CN100535106C - 使间充质干细胞分化为神经细胞的方法 - Google Patents
使间充质干细胞分化为神经细胞的方法 Download PDFInfo
- Publication number
- CN100535106C CN100535106C CNB028083911A CN02808391A CN100535106C CN 100535106 C CN100535106 C CN 100535106C CN B028083911 A CNB028083911 A CN B028083911A CN 02808391 A CN02808391 A CN 02808391A CN 100535106 C CN100535106 C CN 100535106C
- Authority
- CN
- China
- Prior art keywords
- cell
- neurocyte
- stem cell
- bfgf
- egf
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims abstract description 34
- 210000002901 mesenchymal stem cell Anatomy 0.000 title abstract description 7
- 210000003061 neural cell Anatomy 0.000 title abstract description 4
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims abstract description 42
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims abstract description 39
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 19
- 201000010099 disease Diseases 0.000 claims abstract description 18
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 claims abstract description 6
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 claims abstract 3
- 210000004027 cell Anatomy 0.000 claims description 96
- 210000000130 stem cell Anatomy 0.000 claims description 73
- 230000004069 differentiation Effects 0.000 claims description 24
- 210000001130 astrocyte Anatomy 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 8
- 230000004770 neurodegeneration Effects 0.000 claims description 5
- 208000015122 neurodegenerative disease Diseases 0.000 claims description 5
- 208000018737 Parkinson disease Diseases 0.000 claims description 4
- 208000012661 Dyskinesia Diseases 0.000 claims description 3
- 239000006143 cell culture medium Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 201000006474 Brain Ischemia Diseases 0.000 claims description 2
- 201000011240 Frontotemporal dementia Diseases 0.000 claims description 2
- 208000023105 Huntington disease Diseases 0.000 claims description 2
- 206010070511 Hypoxic-ischaemic encephalopathy Diseases 0.000 claims description 2
- 208000000609 Pick Disease of the Brain Diseases 0.000 claims description 2
- 208000020339 Spinal injury Diseases 0.000 claims description 2
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims 1
- 102100021866 Hepatocyte growth factor Human genes 0.000 abstract description 30
- 238000012258 culturing Methods 0.000 abstract description 5
- 230000001537 neural effect Effects 0.000 abstract description 5
- 210000001185 bone marrow Anatomy 0.000 abstract description 4
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 abstract 1
- 210000001616 monocyte Anatomy 0.000 description 15
- 210000002569 neuron Anatomy 0.000 description 13
- 210000001519 tissue Anatomy 0.000 description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 10
- 210000001789 adipocyte Anatomy 0.000 description 9
- 210000001612 chondrocyte Anatomy 0.000 description 9
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 9
- 210000001671 embryonic stem cell Anatomy 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 9
- 102000053171 Glial Fibrillary Acidic Human genes 0.000 description 8
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 230000024245 cell differentiation Effects 0.000 description 8
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 8
- 241000282414 Homo sapiens Species 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 210000003995 blood forming stem cell Anatomy 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 210000001178 neural stem cell Anatomy 0.000 description 5
- 210000000056 organ Anatomy 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 210000001161 mammalian embryo Anatomy 0.000 description 4
- 210000000274 microglia Anatomy 0.000 description 4
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 3
- 102000004877 Insulin Human genes 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 3
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 102000013275 Somatomedins Human genes 0.000 description 3
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 3
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 3
- 229960003957 dexamethasone Drugs 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- CGIGDMFJXJATDK-UHFFFAOYSA-N indomethacin Chemical compound CC1=C(CC(O)=O)C2=CC(OC)=CC=C2N1C(=O)C1=CC=C(Cl)C=C1 CGIGDMFJXJATDK-UHFFFAOYSA-N 0.000 description 3
- 208000014674 injury Diseases 0.000 description 3
- 239000008194 pharmaceutical composition Substances 0.000 description 3
