ml pipette). To collect remaining supematants, vials were centrifuged for another 5 min at 1500 g. Residual supernatant was removed using a 1 ml pipette. Pellets were resuspended in 1 ml NaCl 0.9%. To remove remaining PEG, resuspended precipitates were supplemented with 0.9% NaCl to a total volume of 45 ml and stored at 4°C for about 12h.
The 45 ml suspensions were split and transferred in half into two ultracentrifugation- vials. 0.9% NaCl was added to a total volume of 38 ml. The preparations were centrifuged for 2 h at 110.000 g. The supematants were removed from the sediments as obtained; the supematants were resuspended in 1 ml 0.9%> NaCl solution, and pooled. Then, the sample was filled up to 10 ml using 0.9% NaCl solution, and frozen in 1 ml aliquots in 15 ml reaction vials at -80°C.
Exosome isolation and purification via Monolithic columns
The columns were used according to the instructions of the manufacturer (www.biaseparations.com), with adjustments where necessary.
The supematants were also kept, and stored frozen at -20°C. Then, the aliquots were diluted 1 :5 (0.9%) NaCl solution), and used in the following assays:
- Microbiological test, virulence test, protein content, activity test, pyrogen test, and particle size.
Protein content
The protein contents were determined by the micro BCA assay (Thermo Fisher Scientific). Exosome specific proteins
Exosome specific proteins were identified by Western blot analysis. For this, purified exo- somes were treated with sample buffer (DTT, 0.1 % SDS, 0.1 M Tris HCl, pH 7.0) and spoiled for 5 min at 95°C. Proteins were separated on 12% SDS-PAGE gel and transferred to PVDF membranes. Membranes were blotted with antibodies to TsglOl (Sigma Aldrich), Flotilin 1 (BD Pharmingen), and CD81 (BD Pharmingen), then incubated with appropriate HRP- conjugated secondary antibodies (Dianova) and visualized by enhanced chemiluminescence (Pierce).
Nano particle tracking analysis (NTA)
For particle size determination, NTA was performed with a Nano Sight LM10 instrument equipped with the NTA 2.0 analytical software. All experiments were carried out at 1 :2000 dilutions, resulting in particle concentrations of approx. 3 x 108 per mL. Each experiment was carried out in triplicate. The 50% median value (D50) is given in all cases, and the standard deviation was calculated for all data.
Peripheral blood samples
In order to monitor the cytokine reactivity profiles of peripheral blood mononuclear cells (PBMC), EDTA plasma samples were procured from donors as well as from the respective patient before, during and after the MSC-exosome therapy. PBMC were isolated from blood samples by Lymphoprep (Invitrogen, Karlsruhe, Germany) density gradient centrifugation and cryopreserved until further analysis.
Cytokine and HLA-G determinations
Levels of soluble HLA-G (sHLA-G), cytokines and apoptosis-inducing molecules in MSC- exosome preparations were analyzed by ELISA. Before performing the various ELISA formats, exosomes (approx. 60.5 x 108 particles) were solubilized in 1 ml PBS-Tween (1%) and sonicated for 60 sec. sHLA-G concentrations were determined as described before (Schutt et al, 2010) with minor modifications: The monoclonal antibody (mAb) G233 (Exbio, Praha, Czech Republic) was used as capture reagent and bound sHLA-G was detected by the bioti- nylated mAb w6/32 (Leinco Technologies, St. Louis, Missouri, USA) followed by AMDEX™ Streptavidin HRP (Amersham, Freiburg, Germany). The detection limit of sHLA- G ELISA was 0.1 ng/ml. Concentrations of pro -inflammatory (IFN-γ, TNF-a, IL-lb, IL-2, IL- 6, IL-8, IL-17a, IL-21), anti-inflammatory (IL-l RA, IL-4, IL-10, IL-13, TGF-βΙ) and apoptosis-inducing molecules (soluble FasL) were determined according to the manufacturers' protocols (eBioscience, Frankfurt, Germany or BD Biosciences, San Jose, CA, USA). The assay sensitivities ranged between 2.0 and 20 pg per mL.
Determination of the IL-Ιβ, TNF-a, and IFN-γ cytokine reactivity profile by ELISpot
After thawing, PBMC and NK cells, isolated by negative selection out of these PBMC (Invitrogen, Karlsruhe, Germany), were adjusted to a fixed concentration of lxlO6 cells/mL and used as effector cells. To analyze the influence of the MSC-exosomes on the cytokine response lxlO6 effector cells were mixed with approx. 60.5 x 108 MSC-exosomes. In this case
effector cell suspensions with exosomes and without exosomes were used in cytokine stimulation experiments. HLA-E*01 :03, HLA-B35 or B27 transfected K562 cells and the parental HLA class I neg. K562 cell line served as stimulator cells and were adjusted to a concentration of O.lxlO6 cells/mL. All ELISpot assays were performed in an effector/stimulator ratio of 6: 1. For the IL-Ιβ and the TNF-a ELISpot assays, 12.5 x 104 effector cells and 0.25 x 104 stimulator cells and for the IFN-γ ELISpot assay, 75 x 104 effector cells and 1.5 x 104 stimulator cells were seeded with 200 μΐ culture medium containing 200U/mL IL-2 and 10% fetal calf serum onto MultiScreen-HA ELISpot plates (MAIPS4510, Millipore, Bedford, MA, USA) pre-coated with the respective capture antibody of the ELISpot Kit (eBioscience, Frankfurt, Germany). Effector cells without stimulator cells and stimulator cells without effector cells served as controls. Each stimulation condition was performed in duplicate. After 24h incubation at 37°C under 5% C02 numbers of cytokine producing effector cells were detected according to manufacturer's protocol. Spots were enumerated by the ELISpot reader AID EliSpot Reader Systems (Autoimmun Diagnostika GmbH, Strassberg, Germany) using AID EliSpot Software Version 6.x.
Characteristics of exosomes that were prepared from propagated MSCs of four different donors
Cell numbers, amount of harvested culture media, number of obtained exosomes and features of the obtained exosome fractions (particle numbers, protein amount etc.)
Protein content of the sample: 1.1 mg/ml;
Particle size: 199.5 +/- 70 nm;
Particle number: 6.02 * 1012 Particle/ml
Endotoxin content: 0.588 I.E./ml (+/- 50 %) corresponding to 2.94 I.E./ml of the concentrated solution.
Detection of Flotillin-1 and TsglOl as exosome markers was confirmed using antibodies and Western blot.