- 230000002062 proliferating effect Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 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 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000012981 Hank's balanced salt solution Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000002490 cerebral effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 210000003677 hemocyte Anatomy 0.000 description 2
- 229960000905 indomethacin Drugs 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N lactose group Chemical group OC1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@@H](O)[C@H](O2)CO)[C@H](O1)CO GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- MEIRRNXMZYDVDW-MQQKCMAXSA-N (2E,4E)-2,4-hexadien-1-ol Chemical compound C\C=C\C=C\CO MEIRRNXMZYDVDW-MQQKCMAXSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- APIXJSLKIYYUKG-UHFFFAOYSA-N 3 Isobutyl 1 methylxanthine Chemical compound O=C1N(C)C(=O)N(CC(C)C)C2=C1N=CN2 APIXJSLKIYYUKG-UHFFFAOYSA-N 0.000 description 1
- HSTOKWSFWGCZMH-UHFFFAOYSA-N 3,3'-diaminobenzidine Chemical compound C1=C(N)C(N)=CC=C1C1=CC=C(N)C(N)=C1 HSTOKWSFWGCZMH-UHFFFAOYSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 229930183010 Amphotericin Natural products 0.000 description 1
- QGGFZZLFKABGNL-UHFFFAOYSA-N Amphotericin A Natural products OC1C(N)C(O)C(C)OC1OC1C=CC=CC=CC=CCCC=CC=CC(C)C(O)C(C)C(C)OC(=O)CC(O)CC(O)CCC(O)C(O)CC(O)CC(O)(CC(O)C2C(O)=O)OC2C1 QGGFZZLFKABGNL-UHFFFAOYSA-N 0.000 description 1
- 108010081589 Becaplermin Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 1
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 102000002734 Collagen Type VI Human genes 0.000 description 1
- 108010043741 Collagen Type VI Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 239000004386 Erythritol Substances 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 239000007995 HEPES buffer Substances 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
- 101000888419 Homo sapiens Glial fibrillary acidic protein Proteins 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 102000011931 Nucleoproteins Human genes 0.000 description 1
- 108010061100 Nucleoproteins Proteins 0.000 description 1
- NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 0.000 description 1
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 206010037714 Quadriplegia Diseases 0.000 description 1
- 241000978776 Senegalia senegal Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound 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 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229940009444 amphotericin Drugs 0.000 description 1
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- -1 anti-agglutinant Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- DHCLVCXQIBBOPH-UHFFFAOYSA-N beta-glycerol phosphate Natural products OCC(CO)OP(O)(O)=O DHCLVCXQIBBOPH-UHFFFAOYSA-N 0.000 description 1
- GHRQXJHBXKYCLZ-UHFFFAOYSA-L beta-glycerolphosphate Chemical compound [Na+].[Na+].CC(CO)OOP([O-])([O-])=O GHRQXJHBXKYCLZ-UHFFFAOYSA-L 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 150000001886 cortisols Chemical class 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000013118 diabetic mouse model Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000019414 erythritol Nutrition 0.000 description 1
- 229940009714 erythritol Drugs 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229920000591 gum Polymers 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 229940000351 hemocyte Drugs 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 210000001320 hippocampus Anatomy 0.000 description 1
- 102000051520 human GFAP Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 150000003840 hydrochlorides Chemical class 0.000 description 1
- 230000001709 ictal effect Effects 0.000 description 1
- 238000003365 immunocytochemistry Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 229960004232 linoleic acid Drugs 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 229960002160 maltose Drugs 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 229960001855 mannitol Drugs 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000001259 mesencephalon Anatomy 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- OSWPMRLSEDHDFF-UHFFFAOYSA-N methyl salicylate Chemical class COC(=O)C1=CC=CC=C1O OSWPMRLSEDHDFF-UHFFFAOYSA-N 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 210000002161 motor neuron Anatomy 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 210000000107 myocyte Anatomy 0.000 description 1
- 230000001114 myogenic effect Effects 0.000 description 1
- 210000000478 neocortex Anatomy 0.000 description 1
- 230000001423 neocortical effect Effects 0.000 description 1
- 239000003900 neurotrophic factor Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000004248 oligodendroglia Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 210000005034 parasympathetic neuron Anatomy 0.000 description 1
- 239000003182 parenteral nutrition solution Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 210000004197 pelvis Anatomy 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 229930029653 phosphoenolpyruvate Natural products 0.000 description 1
- DTBNBXWJWCWCIK-UHFFFAOYSA-N phosphoenolpyruvic acid Chemical compound OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical class CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229930002330 retinoic acid Natural products 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 210000003625 skull Anatomy 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229950003937 tolonium Drugs 0.000 description 1
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000011735 vitamin B7 Substances 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0619—Neurons
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/12—Hepatocyte growth factor [HGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1353—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Neurology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Neurosurgery (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Psychology (AREA)
- Hospice & Palliative Care (AREA)
- Psychiatry (AREA)
- Developmental Biology & Embryology (AREA)
- Rheumatology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
一种使骨髓间充质干细胞分化为神经细胞的方法，其包括在含有表皮生长因子(EGF)，碱性成纤维细胞生长因子(bFGF)和肝细胞生长因子(HGF)的培养基中培养间充质干细胞，并且由此生产的神经细胞可用于神经疾病的治疗。
Description
发明领域
本发明涉及通过在含有表皮生长因子(EGF)，碱性成纤维细胞生长因子(bFGF)和肝细胞生长因子(HGF)的培养基中培养它们，使骨髓中的间充质干细胞分化为神经细胞的一种方法，以及含有神经细胞作为有效成分用于治疗神经疾病的组合物。
背景技术
干细胞具有在培养中无限***的能力并且在特异性的分化刺激物的刺激下产生构成组织的特化细胞。
根据它们的分化潜能，干细胞分为胚胎干细胞(ES细胞)和组织特异性干细胞。ES细胞是在胚泡期从胚胎内细胞团(ICM)分离而来，并且是多潜能的，即它们实际上能够分化为在生物中发现的全部类型的细胞。
相反，组织特异性干细胞在胚胎发育过程中器官形成的阶段出现，并且它们是器官特异性的和多能的，即它们通常定向产生构成特定器官的细胞。这些组织特异性干细胞在大多数成年人器官中保留并履行不断补充正常或病理性发生的细胞损失的关键任务。代表性的组织特异性干细胞包括在骨髓中存在的造血干细胞和间充质干细胞。造血干细胞产生各种血细胞如红细胞和白细胞；间充质干细胞产生***细胞，例如成骨细胞，成软骨细胞，脂肪细胞和成肌细胞。
近来，由于人胚胎干细胞的成功分离，干细胞的临床应用已经吸引越来越多的兴趣。干细胞最值得注意的潜在应用是它们作为用于细胞置换治疗的细胞供应的理想来源。几乎不可治愈的疾病，例如神经变性疾病如帕金森氏病和阿尔茨海默氏病，由骨髓损伤，白血病，中风，幼年发作性糖尿病，心梗塞和肝硬变引起的四肢麻痹，是由于构成器官的细胞的破坏和永久性功能失调所导致，其中从外部补充细胞的损失的细胞置换治疗已经作为一种有效治疗出现。
然而，尽管细胞置换治疗的明显益处，在它的临床应用中存在许多限制。具体地，其中将从供体组织分离的完全分化的细胞移植于患者中的常规方法，有难以获得足够量的提供给患者的细胞的问题。为了解决该问题，在细胞置换治疗中可使用从分离的胚胎干细胞分化而来的特定组织的细胞或从分离的和增殖的组织特异性干细胞分化而来的细胞。
到目前为止，已经证明小鼠胚胎干细胞在培养皿上可以分化为各种细胞如造血细胞，心肌细胞，分泌胰岛素的胰细胞和神经细胞。另外，几个报道已经证明，从干细胞分化的细胞的移植在由细胞损失导致的疾病的治疗中有效。例如，当将从胚胎干细胞分化的合成髓鞘质的少突胶质细胞移植于小鼠中时，小鼠中的髓鞘质的合成增加(Brustle等，Science，285：754-756，1999)。通过将从胚胎干细胞分化的分泌胰岛素的细胞移植于糖尿病小鼠模型中调节血糖水平(Soria等，Diabetes，49：157-162，2000)。另外，通过将由胚胎干细胞分化的神经细胞移植于有骨髓损伤的小鼠中显著地治疗了由骨髓损伤导致的运动障碍(McDonald等，Nat.Med.，5(12)：1410-1412，1999)。
然而，由于只是最近才成功分离出人胚胎干细胞，并且没有关于胚胎干细胞在培养皿上分化为除了神经细胞以外的其它特化细胞的报道，由胚胎干细胞分化的特异性的组织细胞在细胞置换治疗中的临床应用还保持在一个可能性的水平上。
另外，由于从胚胎干细胞分化为靶细胞的效率低，在移植中有由和靶细胞混合的其它细胞导致的有害副作用的风险。因此，为了从胚胎干细胞分化的细胞更安全的临床应用，存在开发一种精确分化方法的需要。
另一方面，在当组织特异性干细胞被用于细胞置换治疗中的情形下，在长期培养中，可能出现细胞增殖能力降低或分化为不利的细胞的问题。另外，为了治疗神经变性疾病如帕金森氏病需要移植神经细胞。因为直接从患者获得神经干细胞是困难的，它们通常通过培养从死胎脑组织分离的神经干细胞并且使它们分化为神经细胞而获得。然而，胎脑的使用引起伦理问题并且被不充足的供应所限制，并且可能导致免疫排斥。另外，大部分的神经干细胞易于分化为星形胶质细胞而不是神经元。
因此，如果可能使在患者自身骨髓中的间充质干细胞分化为在细胞置换治疗中使用的神经细胞，可以很容易地供应神经细胞并且象免疫排斥的问题在治疗中将不会出现。
到目前为止，已认为一种类型的干细胞只分化为属于一个特定***的组织细胞。据报道在各种生长因子如血小板衍生生长因子，碱性成纤维细胞生长因子(bFGF)，转化生长因子-β(TGF-β)和表皮生长因子(EGF)的存在下，间充质干细胞体外形成集落(Kuznetsov等，Br.J.Haemato1.，97：561，1997；和van den Bos C.等，Human Cell，10：45，1997)，并且大约三分之一的原始贴壁细胞具有多潜能性，从而分化为***细胞如成骨细胞，成软骨细胞和脂肪细胞(Pittenger MF等，Science，284：143，1999)。另外，Ferrari G.等报道骨髓是形成新肌的生肌前体细胞的来源(Science，279：1528，1998)。
最近的研究报道间充质干细胞还能分化为神经***的细胞。例如，Sanchez-Ramos等报道间充质干细胞在视黄酸和脑衍生神经营养因子(BDNF)的存在下经培养分化为神经元和星形胶质细胞(Exp.Neurology，164：247-256，2000)。Dale Woodbury等报道在骨髓中的间充质干细胞在抗氧化剂如β-巯基乙醇和二甲基亚砜(DMSO)的存在下分化为神经细胞(J.Neuro.Res.，61：364-370，2000)。然而，强分化诱导试剂如DMSO的使用在临床应用中可能导致问题。
本发明者已努力发现高度安全和能够使骨髓中的干细胞分化为神经细胞的物质，并且已发现HGF促进骨髓间充质干细胞分化为神经细胞，还发现将EGF和bFGF加入培养基中，与HGF一起，不但显著地促进干细胞分化为神经细胞，而且增强产生的神经细胞的扩增。
已报道当将它们加入到用于培养从脑组织分离的神经干细胞的无血清培养基中时，EGF和bFGF刺激神经干细胞分化为神经元或星形胶质细胞(Melissa等，Exp.Neurology，158：265-278，1999)。
已报道HGF增强在海马和中脑中的神经元的生存力，并且诱导在新大脑皮质移植(neocortical explant)中轴突的生长(Hamanoue M等，J.Neurosci.Res.，43：554-564，1996)。另外，在周围神经***中，它作为运动神经元的生存因子起作用(Ebens A等，Neuron，17：1157-1172，1996)，并且参与感觉神经元和副交感神经元的生长和生存(Fleur Davey等，Mol.Cell Neurosci.，15：79-87，2000)。
然而，尚未报道通过在含有EGF，bFGF和HGF的培养基中培养它们，能够使间充质干细胞分化为神经细胞。
发明概述
因此，本发明的一个目的是提供一种使骨髓中的间充质干细胞或单核细胞分化为神经细胞的方法。
本发明的另一个目的是提供通过所述方法分化的神经细胞和一种包括神经细胞作为有效成分的用于治疗神经疾病的药物组合物。
本发明的另一个目的是提供一种用于治疗哺乳动物中神经疾病的方法，其包括将通过上述方法生产的神经细胞施用于需要其的受治疗者。
因此，依照本发明的一个方面，提供一种用于使骨髓中的间充质干细胞分化为神经细胞的方法，其包括在含有表皮生长因子(EGF)，碱性成纤维细胞生长因子(bFGF)和肝细胞生长因子(HGF)的培养基中培养间充质干细胞。
附图说明
本发明上述和其它的目的和特征从结合下述附图的本发明的下述描述中将变得明显，其中所述附图分别表示：
图1：来源于在含有10ng/ml EGF，20ng/ml bFGF和20ng/ml HGF的培养基上培养4周的骨髓单核细胞的贴壁细胞的显微照片(x100；以下，使用相同的放大率)；