MSC preparation exosomes exerted donor-dependent immunosuppressive effects in vitro
Exosomes derived from four different MSC preparations were analyzed for the content of anti-, pro-, and apoptosis-inducing molecules: Besides the two pro -inflammatory cytokines IFN-γ and IL-8, MSC-exosomes contained preferentially high quantities of the antiinflammatory and/or immune suppressive molecules IL-10, TGF-βΙ and HLA-G. Especially,
TGF-βΙ levels were found to be ten times higher in the MSC-exosome solution of 60.5 x 10 particles/mL than in plasma levels of healthy controls.
Impact of exosomses on PB-MNCs
The influence of the MSC-exosomes on the cytokine response of PBMC or NK cells of PB donors on K562 leukemic cells was proven by IL-Ιβ, TNF-a and IFN-γ ELISpot using. The donors effector cells were co-cultured with HLA class I negative K562 or genetically engineered K562 cells expressing HLA-E*01 :03 or HLA-B35. It was found that the IL-Ιβ, TNF- α and IFN-γ cytokine responses of the donor's effector cells were reduced towards the different allogenic cells in the presence of MSC-exosomes compared to responses in the absence of MSC-exosomes.
This implies that the MSC-exosomes are able to impair in vitro the capability of PBMC to produce pro -inflammatory cytokines known to be operative in GvHD.
Therapies using exosome preparations according to the present invention
Experiments in rats (Figure 1)
In order to show positive effects of the preparations of the invention, experiments with acquired damages of the brain in newborn rats were undertaken. After lipopolysaccharide (LPS) administration, treated animals do no longer show weight gain compared to control animals (A), furthermore, an increased expression of apoptosis markers is found in the brains of the treated animals (B). Both the loss of weight gain and the amount of the apoptosis marker (Caspase 3) as found is markedly reduced upon the simultaneous administration of MSC derived fractions that are enriched for exosomes (A, B).
Human treatment (see Figures 3 and 4)
History of the patient - The 22-year old female patient received an allogeneic stem cell transplantation (AlloSCT) due to a myelodysplasia syndrome (MDS RAEB-T) with peripheral blood stem cells (PBSC) from a HLA-identical female donor following conditioning with busulfan, cyclophosphamide und melphalan.
After the first AlloSCT she developed mucosal GvHD but recovered hematologically with full donor chimerism. In October 2010 she suffered a relapse as a secondary acute myeloid leukemia (AML) with 57% blasts in the bone marrow examination. FLT3- and NPM-
mutations were detectable. Therefore, in January 2011 a second AlloSCT with PBSC from a HLA-identical male donor following conditioning with hyper- fractionated total body irradiation with 12 Gy and cyclophosphamide was performed. GvHD prophylaxis was standard CSP and a short course of MTX. She suffered a hyperacute GvHD of the skin with general erythema and bullae, grade IV, which initially responded sufficiently to high-dose steroid- therapy. Thus, the patient could be discharged with a dual immunosuppression of CSP and steroids. Unfortunately, the GvHD worsened shortly after discharge and the patient had to be admitted to hospital again in March 2011 with exacerbated skin GvHD and now also severe GIT involvement. She had therapy-refractory nausea and vomiting, abdominal cramping and diarrhea volumes reached up to five liters/day. The intestinal GvHD was repeatedly documented be endoscopy and histological examination.
The severe GvHD did not respond to a number of immunosuppressive lines. The patient immediately upon re-admission received high-dose steroids (5 mg/kg body weight [BW]) with no obvious improvement. Therefore, a course of anti-thymocyte globulin (10 mg/kg BW) was applied over five days which also did not lead to a response. Then, mycophenolate mofetil (MMF) and subsequently tacrolimus were added and since in some colon biopsies HHV-6, adeno and EBV virus DNA was detectable, a therapy course with cidofovir was initiated in late April 2011, too. Unfortunately, all these measures did not lead to a relief in GvHD symptoms. Therefore a course of infliximab (10 mg/kg BW once per week over a four week period) was initiated and 17 sessions of extracorporeal photopheresis were performed over four months. Throughout this time the basic immunosuppression with steroids, mostly in combination with either tacrolimus or MMF, was continued.
Given the history of this refractory and severe GvHD and the continuous suffering of the patient an experimental approach to control the GvHD symptoms was pursued after intensive discussion. A concept for an individual treatment attempt with MSC-exosomes was developed and approved by the Ethical Committee of the University Hospital of Essen, Germany. The patient agreed to the proposed treatment and gave her written consent.
Exosome application in increasing doses
The MSC-exosomes were administered between August, 1st to August, 15th 2011 in increasing doses.
Application Date Dosage
1 2011-08-01 1 ml of a 1 : 10 diluted exosome aliquot (100 μΐ)
2 2011-08-03 2 ml of a 1 : 10 diluted exosome aliquot (200 μΐ)
3 2011-08-05 3 ml of a 1 : 10 diluted exosome aliquot (300 μΐ)
4 2011-08-08 4 ml of a 1 : 10 diluted exosome aliquot (400 μΐ)
5 2011-08-10 1 ml of undiluted exosome aliquot
6 2011-08-12 2 ml of undiluted exosome aliquot
7 2011-08-15 4 ml of undiluted exosome aliquot
The patient received pre-medication with steroids and anti-histamins prior to each MSC- exosome application and was closely monitored in the intensive care unit. The therapy was tolerated very well, and no side effects were detected. After each administration, blood samples were taken and analyzed for their capability to produce IL-Ιβ, TNF-a, and IFN-γ in response towards K562, and K562 transfected with HLA-E*01 :03 or HLA-B27 (see above).
After the third MSC-exosome application the PBMC of the patient started to lessen their cytokine reaction towards the stimulator cells. Compared to the cytokines' responses before MSC-exosome therapy the numbers of IL-Ιβ, TNF-a and IFN-γ producing PBMC were found to be reduced more than fifty percent after the last application (p < 0.0001, One-way ANOVA). Thus, it seems that the MSC-exosomes are able to impair in-vivo the capability of PBMC to produce pro -inflammatory cytokines known to be operative in GvHD.
GvHD in the patient during and following exosome treatment
In line with the reduced cytokine reaction profile during the course of MSC-exosome therapy the clinical GvHD had improved significantly shortly after the beginning of treatment: The skin manifestation clearly lost activity, nausea, vomiting and diarrhea were dramatically reduced. The cutaneous and mucosal GvHD showed a remarkable response within two weeks which was stable even after 16 weeks after exosome therapy. The diarrhea volume, too, was objectively reduced after exosome therapy. Due to the response, the dosage of the immunosuppressive agents could be reduced.