图2：从在含有10ng/ml EGF，20ng/ml bFGF和20ng/ml HGF的培养基上培养8周的骨髓单核细胞分化来的神经细胞的显微照片；
图3A和3B：分别为神经元和星形胶质细胞显微照片，其是从图2的神经细胞分离而来；
图4A到4C：图2分化的神经细胞的免疫细胞化学染色结果，其中图4A是NSE-阳性细胞；图4B，NeuN-阳性细胞和图4C，GFAP-阳性细胞；
图5A至图5C：分别为从间充质干细胞分化的成骨细胞，成软骨细胞和脂肪细胞的显微照片；
图6：在其接种在含有10ng/ml EGF，20ng/ml bFGF和20ng/ml HGF的培养基上之后即刻拍摄的间充质干细胞的显微照片；
图7：由在含有10ng/ml EGF，20ng/ml bFGF和20ng/ml HGF的培养基上培养8周的间充质干细胞分化的神经细胞的显微照片；
图8A至8C：图7分化的神经细胞的免疫细胞化学染色结果，其中图8A是NSE-阳性细胞；图8B，NeuU-阳性细胞和图8C，GFAP-阳性细胞。
发明详述
本发明提供一种使从骨髓分离的间充质干细胞分化为神经细胞的方法，其包括在含有表皮生长因子(EGF)，碱性成纤维细胞生长因子(bFGF)和肝细胞生长因子(HGF)的培养基中培养间充质干细胞。
另外，还提供通过所述方法分化的神经细胞和一种包括神经细胞作为有效成分用于治疗神经疾病的药物组合物。
如在此使用，术语“神经细胞”指包括神经元，星形胶质细胞和小胶质细胞的类神经细胞(nerve-like cell)。
为了使间充质干细胞分化为神经细胞，优选在细胞培养基中培养间充质干细胞超过一周时间，所述培养基含有1-1,000ng/ml，优选5-10ng/ml的EGF，1-1000ng/ml，优选10-20ng/ml的bFGF和1-1000ng/ml，优选5-20ng/ml的HGF。更优选培养干细胞超过4周时间。在培养开始大约4周后，形成几个细胞组成的神经细胞集落，在大约8周后，通过神经细胞集落的连续生长和增殖产生大量的神经细胞。
相反，如果在相同但缺少HGF的培养基中培养间充质干细胞，细胞不能分化为神经细胞。另外，如果只用HGF处理它们，早期分化发生，因而抑制增殖。该结果暗示在培养间充质干细胞以获得神经细胞的本发明的方法中，EGF和bFGF刺激细胞增殖，HGF刺激干细胞分化为神经细胞。另外，当只使用所述因子的一种时不能获得充足量的神经细胞。
在8-周的培养后一旦神经细胞完全分化和增殖，即使在只用EGF和bFGF处理的时候，它们也可以进一步增殖而不丢失它们作为神经细胞的独特特征。在另一方面，如果只用HGF随后处理完全分化和增殖的神经细胞，细胞继续分化而不增殖，因此，细胞量减少。因此，在培养8周后，优选通过在含有EGF和bFGF的培养基中进一步培养它们以使神经细胞增殖。
依照使用EGF，bFGF和HGF的本方法，大于80％的总分化细胞是神经细胞，基于细胞量，其包含60-80％，优选65-75％的神经元和20-40％，优选25-35％的星形胶质细胞，并且不含有小胶质细胞。
在本发明中，间充质干细胞优选从人骨髓获得。源于骨髓的单核细胞包含造血干细胞和间充质干细胞，当培养它们1-2周时，造血干细胞容易地分化为成熟血细胞。因此，此后增殖的干细胞是间充质干细胞，其在20代的传代培养后继续增殖。间充质干细胞分化为包括成骨细胞，成软骨细胞和脂肪细胞的各种***细胞。
此外，当在本发明方法中使用包含间充质干细胞的骨髓单核细胞代替分离的间充质干细胞时，还可以实现神经细胞的大量生产。
由于神经细胞是通过使用在人体内存在的特异的蛋白质，即EGF，bFGF和HGF，而未使用有害的分化诱导物如DMSO，由间充质干细胞分化而来，本方法特别在安全方面是有利的。另外，因为从患者自身骨髓可以产生对于疾病治疗足够量的神经细胞，由于神经细胞迅速的有效性和降低的免疫排斥风险，它的临床应用将是可行的。
依照本发明从间充质干细胞分化的神经细胞可以作为一种细胞组合物的活性成分使用，该组合物用于神经疾病的细胞置换治疗。可以通过使用本发明的神经细胞治疗的神经疾病的非限制性实施例包括神经变性疾病如帕金森氏病，阿尔茨海默氏病，皮克病，亨廷顿舞蹈病，肌萎缩性侧索硬化和局部缺血性脑病。本发明的神经细胞还可以用于由神经细胞的异常损失导致的各种疾病以及由脊髓损伤导致的运动障碍的治疗。
通过将用本发明方法分化的神经细胞和药用赋形剂或载体混和，或通过按照任何常规程序使用药用稀释剂将其稀释，可以制备用于预防或治疗神经疾病的细胞组合物。合适载体，赋形剂和稀释剂的实例是乳糖，葡萄糖，蔗糖，山梨醇，甘露醇，木糖醇，赤藓糖醇，麦芽糖醇，淀粉，***胶，藻酸盐，明胶，磷酸钙，硅酸钙，纤维素，甲基纤维素，微晶纤维素，聚乙烯吡咯烷酮，水，羟基苯甲酸甲酯类，羟基苯甲酸丙酯类，滑石，硬脂酸镁和矿物油。制剂可另外包括填料，抗凝集剂，润滑剂，湿润剂，香料，乳化剂，防腐剂等。可通过使用本领域熟知的任何程序来配制本发明的药物组合物，以便在将它们对哺乳动物给药后，提供活性成分的迅速，持续或延迟的释放。因此，制剂可以是无菌的注射液，混悬液，乳剂，溶液等形式，其中无菌注射液是优选的。
因此，本发明还提供一种在哺乳动物中治疗神经疾病的方法，其包括以治疗疾病有效的量对需要其的受试者给药用本发明方法生产的神经细胞。
通过任何常规方法可以将通过本发明方法生产的神经细胞注射于患者内。例如，可使用Douglas Kondziolka的方法(D.Kondziolka等，Neurology55：556-569，2000)。具体地，将患者的颅骨切除以产生一个具有大约1cm直径的孔，将含有神经细胞的HBSS(Hank’s平衡盐溶液)注射于大约3个位点。注射是通过用于将所需的细胞溶液在正确位置注射于脑的深层部分的具有长针头和立体定位支架的注射器进行的。可以通过包括透皮，皮下，静脉内和肌内输入，外科定向输入，通过血管导管***术的损害内的输入的各种途径给药本发明的细胞组合物。
神经细胞的典型单位剂量可从1×106细胞至1×109细胞变化，并且可每周或每月给药。然而，应该理解实际给药的活性成分的量应该根据各种相关因素确定，所述因素包括待治疗的疾病，患者症状的严重程度，选择的给药途径，个体患者的年龄，性别和体重；因此，上述剂量不应该是想以任何方式限制本发明的范围。
下列实施例是打算进一步举例说明本发明而不限制其范围。
另外，下面给出的对于固体在固体混合物中，液体在液体中和固体在液体中的百分比是分别基于重量/重量(wt/wt)，体积/体积(vol/vol)和重量/体积(wt/vol)，并且除非特别另外说明，所有的反应在室温下进行。
实施例1：分离骨髓中的单核细胞
从每个健康志愿者的骨盆取大约10ml的骨髓，并保存在含肝素的玻璃管中。将30ml磷酸缓冲盐水(PBS)加至10ml骨髓中，将20ml得到的混合物缓慢转移至10ml Ficoll-PaqueTM plus溶液(1.077g/ml，Amersham Pharmacia Biotech)之上，并在2000rpm进行密度梯度离心20分钟。回收位于顶层和Ficoll-PaqueTM plus层之间界面的单核细胞层，并在1800rpm进行离心5分钟以获得单核细胞。
实施例2单核细胞的培养
将实施例1中获得的单核细胞以1×106细胞/cm2的密度接种于含有基本培养基的培养瓶中。在37℃，5％的CO2下孵育培养瓶。在4小时后，用新鲜基本培养基洗涤培养瓶以去除未贴壁细胞。基本培养基为含有3.5μM氢化可的松(Sigma)，以相等的摩尔比与无脂肪酸的牛血清白蛋白(Gibco BRL)混合的50ng/ml的亚油酸(Sigma公司)，0.1μM CuSO4·5H2O(Sigma)，50pM ZnSO4·7H2O(Sigma公司)，3ng/ml H2SeO3(Sigma)，1.05mg/ml NaHCO3(Sigma公司)，1.19mg/ml HEPES(Sigma)，100U/ml青霉素(Gibco BRL)，10mg/ml链霉素(Gibco BRL)和25mg/ml两性霉素(Gibco BRL)的Williams’E培养基(Gibco BRL)。
实施例3：单核细胞分化为神经细胞
为了证实实施例2获得的单核细胞是否能分化为神经细胞，在37℃，5％的CO2下在基本培养基中培养单核细胞，所述培养基含有10ng/ml表皮生长因子(Gibco BRL)，20ng/ml碱性成纤维细胞生长因子(R&D***)和20ng/ml肝细胞生长因子(R&D***)(“分化培养基”)。一周两次补充分化培养基。
大约4周后，神经细胞集落出现并不断增殖(参见表1和图1)。
大约8周后，观察到由长突出物如轴突和短突出物如树突组成的神经元状细胞和只包括短树突的星形胶质细胞状的细胞(参见图2和3)。另外，在8周后，即使在只含有EGF和bFGF的基本培养基上培养，细胞增殖而它们的形状不变(参见表2)。
然而，当在只含有EGF和bFGF的培养基中培养间充质干细胞时，细胞不分化为神经细胞。另外，当在只含有HGF的培养基中培养间充质干细胞时，细胞早期分化，因此它们既不生长也不增殖(参见表1)。
表1.间充质干细胞培养8周后细胞数/ml培养物
| 接种细胞数 | 无生长因子 | 用HGF处理 | 用EGF和bFGF处理 | 用EGF，bFGF和HGF处理 |
| 7.5×10<sup>7</sup> | 未增殖 | 未增殖 | 1×10<sup>5</sup> | 2×10<sup>5</sup> |
表2.通过在含有EGF，bFGF和HGF的培养基上培养8周从间充质干细胞分化的神经细胞培养4或8周后细胞的增殖(接种细胞数：1×105)
| 无生长因子 | 用HGF处理 | 用EGF和bFGF处理 | 用EGF，bFGF和HGF处理 | |
| 4周后 | 未增殖 | 未增殖 | 2×10<sup>5</sup> | 2×10<sup>5</sup> |
| 8周后 | 未增殖 | 未增殖 | 5×10<sup>5</sup> | 1×10<sup>5</sup> |
实施例4：免疫细胞化学
使实施例3中获得的神经细胞以1×104细胞/cm2的密度附着在1cm2的盖玻片上，所述神经细胞是通过在含有EGF，bFGF和HGF的培养基上培养8周从骨髓的单核细胞分化而来。用0.1M磷酸盐缓冲液洗涤细胞5分钟，用含有4％低聚甲醛的0.1M磷酸盐缓冲液固定15分钟，并用0.1M磷酸缓冲盐水(PBS)洗涤两次。用含1％BSA和2％ Triton X-100的0.1M PBS处理细胞5分钟，然后使其与第一抗体小鼠抗人神经元特异性的烯醇化酶(NSE)(Chemico公司)，小鼠抗人神经元特异性的核蛋白(NeuN)(Chemicon公司)，小鼠抗人β-微管蛋白III(Sigma公司)和小鼠抗人胶质细胞原纤维酸性蛋白(GFAP)(Sigma公司)反应16小时。
在与第一抗体的反应完成后，除去残余抗体并用含有0.5％BSA的0.1M PBS洗涤细胞两次，每次15分钟。另外加入第二抗体，兔抗鼠IgG(Sigma公司)并孵育30分钟。用含0.5％BSA的0.1M PBS洗涤细胞，一次5分钟。通过使用含有抗生物素蛋白-生物素的Vectastain Elite ABC试剂盒(Vector Laboratory公司)进行反应30分钟。用0.1M的磷酸盐缓冲液洗涤细胞两次，每次5分钟，另外加入DAB(脱水3，3’-二氨基联苯胺四盐酸盐，Sigma公司)作为显色底物，使混合物反应5分钟。通过用0.1M磷酸盐缓冲液处理反应物5分钟并用该缓冲液洗涤它们两次，每次5分钟来终止反应。将得到的反应物干燥，并用蒸馏水洗涤5分钟。通过依次使用蒸馏水，70％，80％，95％和100％的乙醇处理将细胞脱水和固定。
上述免疫细胞化学染色的结果在图4A至4C中显示，其中，对于神经元标记NeuN，NSE和β-微管蛋白III，以及星形胶质细胞标记GFAP，分化的细胞显示阳性结果。这些结果显示细胞分化为神经元和星形胶质细胞，其是通过它们的生物化学以及形态特征来判断。然而，对于小胶质细胞标记OX-42细胞显示阴性，证明单核细胞不分化为小胶质细胞。
检验神经元(对NeuN和NSE为阳性)和星形胶质细胞(对GFAP为阳性)在细胞中的比例并在在表3中显示，所述细胞是通过在含有EGF+bFGF+HGF或EGF+bFGF的培养基中培养8周从骨髓的单核细胞分化而来。
表3
| NSE | NeuN | GFAP | 阴性细胞 | |
| EGF+bFGF | 约0.9％ | 约0.8％ | 约1.2％ | 约89％ |
| EGF+bFGF+HGF | 约56％ | 约75％ | 约24％ | 约20％ |
从表3可见，当在含有EGF+bFGF+HGF的培养基上培养8周时约80％的总分化细胞是神经细胞。神经细胞由约70％的神经元和约30％的星形胶质细胞组成。
实施例5：间充质干细胞的分离和培养
为了了解在骨髓的单核细胞中的间充质干细胞是否将分化为神经细胞，培养单核细胞并从中分离间充质干细胞。如下检验间充质干细胞分化为各种细胞的能力。
将如在实施例2中培养的单核细胞以1×103细胞/cm2的密度接种于含有用10％FBS(胎牛血清)补充的DMEM(Gibco BRL)的培养瓶中。将细胞在5％CO2的气氛下37℃培养。在1～2周后，将增殖的细胞进行传代培养，在20代传代培养后增殖继续。
由骨髓获得的单核细胞包括成熟白细胞，淋巴细胞，成骨细胞，软骨细胞，肌细胞，成纤维细胞，脂肪细胞以及将分化为这些细胞的干细胞，所述干细胞分为造血和间充质干细胞。产生血细胞如红细胞，白细胞和淋巴细胞的造血干细胞不能增殖，但在普通的培养基中容易分化为成熟血细胞。因此，可以看到如上继续增殖的细胞是间充质干细胞。
为了证实增殖的细胞的确是间充质干细胞，用各种细胞因子和化学试剂处理细胞，并依照Pittenger等，Science，284：143-147，1999的方法检验它们分化为包括成骨细胞，成软骨细胞和脂肪细胞的各种***细胞。
为了使干细胞分化为成骨细胞，使用100mM***和10mM β-甘油磷酸(β-glycerol phosphate)和50nM抗坏血酸-2-磷酸酯和10％FBS处理细胞。
另外，为了证实干细胞分化为成软骨细胞的能力，将培养的干细胞离心以获得细胞沉淀，用不含血清的100nM***和10ng/ml TGF-β3处理。
通过用0.5mM 1-甲基3-异丁基黄嘌呤，1mM***，10g/nd胰岛素，100nM吲哚美辛(indomethacine)和10％FBS处理干细胞诱导干细胞分化为脂肪细胞。
通过碱性磷酸酶染色(Jaiswal等，J.Cell Biochem.，64(2)：295-312，1997)检验成骨细胞；通过II型胶原的RT-PCR(Mackay等，Tissue Eng.，4(4)：415-428，1998)和用甲苯胺蓝染色检验成软骨细胞；通过用油红O染色检验脂肪细胞。
结果，从图5A，5B和5C可见，在光学显微镜下在所有样品中观察到阳性结果。这些结果证明体外培养和增殖的间充质干细胞还保持能够分化为各种***细胞如成骨细胞，成软骨细胞和脂肪细胞的干细胞性能。
实施例6：间充质干细胞分化为神经细胞
为了检验实施例5中分离的间充质干细胞是否能分化为神经细胞，在用EGF，bFGF和HGF补充的培养基中，按照实施例3的方法培养间充质干细胞。
结果，与源于骨髓的单核细胞的神经细胞分化类似，在4周后形成神经细胞集落，并且神经细胞继续生长和增殖直至第8周。图6和图7举例说明了分别在接种后和在8周培养后即刻拍摄的细胞的显微照片。
在第8周后，即使只用EGF和bFGF处理，细胞继续增殖而保持神经细胞的形态特征。
同样，按照实施例4的方法，对分化细胞进行免疫细胞化学染色。
结果，与源于骨髓的单核细胞的神经细胞获得的结果类似，对于神经元标记(neural marker)NeuN，NSE和β-微管蛋白III以及星形胶质细胞标记GFAP，分化细胞显示阳性结果。因此，证实了间充质干细胞不仅分化为神经元而且分化为星形胶质细胞(参见图8A至8C)。图8A至图8C显示免疫细胞化学染色的结果，图8A代表NSE-阳性细胞；图8B，NeuN-阳性细胞；和图8C，GFAP-阳性细胞。
虽然关于上述具体的实施方案已经描述了本发明，但应承认那些本领域的技术人员可以对本发明进行各种修改和改变，这些修改和改变也属于如后附的权利要求所限定的本发明的范围内。
Claims (10)
1.一种使骨髓的间充质干细胞分化为神经细胞的方法，其包括在含有1-1000ng/ml表皮生长因子(EGF)，1-1000ng/ml碱性成纤维细胞生长因子(bFGF)和1-1000ng/ml肝细胞生长因子(HGF)的细胞培养基中培养所述间充质干细胞多于4周。
2.权利要求1的方法，其中所述间充质干细胞被培养4-8周的时间。
3.权利要求1的方法，其还包括将产生的分化神经细胞在含有EGF和bFGF的细胞培养基中增殖的步骤。
4.权利要求1的方法，其中所述神经细胞包含神经元和星形胶质细胞。
5.通过权利要求1至4任何一项的方法生产的一组神经细胞。
6.权利要求5所述的一组神经细胞，其基于细胞数，含有60-80％神经元和20-40％星形胶质细胞。
7.一种治疗神经疾病的细胞组合物，其包含权利要求5的神经细胞。
8.权利要求1的方法生产的神经细胞在制备用于治疗神经疾病的药物中的应用。
9.权利要求8的应用，其中所述神经疾病是一种神经变性疾病或由于脊髓损伤导致的运动障碍。
10.权利要求9的应用，其中所述神经变性疾病选自帕金森氏病，阿尔茨海默氏病，皮克病，亨廷顿舞蹈病，肌萎缩性侧索硬化和局部缺血性脑病。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR2001/21064 | 2001-04-19 | ||
| KR10-2001-0021064A KR100449141B1 (ko) | 2001-04-19 | 2001-04-19 | 간엽 간세포를 신경세포로 분화시키는 방법 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1549856A CN1549856A (zh) | 2004-11-24 |
| CN100535106C true CN100535106C (zh) | 2009-09-02 |
Family
ID=19708462
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB028083911A Expired - Fee Related CN100535106C (zh) | 2001-04-19 | 2002-04-19 | 使间充质干细胞分化为神经细胞的方法 |
Country Status (9)
| Country | Link |
|---|---|
| US (1) | US7229827B2 (zh) |
| EP (1) | EP1379627B1 (zh) |
| JP (1) | JP3976190B2 (zh) |
| KR (1) | KR100449141B1 (zh) |
| CN (1) | CN100535106C (zh) |
| CA (1) | CA2444724C (zh) |
| DE (1) | DE60228067D1 (zh) |
| HK (1) | HK1071162A1 (zh) |
| WO (1) | WO2002086108A1 (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978156A (zh) * | 2012-11-13 | 2013-03-20 | 湖州市中心医院 | 一种骨髓间充质干细胞的体外扩增纯化培养方法及培养基 |
Families Citing this family (72)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8075881B2 (en) * | 1999-08-05 | 2011-12-13 | Regents Of The University Of Minnesota | Use of multipotent adult stem cells in treatment of myocardial infarction and congestive heart failure |
| US10638734B2 (en) | 2004-01-05 | 2020-05-05 | Abt Holding Company | Multipotent adult stem cells, sources thereof, methods of obtaining and maintaining same, methods of differentiation thereof, methods of use thereof and cells derived thereof |
| US7015037B1 (en) | 1999-08-05 | 2006-03-21 | Regents Of The University Of Minnesota | Multiponent adult stem cells and methods for isolation |
| US8609412B2 (en) * | 1999-08-05 | 2013-12-17 | Regents Of The University Of Minnesota | Mapc generation of lung tissue |
| CA2852534A1 (en) * | 1999-08-05 | 2001-02-15 | Abt Holding Company | Multipotent adult stem cells and methods for isolation |