Example III
Comparison of immune modulatory activities of Exosome-enriched fractions obtained from different MSC preparations.
The inventors compared the immune-mo dulative activity of exosome-enriched fractions obtained from 10 independent MSC preparations within T cell assays. To measure the exo- somes' impact on the proliferation of phytohemagglutinin (PHA) stimulated T cells, exo- somes equivalents of 800.000 MSCs (corresponds to 0.02 Units of the exosome enriched fractions) were added into each reaction. Apart of the control, approx. 200.000 CFSE stained, living PB-MNCs of given donors were stimulated with the lectin PHA (final concentration: 200 ng/ml) for 5 days. Thereafter, cells were stained with anti-CD3 antibodies and analyzed by flow cytometry in two independent experiments. As highlighted in Figure 6, all non- stimulated T cells retained their CFSE label. In contrast, a large proportion of PHA stimulated T cells showed reduced CFSE intensities indicating that these cells had been activated to divide.
Depending on the exosome fraction, MSC-enriched exosomes suppressed, did not modulate, or increased the proliferation rates of treated T cells, respectively (results of a characteristic experiment is given in Figure 6; increments of all experiments are given as black bars in Figure 7). These results demonstrate that exosome-enriched fractions obtained from independent MSC preparations differ in their ability to modulate the proliferative activity of PHA stimulated T cells.
In a second approach, PB-MNCs of 10 different peripheral blood donors were pooled. Ali- quots of 400.000 cells were cultured in triplicates each in a total volume of 200 μΐ, either in the presence or absence of 0.02 U of the same exosome fractions presented in Figure 6.
The release of the pro -inflammatory cytokines IL-lbeta and TNF-alpha were documented by ELISA after 3 days. Increments of the release (%) in comparison to cultures without exosomes are documented in Figure 7. Comparing the three parameters it becomes evident that certain exosome fractions such as Exo MSC A (fraction 3 in Fig. 7) and MSC Exo F (fraction 7 in Fig. 7) suppress proliferation of PHA stimulated T cells and IL-lbeta and TNF alpha release of stimulated T cells. In contrast MSC Exol promote proliferation of PHA stimulated T cells and forces and IL-lbeta and TNF alpha release within the mixed lymphocyte reaction assay.
Thus, these results demonstrate that exosome enriched- fractions of different MSCs exert different immune modulatory functions.
Literature as cited
Baron F, Storb R. Mesenchymal stromal cells: a new tool against graft-versus-host disease? Biol Blood Marrow Transplant. 2012 Jun;18(6):822-40.
Lai, R.C., Arslan, F., Lee, M.M., Sze, N.S., Choo, A., Chen, T.S., Salto-Tellez, M., Timmers, L., Lee, C.N., El Oakley, R.M., et al. (2010). Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Res 4, 214-222.
Lee, R.H., Pulin, A.A., Seo, M.J., Kota, D.J., Ylostalo, J., Larson, B.L., Semprun-Prieto, L., Delafontaine, P., and Prockop, D.J. (2009). Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti- inflammatory protein TSG-6. Cell Stem Cell 5, 54-63.
Schutt, P., Schutt, B., Switala, M., Bauer, S., Stamatis, G., Opalka, B., Eberhardt, W., Schuler, M., Horn, P. A., and Rebmann, V. (2010). Prognostic relevance of soluble human leukocyte antigen-G and total human leukocyte antigen class I molecules in lung cancer patients. Hum Immunol 71, 489-495.
Claims
1. Pharmaceutical preparation comprising exosomes, obtainable by a method comprising the following steps:
a) providing a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs) comprising exosomes,
b) enriching said exosomes, optionally comprising polyethylene glycol precipitation, c) determining an in vitro immunomodulatory effect, in particular an antiinflammatory effect and/or immune suppressive effect, of said enriched exosomes by, for example, a reduced IL-Ιβ, TNF-a, T-cell proliferation, and/or IFN-γ cytokine response of effector cells of a donor, and
d) selecting those enriched exosomes that exhibit an immunomodulatory effect, in particular an anti-inflammatory effect and/or immune suppressive effect.
2. The pharmaceutical preparation according to claim 1, wherein said MSCs are selected from human MSCs, and preferably from human MSCs derived from umbilical cord blood, umbilical cord tissue, placenta, bone or adipose tissue, and MSCs that have been cultured in platelet lysate.
3. The pharmaceutical preparation according to claim 1 or 2, wherein said exosomes in said enriched preparation comprise biological factors, such as, for example, proteins, such as cytokines, IFN-γ, IL-8, IL-10, TGF-βΙ, and HLA-G, and/or nucleic acids, such as, for example, microRNAs.
4. The pharmaceutical preparation according to claim 3, wherein said levels of TGF-βΙ are ten times higher (at 60.5 x 108 particles/mL) in the preparation than in plasma levels of healthy controls.
5. The pharmaceutical preparation according to any of claims 1 to 4, wherein said exosomes have a size of between about 70 to 200 nm, preferably between about 70 to 140 nm, are positive for cellular exosome markers, and wherein the protein content of the pharmaceutical preparation is higher than 1 mg/ml.
6. The pharmaceutical preparation according to any of claims 1 to 5, wherein said preparation is suitable for i.v. administration, such as for example, intravenous administration or infusion.
7. A method for producing a pharmaceutical preparation according to any of claims 1 to 6, comprising the following steps:
a) providing a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs), optionally from MSCs that have been cultured in platelet lysate, comprising exosomes,
b) enriching said exosomes, optionally comprising polyethylene glycol precipitation, c) determining an in vitro immunomodulatory effect, in particular an antiinflammatory effect and/or immune suppressive effect, of said enriched exosomes by, for example, a reduced IL-Ιβ, TNF-a, T-cell proliferation, and/or IFN-γ cytokine response of effector cells of a donor,
d) selecting those enriched exosomes that exhibit an immunomodulatory effect, in particular an anti-inflammatory effect and/or immune suppressive effect, and
e) admixing said enriched exosomes of step d) with at least one suitable pharmaceutical excipient and/or carrier.
8. The method according to claim 7, wherein said preparation as produced is suitable for i.v. administration, such as for example, intravenous administration or infusion.