| US8252280B1 (en) | 1999-08-05 | 2012-08-28 | Regents Of The University Of Minnesota | MAPC generation of muscle |
| JP2004529621A (ja) * | 2001-02-14 | 2004-09-30 | ティー ファークト，レオ | 多能性成体幹細胞、その起源、それを得る方法および維持する方法、それを分化させる方法、その使用法、ならびにそれ由来の細胞 |
| DE10144326B4 (de) * | 2001-09-10 | 2005-09-22 | Siemens Ag | Verfahren und System zur Überwachung eines Reifenluftdrucks |
| NZ533688A (en) * | 2002-01-14 | 2008-06-30 | Ford Henry Health System | Materials from bone marrow stromal cells for use in forming blood vessels and producing angiogenic and trophic factors |
| EP2270146A1 (en) | 2002-02-06 | 2011-01-05 | Sanbio, Inc. | Method of inducing differentiation of bone marrow stromal cells to neural cells or skeletal muscle cells by introduction of notch gene |
| KR100519227B1 (ko) * | 2002-08-17 | 2005-10-07 | 서해영 | 간엽 줄기세포를 신경세포로 분화시키는 방법 |
| KR100495532B1 (ko) * | 2002-09-18 | 2005-06-14 | 에프씨비파미셀 주식회사 | 간엽 간세포를 신경세포로 분화 및 증식시키는 방법 |
| US20060228798A1 (en) | 2002-11-27 | 2006-10-12 | Catherine Verfaillie | Homologous recombination in multipotent adult progenitor cells |
| WO2004050865A1 (en) * | 2002-12-02 | 2004-06-17 | Anges Mg, Inc. | Method for culturing neural stem cells using hepatocyte growth factor |
| ZA200404101B (en) * | 2003-05-26 | 2005-03-07 | Reliance Life Sciences Pvt Ltd | In vitro culture of Mesenchymal Stem Cells (MSC) and a process for the preparation thereof for therapeutic use. |
| WO2005003320A2 (en) * | 2003-07-02 | 2005-01-13 | Regents Of The University Of Minnesota | Neuronal differentiation of stem cells |
| CA2543118C (en) | 2003-10-29 | 2009-10-06 | Fcb Pharmicell Co., Ltd. | Method for differentiating mesenchymal stem cell into neural cell and pharmaceutical composition containing the neural cell for neurodegenerative disease |
| JP2005176659A (ja) * | 2003-12-17 | 2005-07-07 | Ikushu Fu | 幹細胞からニューロンを生成する方法および幹細胞を培養するための培地 |
| US7534606B2 (en) | 2004-01-12 | 2009-05-19 | National Health Research Institutes | Placental stem cell and methods thereof |
| CN1973031B (zh) | 2004-04-12 | 2011-11-02 | 桑比欧公司 | 具有神经元祖细胞特性的细胞 |
| JP4624033B2 (ja) * | 2004-08-11 | 2011-02-02 | オリンパス株式会社 | 間葉系幹細胞の培養方法および肝細胞増殖因子の使用方法 |
| EP1859028B1 (en) * | 2005-02-10 | 2015-12-02 | Regents Of The University Of Minnesota | Vascular endothelial cells |
| CA2607218C (en) * | 2005-05-05 | 2016-08-23 | Regents Of The University Of Minnesota | Use of mapc or progeny therefrom to populate lymphohematopoietic tissues |
| CN101218341A (zh) * | 2005-05-05 | 2008-07-09 | 明尼苏达大学董事会 | Nk细胞抑制作用用于促进定植的mhc-i阴性细胞持续存在的用途 |
| CN101243178A (zh) | 2005-06-16 | 2008-08-13 | 特拉维夫大学拉莫特有限公司 | 用于治疗cns疾病的分离细胞和包含这种细胞的细胞群 |
| WO2007117262A2 (en) * | 2005-07-29 | 2007-10-18 | Athersys, Inc. | Culture of non-embryonic cells at high cell density |
| CA2625883A1 (en) * | 2005-10-14 | 2007-04-26 | Regents Of The University Of Minnesota | Differentiation of non-embryonic stem cells to cells having a pancreatic phenotype |
| CN101070532B (zh) * | 2006-05-12 | 2011-03-30 | 上海国睿生命科技有限公司 | 一种诱导干细胞向血管平滑肌细胞分化的方法 |
| US8277795B2 (en) | 2006-05-24 | 2012-10-02 | Corestem Co., Ltd. | Methods and compositions for treating motor neuron diseases comprising mesenchymal stem cells |
| US20080118477A1 (en) * | 2006-11-09 | 2008-05-22 | Rush University Medical Center | Umbilical cord mesenchymal stem cells support cord blood hematopoiesis |
| CN101541954B (zh) * | 2006-11-30 | 2012-12-26 | Medipost株式会社 | 含有人脐带血衍生的间充质干细胞的组合物诱导神经前体细胞或神经干细胞分化和增殖为神经细胞的用途 |
| JP2010533488A (ja) * | 2007-07-19 | 2010-10-28 | エージェンシー フォー サイエンス， テクノロジー アンド リサーチ | 胚性幹細胞をａｑｐ−１発現細胞へ分化させるための方法 |
| KR100935639B1 (ko) * | 2007-09-07 | 2010-01-07 | 부산대학교 산학협력단 | 조직재생능이 향상된 중간엽 줄기세포 및 그의 제조방법 |
| CA2712496C (en) | 2008-01-18 | 2021-01-12 | Wei-Shou Hu | Stem cell aggregates and methods for making and using |
| DK2620493T3 (da) | 2008-05-28 | 2019-07-01 | Univ Ramot | Mesenkymale stamceller til behandling af CNS-sygdomme |
| US9057051B2 (en) | 2008-10-31 | 2015-06-16 | Katholieke Universiteit Leuven | Optimized methods for differentiation of cells into cells with hepatocyte progenitor phenotypes, cells produced by the methods, and methods of using the cells |
| KR20100054711A (ko) | 2008-11-14 | 2010-05-25 | 메디포스트(주) | 간엽 줄기세포 또는 이의 배양액을 포함하는 신경질환의 예방 또는 치료용 조성물 |
| CA2768576C (en) * | 2009-07-21 | 2022-12-06 | Abt Holding Company | Use of stem cells to reduce leukocyte extravasation |
| SG10201913618WA (en) * | 2009-07-21 | 2020-03-30 | Abt Holding Co | Use of stem cells to reduce leukocyte extravasation |
| US20110206647A1 (en) * | 2010-02-25 | 2011-08-25 | Abt Holding Company | Modulation of Angiogenesis |
| WO2011106476A1 (en) * | 2010-02-25 | 2011-09-01 | Abt Holding Company | Modulation of microglia activation |
| JPWO2011118795A1 (ja) | 2010-03-26 | 2013-07-04 | 国立大学法人名古屋大学 | 損傷部治療用組成物 |
| DK2568991T6 (en) | 2010-05-12 | 2019-03-18 | Abt Holding Co | MODULATION OF SPLENOCYTES IN CELL THERAPY |
| JP2010207245A (ja) * | 2010-06-01 | 2010-09-24 | Ikushu Fu | 幹細胞からニューロンを生成する方法および幹細胞を培養するための培地 |
| US9090878B2 (en) | 2010-06-17 | 2015-07-28 | Katholieke Universiteit Leuven | Methods for differentiating cells into hepatic stellate cells and hepatic sinusoidal endothelial cells, cells produced by the methods, and methods for using the cells |
| CN103237886B (zh) | 2010-08-24 | 2018-10-30 | 明尼苏达大学董事会 | 细胞集合体的非静态悬浮培养 |
| US8895291B2 (en) | 2010-10-08 | 2014-11-25 | Terumo Bct, Inc. | Methods and systems of growing and harvesting cells in a hollow fiber bioreactor system with control conditions |
| JP6286689B2 (ja) | 2012-02-10 | 2018-03-14 | 株式会社ジャパニック | 非ヒト幹細胞の培養上清を原材料とする化粧品又は皮膚再生促進剤、及びタンパク質のイオン導入方法 |
| US10034902B2 (en) * | 2012-02-22 | 2018-07-31 | Exostem Biotec Ltd. | MicroRNAs for the generation of astrocytes |
| PL2983680T3 (pl) | 2013-04-12 | 2021-03-08 | Houston Methodist Hospital | Ulepszanie narządów do przeszczepu |
| EP3354725B1 (en) * | 2013-10-14 | 2019-12-04 | Hadasit Medical Research Services and Development Ltd. | Terminally differentiated neuronal lineages and uses thereof |
| KR101542848B1 (ko) * | 2013-11-01 | 2015-08-10 | 주식회사 비비에이치씨 | 중간엽 줄기세포로부터 유도된 만능 줄기세포를 이용하여 골아세포로 분화시키는 방법 |
| CN105992816B (zh) | 2013-11-16 | 2018-04-17 | 泰尔茂比司特公司 | 生物反应器中的细胞扩增 |
| KR20150062817A (ko) * | 2013-11-29 | 2015-06-08 | 가톨릭대학교 산학협력단 | 태반의 융모막 또는 와튼제대교질 유래 간엽줄기세포로부터 신경세포 및 유모세포를 분화시키는 방법 |
| EP3613841B1 (en) | 2014-03-25 | 2022-04-20 | Terumo BCT, Inc. | Passive replacement of media |
| JP6830059B2 (ja) | 2014-09-26 | 2021-02-17 | テルモ ビーシーティー、インコーポレーテッド | スケジュール化された細胞フィーディング |
| WO2017004592A1 (en) | 2015-07-02 | 2017-01-05 | Terumo Bct, Inc. | Cell growth with mechanical stimuli |
| US10478458B2 (en) * | 2015-08-03 | 2019-11-19 | The Johns Hopkins University | In vitro pre-conditioned bone marrow-derived mesenchymal stem cells and uses thereof |
| KR20180102661A (ko) | 2016-01-21 | 2018-09-17 | 에이비티 홀딩 컴퍼니 | 상처 치유를 위한 줄기 세포 |
| CN105505875A (zh) * | 2016-02-16 | 2016-04-20 | 杨廷稳 | 一种高效诱导干细胞定向分化的培养基和培养方法 |
| CN105586314A (zh) * | 2016-02-16 | 2016-05-18 | 赵顺英 | 用于诱导干细胞定向分化的方法 |
| WO2017205667A1 (en) | 2016-05-25 | 2017-11-30 | Terumo Bct, Inc. | Cell expansion |
| US11104874B2 (en) | 2016-06-07 | 2021-08-31 | Terumo Bct, Inc. | Coating a bioreactor |
| US11685883B2 (en) | 2016-06-07 | 2023-06-27 | Terumo Bct, Inc. | Methods and systems for coating a cell growth surface |
| CN106190963A (zh) * | 2016-07-13 | 2016-12-07 | 浙江大学 | 一种采用线粒体移植促进损伤神经元存活的方法 |
| CN106244550A (zh) * | 2016-09-30 | 2016-12-21 | 广州赛莱拉干细胞科技股份有限公司 | 一种细胞培养液及其应用和诱导骨骼肌干细胞向神经样细胞分化的方法 |
| CN117247899A (zh) | 2017-03-31 | 2023-12-19 | 泰尔茂比司特公司 | 细胞扩增 |
| US11624046B2 (en) | 2017-03-31 | 2023-04-11 | Terumo Bct, Inc. | Cell expansion |
| CN109694851B (zh) * | 2019-01-16 | 2021-03-16 | 吉林省拓华生物科技有限公司 | 一种人间充质干细胞的诱导组合物、诱导分化培养液及其体外诱导方法和应用 |
| WO2020153687A1 (ko) * | 2019-01-22 | 2020-07-30 | 고려대학교 산학협력단 | 직접 세포전환을 기반으로한 신경줄기세포의 성상교세포로의 분화방법 |
| CN111733133B (zh) * | 2020-07-22 | 2020-12-01 | 华夏源(上海)生命科技有限公司 | 一种促进表皮干细胞分化和生长的方法 |
| CN112481213B (zh) * | 2020-11-30 | 2022-08-09 | 德州蓝力生物技术有限公司 | 一种小分子活性肽在诱导hUC-MSCs分化方面的应用 |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5980885A (en) * | 1991-07-08 | 1999-11-09 | Neurospheres Holdings Ltd. | Growth factor-induced proliferation of neural precursor cells in vivo |
| US6143714A (en) * | 1994-10-24 | 2000-11-07 | Regeneron Pharmaceuticals, Inc. | Methods of using hepatocyte growth factor to promote survival, growth and differentiation of motor neurons |
| US5736396A (en) * | 1995-01-24 | 1998-04-07 | Case Western Reserve University | Lineage-directed induction of human mesenchymal stem cell differentiation |
| US5753506A (en) * | 1996-05-23 | 1998-05-19 | Cns Stem Cell Technology, Inc. | Isolation propagation and directed differentiation of stem cells from embryonic and adult central nervous system of mammals |
| WO1999011287A1 (en) * | 1997-09-04 | 1999-03-11 | Osiris Therapeutics, Inc. | Ligands that modulate differentiation of mesenchymal stem cells |
| EP1218487A2 (en) * | 1999-09-23 | 2002-07-03 | Cell Science Therapeutics | Methods and devices for obtaining non-hematopoietic lineage cells from hematopoietic progenitor cells |
-
2001
- 2001-04-19 KR KR10-2001-0021064A patent/KR100449141B1/ko not_active IP Right Cessation
-
2002
- 2002-04-19 DE DE60228067T patent/DE60228067D1/de not_active Expired - Lifetime
- 2002-04-19 US US10/475,056 patent/US7229827B2/en not_active Expired - Lifetime