9. A pharmaceutical preparation comprising exosomes derived from a cell culture medium supernatant from neonatal or adult tissue-derived mesenchymal stem-cells (MSCs), preferably according to any of claims 1 to 6 for use in the prevention and/or treatment of diseases.
10 The pharmaceutical preparation according to claim 9, wherein said diseases are selected from the group consisting of inflammatory diseases, neuronal diseases, transplant rejections, stroke, and ischemia.
11. The pharmaceutical preparation according to claim 10, wherein said diseases are selected from pre- or postnatal damages of the nervous system, such as for example, brain damages related to hypoxia, inflammation, and/or ischemia, or wherein said dis-
eases are selected from graft-versus-host disease, and transplant rejections following organ or bone marrow transplantation.
12. A method for preventing and/or the treatment of a disease selected from the group consisting of inflammatory diseases, neuronal diseases, transplant rejections, stroke, and ischemia in a patient, comprising administering to said patient an effective amount of a pharmaceutical preparation according to any of claims 1 to 6.
13. The method according to claim 12, wherein said disease is selected from pre- or postnatal damages of the nervous system, such as for example, brain damages related to hypoxia, inflammation, ischemia, graft-versus-host disease, and transplant rejections following organ or bone marrow transplantation.
14. The method according to claim 12 or 13, wherein said administration is through i.v. administration, such as for example, intravenous administration or infusion.
15. The method according to any of claims 12 to 14, wherein said patient is a newborn.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP13737623.2A EP2874634B1 (en) | 2012-07-18 | 2013-07-18 | Use of preparations comprising exosomes derived from mesenchymal stem cells (mscs) in the prevention and therapy of inflammatory conditions |
| US14/414,843 US9877989B2 (en) | 2012-07-18 | 2013-07-18 | Use of preparations comprising exosomes derived from mesenchymal stem cells (MSCs) in the prevention and therapy of inflammatory conditions |
| ES13737623T ES2908429T3 (en) | 2012-07-18 | 2013-07-18 | Use of preparations comprising exosomes derived from mesenchymal stem cells (msc) in the prevention and therapy of inflammatory conditions |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP12176968.1A EP2687219A1 (en) | 2012-07-18 | 2012-07-18 | Use of preparations comprising exosomes derived from mesenchymal stem cells (MSCs) in the prevention and therapy of inflammatory conditions |
| EP12176968.1 | 2012-07-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2014013029A1 true WO2014013029A1 (en) | 2014-01-23 |
Family
ID=48794113
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/EP2013/065219 WO2014013029A1 (en) | 2012-07-18 | 2013-07-18 | Use of preparations comprising exosomes derived from mesenchymal stem cells (mscs) in the prevention and therapy of inflammatory conditions |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US9877989B2 (en) |
| EP (2) | EP2687219A1 (en) |
| ES (1) | ES2908429T3 (en) |
| WO (1) | WO2014013029A1 (en) |
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015142061A1 (en) * | 2014-03-18 | 2015-09-24 | 사회복지법인 삼성생명공익재단 | Composition for treating inflammatory brain disease comprising stem-cell-derived exosome as an active ingredient |
| WO2015153880A3 (en) * | 2014-04-02 | 2015-12-10 | The Trustees Of Columbia University In The City Of New York | Modulation of hotair and adipogenesis |
| WO2016043654A1 (en) | 2014-09-15 | 2016-03-24 | Agency For Science, Technology And Research | Methods of treating graft versus host disease (gvhd) or epidermolysis bullosa (eb) with exosomes |
| WO2016104911A1 (en) * | 2014-12-23 | 2016-06-30 | 영남대학교 산학협력단 | Pharmaceutical composition for prevention or treatment of cellular senescence, comprising exosome as active ingredient |
| WO2016157142A1 (en) * | 2015-04-02 | 2016-10-06 | Stegi-Ra Trust | Composition for use in treating celiac disease |
| US20170112879A1 (en) * | 2014-06-09 | 2017-04-27 | University Of Washington | Methods of protection against ischemia reperfusion injury |
| JP2017526723A (en) * | 2013-09-16 | 2017-09-14 | エイジェンシー・フォー・サイエンス，テクノロジー・アンド・リサーチ | Method for treating graft-versus-host disease (GVHD) or epidermolysis bullosa (EB) with exosomes |
| EP3254684A1 (en) | 2016-06-08 | 2017-12-13 | Kyra Natalia Matahari de Miroschedji | Human platelet lysate or a fraction that is enriched for human platelet lysate derived extracellular vesicles for use in medicine |
| WO2018004145A1 (en) * | 2016-06-30 | 2018-01-04 | (주)아모레퍼시픽 | Anti-inflammatory composition containing yeast-derived extracellular vesicle |
| WO2018071677A1 (en) * | 2016-10-13 | 2018-04-19 | Vbc Holdings Llc | Medical uses of exosomes |
| WO2018071682A1 (en) * | 2016-10-13 | 2018-04-19 | Vbc Holdings Llc | Anti-inflammatory exosomes from inflamed cells or tissues |
| WO2018109525A1 (en) * | 2016-12-14 | 2018-06-21 | Università Degli Studi Di Padova | New anti-angiogenic extracellurlar vesicles |
| US20180318354A1 (en) * | 2015-11-02 | 2018-11-08 | Tigenix, S.A.U. | Mesenchymal stem cell-derived exosomes and their uses |
| KR20180131158A (en) * | 2017-05-31 | 2018-12-10 | 충북대학교 산학협력단 | Method for stimulating the secretion of exosome by stem cell and cosmetic composition comprising thereof |
| EP3328403A4 (en) * | 2015-07-31 | 2019-04-17 | Zen-Bio, Inc. | Exosome compositions and use thereof for soft tissue repair |
| WO2021020726A1 (en) * | 2019-07-30 | 2021-02-04 | 주식회사 엑소코바이오 | Novel exosome production method and application thereof |
| EP3654995A4 (en) * | 2017-07-17 | 2021-04-14 | Codiak BioSciences, Inc. | Nanovesicles produced from mesenchymal stromal cells for anti-inflammatory applications |
| CN115804847A (en) * | 2022-07-26 | 2023-03-17 | 四川省医学科学院·四川省人民医院 | PH/hydrogen peroxide/MMP 9 time-ordered response microsphere, exosome-carrying biological carrier and application |
Families Citing this family (52)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11660317B2 (en) | 2004-11-08 | 2023-05-30 | The Johns Hopkins University | Compositions comprising cardiosphere-derived cells for use in cell therapy |
| US11286463B2 (en) | 2012-03-08 | 2022-03-29 | Advanced ReGen Medical Technologies, LLC | Reprogramming of aged adult stem cells |
| JP6433896B2 (en) | 2012-08-13 | 2018-12-05 | シーダーズ−サイナイ・メディカル・センターＣｅｄａｒｓ−Ｓｉｎａｉ Ｍｅｄｉｃａｌ Ｃｅｎｔｅｒ | Exosomes and microribonucleic acids for tissue regeneration |
| JP6562897B2 (en) * | 2013-03-13 | 2019-08-21 | ユニバーシティー オブ マイアミＵｎｉｖｅｒｓｉｔｙ Ｏｆ Ｍｉａｍｉ | Methods for isolation and purification of microvesicles from cell culture supernatants and biological fluids |
| US10772911B2 (en) | 2013-12-20 | 2020-09-15 | Advanced ReGen Medical Technologies, LLC | Cell free compositions for cellular restoration and methods of making and using same |
| CR20160307A (en) | 2013-12-20 | 2016-11-08 | Advanced Regen Medical Tech Llc | Compositions for cellular restoration and methods of making and using same |
| AU2015313211B2 (en) * | 2014-09-09 | 2018-10-18 | Morinaga Milk Industry Co., Ltd. | Anti-inflammatory agent |
| JP6878274B2 (en) | 2014-10-03 | 2021-05-26 | シーダーズ−サイナイ・メディカル・センターＣｅｄａｒｓ−Ｓｉｎａｉ Ｍｅｄｉｃａｌ Ｃｅｎｔｅｒ | Myocardial cell-derived cells and exosomes secreted by such cells in the treatment of muscular dystrophy |
| WO2016057560A1 (en) * | 2014-10-06 | 2016-04-14 | Cedars-Sinai Medical Center | Polarization of macrophages to a healing phenotype by cardiosphere-derived cells and by the exosomes secreted by such cells |
| CN111773173B (en) * | 2014-11-07 | 2024-03-22 | 胞外体干细胞株式会社 | Composition for inducing adipogenic differentiation, regenerating adipose tissue, whitening skin or improving wrinkles comprising stem cell-derived exosomes |
| RU2017122817A (en) * | 2014-12-01 | 2019-01-10 | Самма Хелт | CAMKK1 AS A NEW REGENERATIVE THERAPY |
| WO2017064688A1 (en) * | 2015-10-15 | 2017-04-20 | Cells For Cells, S.P.A. | Neurite regeneration therapy based on exosomes derived from menstrual stem cells |
| EP3402543B1 (en) | 2016-01-11 | 2021-09-08 | Cedars-Sinai Medical Center | Cardiosphere-derived cells and exosomes secreted by such cells in the treatment of heart failure with preserved ejection fraction |
| WO2017152035A1 (en) * | 2016-03-03 | 2017-09-08 | Henry Ford Health System | 3-d collagen scaffold-generated exosomes and uses thereof |
| CA3023468A1 (en) | 2016-04-29 | 2017-11-02 | Advanced ReGen Medical Technologies, LLC | Microrna compositions and methods of making and using same |
| US11351200B2 (en) | 2016-06-03 | 2022-06-07 | Cedars-Sinai Medical Center | CDC-derived exosomes for treatment of ventricular tachyarrythmias |
| EP3494977A4 (en) * | 2016-08-05 | 2020-03-18 | Exostemtech Co., Ltd. | Composition for preventing or treating pulmonary fibrosis including exosome extracted from adipose-derived stem cell as active component |
| US11541078B2 (en) | 2016-09-20 | 2023-01-03 | Cedars-Sinai Medical Center | Cardiosphere-derived cells and their extracellular vesicles to retard or reverse aging and age-related disorders |
| CN106265741A (en) * | 2016-10-11 | 2017-01-04 | 青岛大学 | A kind of biological preparation promoting skin wound healing |
| EP3308795A1 (en) * | 2016-10-12 | 2018-04-18 | Unicyte EV AG | A composition of extracellular vesicles (evs) and medical uses thereof |
| IT201600109148A1 (en) * | 2016-10-28 | 2018-04-28 | Pharmaexceed Srl | Process to isolate and freeze-dry extracellular vesicles |
| CN106754668B (en) * | 2016-11-16 | 2020-08-25 | 沈阳细胞治疗工程技术研发中心有限公司 | Stem cell culture solution and injection |
| WO2018182356A1 (en) * | 2017-03-31 | 2018-10-04 | (주)안트로젠 | Culture containing high concentration of mesenchymal stem cell-derived high-purity exosome, and preparation method therefor |
| EP3612191A4 (en) | 2017-04-19 | 2020-12-30 | Cedars-Sinai Medical Center | Methods and compositions for treating skeletal muscular dystrophy |
| US20200392219A1 (en) * | 2017-05-08 | 2020-12-17 | Trustees Of Tufts College | Extracellular vesicles comprising membrane-tethered tgf-beta, compositions and methods of use thereof |
| CN107007541B (en) * | 2017-05-23 | 2020-11-13 | 北京希诺赛尔健康科技推广有限公司 | Application of exosome in skin whitening preparation |
| KR20190011213A (en) * | 2017-07-24 | 2019-02-01 | 한양대학교 에리카산학협력단 | Composition for preventing or treating osteoporosis comprising stem cell-derived exosomes |
| CA3073879A1 (en) * | 2017-08-24 | 2019-02-28 | The Texas A&M University System | Mesenchymal stem/stromal cell-derived extracellular vesicles and uses thereof in autoimmune diseases |
| CN107854418A (en) * | 2017-11-10 | 2018-03-30 | 南京九圣生物科技股份有限公司 | A kind of human umbilical cord mesenchymal stem cells excretion external use creams and preparation method thereof |
| CN111433352A (en) * | 2017-11-16 | 2020-07-17 | 细胞结构公司 | Placental culture to isolate exosomes |
| JP2021503286A (en) * | 2017-11-16 | 2021-02-12 | ボード・オブ・リージエンツ，ザ・ユニバーシテイ・オブ・テキサス・システム | Method for producing MSC-derived exosomes |
| EP3727351A4 (en) | 2017-12-20 | 2021-10-06 | Cedars-Sinai Medical Center | Engineered extracellular vesicles for enhanced tissue delivery |
| EP3731849A4 (en) * | 2017-12-28 | 2021-12-01 | Codiak BioSciences, Inc. | Exosomes for immuno-oncology and anti-inflammatory therapy |
| US10717981B2 (en) | 2018-01-18 | 2020-07-21 | Advanced ReGen Medical Technologies, LLC | Therapeutic compositions and methods of making and using the same |
| KR102015502B1 (en) | 2018-03-29 | 2019-08-28 | 아키소스템바이오스트래티지스(주) | A method for the isolation of stem cells from human umbilical cords |
| JP2021520790A (en) | 2018-04-10 | 2021-08-26 | ブレインストーム セル セラペウティクス リミテッド | Cell type-specific exosomes and their use |
| JP2021521193A (en) * | 2018-04-18 | 2021-08-26 | スンマ ヘルスＳｕｍｍａ Ｈｅａｌｔｈ | Compositions and methods for the treatment of ischemia and cardiomyopathy |
| CA3099042A1 (en) * | 2018-05-09 | 2019-11-14 | Children's Medical Center Corporation | Mesenchymal stromal cell exosome -treated monocytes and uses thereof |
| US20210228643A1 (en) * | 2018-07-26 | 2021-07-29 | Uniwersytet Jagiellonski | N vivo delivery system of the genome dna modifying enzymes and the use thereof |
| RU2707281C1 (en) * | 2018-09-17 | 2019-11-26 | Гордейчук Владимир Евгеньевич | Cancer immunotherapy method |
| CA3130744A1 (en) * | 2019-02-19 | 2020-08-27 | Direct Biologics Llc | Acellular intravenous infusion including mesenchymal stem cell growth factors and exosomes |
| CN110124058B (en) * | 2019-06-06 | 2021-11-26 | 福建医科大学附属第一医院 | Preparation of exosome-adriamycin nano-targeting drug derived from mesenchymal stem cells and research on in-vitro anti-osteosarcoma |
| CN110499287B (en) * | 2019-08-30 | 2021-07-23 | 博雅干细胞科技有限公司 | Method for simply preparing placenta mesenchymal stem cell exosome |
| CN112831464A (en) * | 2019-11-23 | 2021-05-25 | 路春光 | Industrial preparation method, quality research and application of human cell exosomes |
| WO2021251616A1 (en) * | 2020-06-10 | 2021-12-16 | 재단법인 대구경북첨단의료산업진흥재단 | Composition comprising exosomes derived from feline mesenchymal stem cells and method of treating inflammatory diseases by using same |
| CN111925983A (en) * | 2020-08-14 | 2020-11-13 | 福建医科大学附属协和医院 | Preparation method of high-IL-10-expression human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction |
| CN112007049A (en) * | 2020-09-21 | 2020-12-01 | 济南磐升生物技术有限公司 | Stem cell exosome composition for treating knee osteoarthritis |
| CN115212230A (en) * | 2021-04-21 | 2022-10-21 | 西比曼生物科技（上海）有限公司 | Pharmaceutical composition containing stem cell extracellular vesicles and application of pharmaceutical composition in treatment of respiratory inflammation |
| WO2023021150A1 (en) | 2021-08-18 | 2023-02-23 | Universität Duisburg-Essen | A biomarker indicating the therapeutic efficacy of extracellular vesicle (ev)-preparations |
| WO2023201195A2 (en) * | 2022-04-10 | 2023-10-19 | ThoraGenix Innovations, Inc. | Systems, methods, and devices of exosome delivery for bone healing |
| CN115364120B (en) * | 2022-09-29 | 2023-04-07 | 山东第一医科大学第一附属医院(山东省千佛山医院) | MSC (mesenchymal stem cell) source exosome cultured by hydrogel 3D and application thereof in cerebral ischemia repair |
| CN117511870B (en) * | 2024-01-03 | 2024-04-19 | 广东先康达生物科技有限公司 | Preparation method and application of composite extracellular body preparation with immune regulation |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009105044A1 (en) | 2008-02-22 | 2009-08-27 | Agency For Science, Technology And Research (A*Star) | Mesenchymal stem cell particles |
| WO2012020308A2 (en) | 2010-08-13 | 2012-02-16 | The University Court Of The University Of Glasgow | Cellular and molecular therapies |
| WO2012053976A1 (en) | 2010-10-18 | 2012-04-26 | Agency For Science, Technology And Research | Use of exosomes to promote or enhance hair growth |
| WO2012087241A1 (en) | 2010-12-20 | 2012-06-28 | Agency For Science, Technology And Research | Method of purifying exosomes |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6812023B1 (en) * | 2000-04-27 | 2004-11-02 | Anosys, Inc. | Methods of producing membrane vesicles |
| EP1773381A4 (en) * | 2004-07-01 | 2008-07-02 | Univ Pittburgh Of The Commonwe | Immunosuppressive exosomes |
| MX2011013452A (en) * | 2009-07-02 | 2012-04-30 | Ith Immune Therapy Holdings Ab | Exosome based treatment of cancer. |
| US9427450B2 (en) * | 2012-01-31 | 2016-08-30 | Xon Cells, Inc. | Therapeutic immune modulation by stem cell secreted exosomes |
-
2012
- 2012-07-18 EP EP12176968.1A patent/EP2687219A1/en not_active Withdrawn
-
2013
- 2013-07-18 WO PCT/EP2013/065219 patent/WO2014013029A1/en active Application Filing
- 2013-07-18 EP EP13737623.2A patent/EP2874634B1/en active Active
- 2013-07-18 ES ES13737623T patent/ES2908429T3/en active Active
- 2013-07-18 US US14/414,843 patent/US9877989B2/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009105044A1 (en) | 2008-02-22 | 2009-08-27 | Agency For Science, Technology And Research (A*Star) | Mesenchymal stem cell particles |
| WO2012020308A2 (en) | 2010-08-13 | 2012-02-16 | The University Court Of The University Of Glasgow | Cellular and molecular therapies |
| WO2012020307A2 (en) | 2010-08-13 | 2012-02-16 | The University Court Of The University Of Glasgow | Therapeutic uses of microvesicles and related micrornas |
| WO2012053976A1 (en) | 2010-10-18 | 2012-04-26 | Agency For Science, Technology And Research | Use of exosomes to promote or enhance hair growth |
| WO2012087241A1 (en) | 2010-12-20 | 2012-06-28 | Agency For Science, Technology And Research | Method of purifying exosomes |
Non-Patent Citations (15)
| Title |
|---|
| BARON F; STORB R: "Mensenchymal Stromal Cells: A New Tool against Graft-versus-Host Disease?", BIOL BLOOD MARROW TRANSPLANT, 29 September 2011 (2011-09-29) |
| BARON F; STORB R: "Mesenchymal stromal cells: a new tool against graft-versus-host disease?", BIOL BLOOD MARROW TRANSPLANT, vol. 18, no. 6, June 2012 (2012-06-01), pages 822 - 40, XP028919027, DOI: doi:10.1016/j.bbmt.2011.09.003 |
| BIEBACK K.: "Basic Biology of mesenchymal Stem Cells", TRANSFUS MED HEMOTHER, vol. 35, no. 3, 2008, pages 151 - 2 |
| BIEBACK K; BRINKMANN I: "Mesenchymal stromal cells from human perinatal tissues: From biology to cell therapy", WORLD J STEM CELLS, vol. 2, no. 4, 26 August 2010 (2010-08-26), pages 81 - 92, XP055101589, DOI: doi:10.4252/wjsc.v2.i4.81 |
| FLEISSNER F; GOERZIG Y; HAVERICH A; THUM T: "Microvesicles as novel bi- omarkers and therapeutic targets in transplantation medicine", AM J TRANSPLANT., vol. 12, no. 2, 14 November 2011 (2011-11-14), pages 289 - 97, XP055412640, DOI: doi:10.1111/j.1600-6143.2011.03790.x |
| HORSTMAN LL; JY W; MINAGAR A; BIDOT CJ; JIMENEZ JJ; ALEXANDER JS; AHN YS: "Cell-derived microparticles (MPs) and exosomes in neuro inflammatory disorders", INT REV NEUROBIOL., vol. 79, 2007, pages 227 - 68 |
| LAI RC; ARSLAN F; LEE MM; SZE NS; CHOO A; CHEN TS ET AL.: "Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury", STEM CELL RES, vol. 4, no. 3, May 2010 (2010-05-01), pages 214 - 22, XP027054731 |
| LAI, R.C.; ARSLAN, F.; LEE, M.M.; SZE, N.S.; CHOO, A.; CHEN, T.S.; SALTO-TELLEZ, M.; TIMMERS, L.; LEE, C.N.; EL OAKLEY, R.M. ET AL: "Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury", STEM CELL RES, vol. 4, 2010, pages 214 - 222, XP027054731 |
| LE BLANC K; RASMUSSON I; SUNDBERG B; GOTH- ERSTROM C; HASSAN M; UZUNEL M ET AL.: "Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells", LANCET, vol. 363, no. 9419, 1 May 2004 (2004-05-01), pages 1439 - 41, XP029609065, DOI: doi:10.1016/S0140-6736(04)16104-7 |
| LEE, R.H.; PULIN, A.A.; SEO, M.J.; KOTA, D.J.; YLOSTALO, J.; LARSON, B.L.; SEMPRUN-PRIETO, L.; DELAFONTAINE, P.; PROCKOP, D.J.: "Intravenous hMSCs improve myocardial infarc- tion in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6", CELL STEM CELL, vol. 5, 2009, pages 54 - 63 |
| LUDWIG AK; GIEBEL B: "Exosomes: Small vesicles partici- pating in intercellular communication", INT J BIOCHEM CELL BIOL, 19 October 2011 (2011-10-19) |
| LUDWIG AK; GIEBEL B: "Exosomes: Small vesicles participating in intercellular communication", INT J BIOCHEM CELL BIOL, 19 October 2011 (2011-10-19) |
| SCHUTT, P.; SCHUTT, B.; SWITALA, M.; BAUER, S.; STAMATIS, G.; OPALKA, B.; EBERHARDT, W.; SCHULER, M.; HORN, P.A.; REBMANN, V.: "Prognostic relevance of soluble human leukocyte antigen-G and total human leukocyte antigen class I molecules in lung cancer pa- tients", HUM IMMUNOL, vol. 71, 2010, pages 489 - 495, XP027006099 |
| TIMMERS L; LIM SK; ARSLAN F; ARMSTRONG JS; HOEFER IE; DOEVENDANS PA; PIEK JJ; EL OAKLEY RM; CHOO A; LEE CN: "Reduction of myocar- dial infarct size by human mesenchymal stem cell conditioned medium", STEM CELL RES., vol. 1, no. 2, November 2007 (2007-11-01), pages 129 - 37 |
| TOLAR J; HIPPEN KL; BLAZAR BR: "Immune regulatory cells in umbilical cord blood: T regulatory cells and mesenchymal stromal cells", BR J HAEMATOL, vol. 147, no. 2, October 2009 (2009-10-01), pages 200 - 6, XP055368434, DOI: doi:10.1111/j.1365-2141.2009.07781.x |
Cited By (31)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2017526723A (en) * | 2013-09-16 | 2017-09-14 | エイジェンシー・フォー・サイエンス，テクノロジー・アンド・リサーチ | Method for treating graft-versus-host disease (GVHD) or epidermolysis bullosa (EB) with exosomes |
| KR20150108795A (en) * | 2014-03-18 | 2015-09-30 | 사회복지법인 삼성생명공익재단 | Composition for treating inflammatory disease of brain comprising stem cell-derived exosome |
| US9919011B2 (en) | 2014-03-18 | 2018-03-20 | Samsung Life Public Welfare Foundation | Method for treating an inflammatory brain disease comprising administering a stem cell-derived exosome |
| WO2015142061A1 (en) * | 2014-03-18 | 2015-09-24 | 사회복지법인 삼성생명공익재단 | Composition for treating inflammatory brain disease comprising stem-cell-derived exosome as an active ingredient |
| KR101661847B1 (en) * | 2014-03-18 | 2016-09-30 | 사회복지법인 삼성생명공익재단 | Composition for treating inflammatory disease of brain comprising stem cell-derived exosome |
| EP3120857A4 (en) * | 2014-03-18 | 2017-12-13 | Samsung Life Public Welfare Foundation | Composition for treating inflammatory brain disease comprising stem-cell-derived exosome as an active ingredient |
| JP2017521357A (en) * | 2014-03-18 | 2017-08-03 | サムソン ライフ パブリック ウェルフェア ファウンデーション | Composition for treating brain inflammatory disease comprising stem cell-derived exosome as an active ingredient |
| WO2015153880A3 (en) * | 2014-04-02 | 2015-12-10 | The Trustees Of Columbia University In The City Of New York | Modulation of hotair and adipogenesis |
| US20170112879A1 (en) * | 2014-06-09 | 2017-04-27 | University Of Washington | Methods of protection against ischemia reperfusion injury |
| EP3193890A4 (en) * | 2014-09-15 | 2018-03-14 | Agency For Science, Technology And Research | Methods of treating graft versus host disease (gvhd) or epidermolysis bullosa (eb) with exosomes |
| WO2016043654A1 (en) | 2014-09-15 | 2016-03-24 | Agency For Science, Technology And Research | Methods of treating graft versus host disease (gvhd) or epidermolysis bullosa (eb) with exosomes |
| WO2016104911A1 (en) * | 2014-12-23 | 2016-06-30 | 영남대학교 산학협력단 | Pharmaceutical composition for prevention or treatment of cellular senescence, comprising exosome as active ingredient |
| WO2016157142A1 (en) * | 2015-04-02 | 2016-10-06 | Stegi-Ra Trust | Composition for use in treating celiac disease |
| EP3328403A4 (en) * | 2015-07-31 | 2019-04-17 | Zen-Bio, Inc. | Exosome compositions and use thereof for soft tissue repair |
| US20180318354A1 (en) * | 2015-11-02 | 2018-11-08 | Tigenix, S.A.U. | Mesenchymal stem cell-derived exosomes and their uses |
| US11857575B2 (en) * | 2015-11-02 | 2024-01-02 | Takeda Pharmaceutical Company Limited | Mesenchymal stem cell-derived exosomes and their uses |
| EP3468568B1 (en) | 2016-06-08 | 2020-09-02 | Lysatpharma GmbH | Human platelet lysate derived extracellular vesicles for use in medicine |
| WO2017211906A1 (en) | 2016-06-08 | 2017-12-14 | De Miroschedji Kyra Natalia Matahari | Human platelet lysate derived extracellular vesicles for use in medicine |
| EP3254684A1 (en) | 2016-06-08 | 2017-12-13 | Kyra Natalia Matahari de Miroschedji | Human platelet lysate or a fraction that is enriched for human platelet lysate derived extracellular vesicles for use in medicine |
| WO2018004145A1 (en) * | 2016-06-30 | 2018-01-04 | (주)아모레퍼시픽 | Anti-inflammatory composition containing yeast-derived extracellular vesicle |
| WO2018071677A1 (en) * | 2016-10-13 | 2018-04-19 | Vbc Holdings Llc | Medical uses of exosomes |
| WO2018071682A1 (en) * | 2016-10-13 | 2018-04-19 | Vbc Holdings Llc | Anti-inflammatory exosomes from inflamed cells or tissues |
| CN110072991A (en) * | 2016-10-13 | 2019-07-30 | Vbc控股有限责任公司 | From the anti-inflammatory excretion body of the cell or tissue of inflammation |
| WO2018109525A1 (en) * | 2016-12-14 | 2018-06-21 | Università Degli Studi Di Padova | New anti-angiogenic extracellurlar vesicles |
| WO2018109700A1 (en) * | 2016-12-14 | 2018-06-21 | Università Degli Studi Di Padova | New anti-angiogenic extracellurlar vesicles |
| KR101985941B1 (en) * | 2017-05-31 | 2019-06-04 | 충북대학교 산학협력단 | Method for stimulating the secretion of exosome by stem cell and cosmetic composition comprising thereof |
| KR20180131158A (en) * | 2017-05-31 | 2018-12-10 | 충북대학교 산학협력단 | Method for stimulating the secretion of exosome by stem cell and cosmetic composition comprising thereof |
| EP3654995A4 (en) * | 2017-07-17 | 2021-04-14 | Codiak BioSciences, Inc. | Nanovesicles produced from mesenchymal stromal cells for anti-inflammatory applications |
| WO2021020726A1 (en) * | 2019-07-30 | 2021-02-04 | 주식회사 엑소코바이오 | Novel exosome production method and application thereof |
| CN115804847A (en) * | 2022-07-26 | 2023-03-17 | 四川省医学科学院·四川省人民医院 | PH/hydrogen peroxide/MMP 9 time-ordered response microsphere, exosome-carrying biological carrier and application |
| CN115804847B (en) * | 2022-07-26 | 2023-08-15 | 四川省医学科学院·四川省人民医院 | PH/hydrogen peroxide/MMP 9 time sequence response microsphere, exosome-carrying biological carrier and application |
Also Published As
| Publication number | Publication date |
|---|---|
| EP2687219A1 (en) | 2014-01-22 |
| US20150190429A1 (en) | 2015-07-09 |
| EP2874634B1 (en) | 2021-12-01 |
| EP2874634A1 (en) | 2015-05-27 |
| ES2908429T3 (en) | 2022-04-29 |
| US9877989B2 (en) | 2018-01-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2874634B1 (en) | Use of preparations comprising exosomes derived from mesenchymal stem cells (mscs) in the prevention and therapy of inflammatory conditions | |
| US11730768B2 (en) | Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids | |
| EP3527981A2 (en) | Method for sorting highly effective stem cells for treating immune disorder | |
| JP7249951B2 (en) | Generation of therapeutic cells using extracellular components of target organs | |
| US8647678B2 (en) | Anti-inflammatory macrophages and uses thereof | |
| KR20220024060A (en) | Exosomes for disease treatment | |
| Gortner et al. | Regenerative therapies in neonatology: clinical perspectives | |
| WO2014089397A1 (en) | Compositions and methods of treating and preventing pulmonary fibrosis | |
| US10406181B2 (en) | Method for reducing the inflammatory activity of a stem cell transplant and use thereof | |
| Yu et al. | The potential of mesenchymal stem cells to induce immune tolerance to allogeneic transplants | |
| US20230414673A1 (en) | Extracellular vesicles derived from cardiosphere-derived cells as anti-shock therapeutics | |
| Van Riet et al. | Persistence of residual tumour cells after cytokine‐mediated ex vivo expansion of mobilized CD34+ blood cells in multiple myeloma | |
| Estrela | Delineation of optimal culture conditions for the production of therapeutic human mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) in static cultures | |
| de Witte et al. | Immunomodulation Induced by Mesenchymal Stem Cells (MSC) is Triggered through Phagocytosis of MSC by Innate Immune Cells | |
| Vinci | From understanding the molecular basis of Graft-versus-Host Disease (GvHD), to new diagnostic tools and innovative treatments for improving the management of patients undergoing allogeneic Hematopoietic Stem Cell Transplantation (HSCT) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 13737623 Country of ref document: EP Kind code of ref document: A1 |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 14414843 Country of ref document: US |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 2013737623 Country of ref document: EP |