- 2002-04-19 JP JP2002583623A patent/JP3976190B2/ja not_active Expired - Fee Related
- 2002-04-19 CN CNB028083911A patent/CN100535106C/zh not_active Expired - Fee Related
- 2002-04-19 EP EP02764104A patent/EP1379627B1/en not_active Expired - Fee Related
- 2002-04-19 CA CA2444724A patent/CA2444724C/en not_active Expired - Fee Related
- 2002-04-19 WO PCT/KR2002/000718 patent/WO2002086108A1/en active Application Filing
-
2005
- 2005-05-20 HK HK05104260.0A patent/HK1071162A1/xx not_active IP Right Cessation
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978156A (zh) * | 2012-11-13 | 2013-03-20 | 湖州市中心医院 | 一种骨髓间充质干细胞的体外扩增纯化培养方法及培养基 |
Also Published As
| Publication number | Publication date |
|---|---|
| EP1379627A4 (en) | 2005-08-31 |
| CN1549856A (zh) | 2004-11-24 |
| WO2002086108A1 (en) | 2002-10-31 |
| JP3976190B2 (ja) | 2007-09-12 |
| HK1071162A1 (en) | 2005-07-08 |
| EP1379627A1 (en) | 2004-01-14 |
| EP1379627B1 (en) | 2008-08-06 |
| CA2444724C (en) | 2011-07-05 |
| KR100449141B1 (ko) | 2004-09-21 |
| JP2004527250A (ja) | 2004-09-09 |
| KR20020082239A (ko) | 2002-10-31 |
| DE60228067D1 (de) | 2008-09-18 |
| US20040151701A1 (en) | 2004-08-05 |
| US7229827B2 (en) | 2007-06-12 |
| CA2444724A1 (en) | 2002-10-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100535106C (zh) | 使间充质干细胞分化为神经细胞的方法 | |
| Cao et al. | Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage | |
| Rossi et al. | Neural stem cell therapy for neurological diseases: dreams and reality | |
| Cao et al. | Stem cell repair of central nervous system injury | |
| Klassen et al. | Stem cells and retinal repair | |
| Sanchez‐Ramos | Neural cells derived from adult bone marrow and umbilical cord blood | |
| Roisen et al. | Adult human olfactory stem cells | |
| US20100021434A1 (en) | Isolated Oligodendrocyte-Like Cells and Populations Comprising Same for the Treatment of CNS Diseases | |
| Takagi et al. | Survival and differentiation of neural progenitor cells derived from embryonic stem cells and transplanted into ischemic brain | |
| US20130108669A1 (en) | Dopaminergic neurons differentiated from pluripotent stem cells and uses of thereof | |
| US10724000B2 (en) | Small molecule based conversion of somatic cells into neural crest cells | |
| US20150147301A1 (en) | Methods and compositions of producing patient-specific multipotent neuronal stem cells | |
| Newman et al. | Therapeutic potentials of human embryonic stem cells in Parkinson’s disease | |
| CN101831401A (zh) | 体外诱导间充质干细胞分化为神经干细胞的方法 | |
| US7635591B2 (en) | Method for differentiating mesenchymal stem cell into neural cell and pharmaceutical composition containing the neural cell for neurodegenerative disease | |
| Dai et al. | The Human Skin‐Derived Precursors for Regenerative Medicine: Current State, Challenges, and Perspectives | |
| Valenzuela et al. | Neural stem cell therapy for neuropsychiatric disorders | |
| KR100833612B1 (ko) | 간엽 간세포로부터 분화된 신경세포를 유효성분으로함유하는 신경계 질환 치료용 약학 조성물 | |
| Guan et al. | Ischemia, immunosuppression, and SSEA‐1‐negative cells all contribute to tumors resulting from mouse embryonic stem cell‐derived neural progenitor transplantation | |
| El-Hashash | Neural crest stem cells: a therapeutic hope machine for neural regeneration | |
| KR100495532B1 (ko) | 간엽 간세포를 신경세포로 분화 및 증식시키는 방법 | |
| WO2010030199A1 (en) | Stem cell culture | |
| WO2009024748A1 (en) | Stem cell derived neurotrophic factors | |
| Chen et al. | Characterization of glial-restricted precursors from rhesus monkey embryonic stem cells | |
| Tamada et al. | Recapitulation and investigation of human brain development with neural organoids |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1071162 Country of ref document: HK |
|
| ASS | Succession or assignment of patent right |
Owner name: JIN XUANSHOU; APPLICANT Free format text: FORMER OWNER: JIN XUANSHOU Effective date: 20070706 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20070706 Address after: Gyeonggi Do, South Korea Applicant after: Jin Xuanshou Co-applicant after: FCB Medical Cell Co.,Ltd. Address before: Gyeonggi Do, South Korea Applicant before: Jin Xuanshou |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: GR Ref document number: 1071162 Country of ref document: HK |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20120308 Address after: Gyeonggi Do, South Korea Co-patentee after: Drug cell Ltd Patentee after: Jin Xuanshou Address before: Gyeonggi Do, South Korea Co-patentee before: FCB Medical Cell Co.,Ltd. Patentee before: Jin Xuanshou |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20090902 Termination date: 20170419 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |