JP5608927B2 - 歯髄幹細胞を用いた神経疾患治療用組成物 - Google Patents
歯髄幹細胞を用いた神経疾患治療用組成物 Download PDFInfo
- Publication number
- JP5608927B2 JP5608927B2 JP2010092585A JP2010092585A JP5608927B2 JP 5608927 B2 JP5608927 B2 JP 5608927B2 JP 2010092585 A JP2010092585 A JP 2010092585A JP 2010092585 A JP2010092585 A JP 2010092585A JP 5608927 B2 JP5608927 B2 JP 5608927B2
- Authority
- JP
- Japan
- Prior art keywords
- cells
- stem cells
- dental pulp
- pulp stem
- composition
- 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.)
- Active
Links
- 210000005258 dental pulp stem cell Anatomy 0.000 title claims description 74
- 239000000203 mixture Substances 0.000 title claims description 41
- 208000012902 Nervous system disease Diseases 0.000 title description 31
- 208000025966 Neurological disease Diseases 0.000 title description 30
- 238000011282 treatment Methods 0.000 title description 14
- 208000020431 spinal cord injury Diseases 0.000 claims description 24
- 230000004069 differentiation Effects 0.000 claims description 22
- 108010025020 Nerve Growth Factor Proteins 0.000 claims description 12
- 210000004489 deciduous teeth Anatomy 0.000 claims description 11
- 210000004556 brain Anatomy 0.000 claims description 7
- 102000015336 Nerve Growth Factor Human genes 0.000 claims description 6
- 102000008730 Nestin Human genes 0.000 claims description 6
- 108010088225 Nestin Proteins 0.000 claims description 6
- 229940053128 nerve growth factor Drugs 0.000 claims description 6
- 210000005055 nestin Anatomy 0.000 claims description 6
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 claims description 5
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 claims description 5
- 101000720704 Homo sapiens Neuronal migration protein doublecortin Proteins 0.000 claims description 5
- 101001092197 Homo sapiens RNA binding protein fox-1 homolog 3 Proteins 0.000 claims description 5
- 102100025929 Neuronal migration protein doublecortin Human genes 0.000 claims description 5
- 102100035530 RNA binding protein fox-1 homolog 3 Human genes 0.000 claims description 5
- 102000004243 Tubulin Human genes 0.000 claims description 5
- 108090000704 Tubulin Proteins 0.000 claims description 5
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 claims description 5
- 102000012438 2',3'-Cyclic-Nucleotide Phosphodiesterases Human genes 0.000 claims description 4
- 108010022794 2',3'-Cyclic-Nucleotide Phosphodiesterases Proteins 0.000 claims description 4
- 238000011419 induction treatment Methods 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 description 50
- 210000000130 stem cell Anatomy 0.000 description 31
- 210000002569 neuron Anatomy 0.000 description 23
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 22
- 238000000034 method Methods 0.000 description 21
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 20
- 210000000278 spinal cord Anatomy 0.000 description 18
- 206010008118 cerebral infarction Diseases 0.000 description 17
- 201000010099 disease Diseases 0.000 description 16
- 210000001178 neural stem cell Anatomy 0.000 description 15
- 210000004248 oligodendroglia Anatomy 0.000 description 15
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 14
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 14
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 description 14
- 210000001130 astrocyte Anatomy 0.000 description 13
- 238000002054 transplantation Methods 0.000 description 13
- 239000002609 medium Substances 0.000 description 11
- 230000004770 neurodegeneration Effects 0.000 description 11
- 208000026106 cerebrovascular disease Diseases 0.000 description 10
- 230000001225 therapeutic effect Effects 0.000 description 10
- 241000700159 Rattus Species 0.000 description 9
- 230000014509 gene expression Effects 0.000 description 9
- 210000004126 nerve fiber Anatomy 0.000 description 8
- 230000001537 neural effect Effects 0.000 description 8
- 229940076279 serotonin Drugs 0.000 description 8
- 201000006474 Brain Ischemia Diseases 0.000 description 7
- 206010008120 Cerebral ischaemia Diseases 0.000 description 7
- 102000008186 Collagen Human genes 0.000 description 7
- 108010035532 Collagen Proteins 0.000 description 7
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 7
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 7
- 206010033799 Paralysis Diseases 0.000 description 7
- 229920001436 collagen Polymers 0.000 description 7
- 239000000499 gel Substances 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 230000007659 motor function Effects 0.000 description 7
- 210000003061 neural cell Anatomy 0.000 description 7
- 102000007072 Nerve Growth Factors Human genes 0.000 description 6
- 102000004230 Neurotrophin 3 Human genes 0.000 description 6
- 108090000742 Neurotrophin 3 Proteins 0.000 description 6
- 208000017442 Retinal disease Diseases 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 210000003141 lower extremity Anatomy 0.000 description 6
- 239000003900 neurotrophic factor Substances 0.000 description 6
- 229940032018 neurotrophin 3 Drugs 0.000 description 6
- 206010008025 Cerebellar ataxia Diseases 0.000 description 5
- 208000018737 Parkinson disease Diseases 0.000 description 5
- 208000010112 Spinocerebellar Degenerations Diseases 0.000 description 5
- 239000002771 cell marker Substances 0.000 description 5
- 210000001968 dental pulp cell Anatomy 0.000 description 5
- 238000012744 immunostaining Methods 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 230000001939 inductive effect Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 230000001172 regenerating effect Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 208000024827 Alzheimer disease Diseases 0.000 description 4
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 108010005939 Ciliary Neurotrophic Factor Proteins 0.000 description 4
- 102100031614 Ciliary neurotrophic factor Human genes 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 208000023105 Huntington disease Diseases 0.000 description 4
- 239000012580 N-2 Supplement Substances 0.000 description 4
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 description 4
- 230000006907 apoptotic process Effects 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 210000003074 dental pulp Anatomy 0.000 description 4
- 210000001671 embryonic stem cell Anatomy 0.000 description 4
- 238000011532 immunohistochemical staining Methods 0.000 description 4
- 210000005036 nerve Anatomy 0.000 description 4
- 210000000578 peripheral nerve Anatomy 0.000 description 4
- 230000000750 progressive effect Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000000472 traumatic effect Effects 0.000 description 4
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 3
- 206010008111 Cerebral haemorrhage Diseases 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 208000001089 Multiple system atrophy Diseases 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000001684 chronic effect Effects 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 210000005064 dopaminergic neuron Anatomy 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 229920002674 hyaluronan Polymers 0.000 description 3
- 229960003160 hyaluronic acid Drugs 0.000 description 3
- 208000014674 injury Diseases 0.000 description 3
- 208000020658 intracerebral hemorrhage Diseases 0.000 description 3
- 208000015122 neurodegenerative disease Diseases 0.000 description 3
- 210000004498 neuroglial cell Anatomy 0.000 description 3
- 230000003961 neuronal insult Effects 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 206010003694 Atrophy Diseases 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 2
- 108010080379 Fibrin Tissue Adhesive Proteins 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- 108010049003 Fibrinogen Proteins 0.000 description 2
- 102000008946 Fibrinogen Human genes 0.000 description 2
- 102100037680 Fibroblast growth factor 8 Human genes 0.000 description 2
- 102100037362 Fibronectin Human genes 0.000 description 2
- 108010067306 Fibronectins Proteins 0.000 description 2
- 101001027382 Homo sapiens Fibroblast growth factor 8 Proteins 0.000 description 2
- 208000028389 Nerve injury Diseases 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 206010058530 Peripheral nerve palsy Diseases 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 238000004115 adherent culture Methods 0.000 description 2
- 238000011316 allogeneic transplantation Methods 0.000 description 2
- 238000002266 amputation Methods 0.000 description 2
- 239000003125 aqueous solvent Substances 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 230000037444 atrophy Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 210000000133 brain stem Anatomy 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000002458 cell surface marker Substances 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 230000004186 co-expression Effects 0.000 description 2
- 229960005188 collagen Drugs 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 210000004002 dopaminergic cell Anatomy 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- 229940012952 fibrinogen Drugs 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 230000033001 locomotion Effects 0.000 description 2
- 239000002906 medical waste Substances 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000008764 nerve damage Effects 0.000 description 2
- 230000019581 neuron apoptotic process Effects 0.000 description 2
- 230000016273 neuron death Effects 0.000 description 2
- 238000012758 nuclear staining Methods 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 210000001328 optic nerve Anatomy 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 108010055896 polyornithine Proteins 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000001525 retina Anatomy 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000008733 trauma Effects 0.000 description 2
- CPKVUHPKYQGHMW-UHFFFAOYSA-N 1-ethenylpyrrolidin-2-one;molecular iodine Chemical compound II.C=CN1CCCC1=O CPKVUHPKYQGHMW-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 239000012583 B-27 Supplement Substances 0.000 description 1
- 206010005176 Blindness congenital Diseases 0.000 description 1
- 208000010392 Bone Fractures Diseases 0.000 description 1
- 102100024506 Bone morphogenetic protein 2 Human genes 0.000 description 1
- 208000014644 Brain disease Diseases 0.000 description 1
- 206010048962 Brain oedema Diseases 0.000 description 1
- 208000016615 Central areolar choroidal dystrophy Diseases 0.000 description 1
- GHXZTYHSJHQHIJ-UHFFFAOYSA-N Chlorhexidine Chemical compound C=1C=C(Cl)C=CC=1NC(N)=NC(N)=NCCCCCCN=C(N)N=C(N)NC1=CC=C(Cl)C=C1 GHXZTYHSJHQHIJ-UHFFFAOYSA-N 0.000 description 1
- 208000033810 Choroidal dystrophy Diseases 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 1
- 101100203200 Danio rerio shha gene Proteins 0.000 description 1
- 206010012689 Diabetic retinopathy Diseases 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
- 241000483002 Euproctis similis Species 0.000 description 1
- 208000037312 Familial drusen Diseases 0.000 description 1
- 206010017076 Fracture Diseases 0.000 description 1
- 208000003098 Ganglion Cysts Diseases 0.000 description 1
- 208000010412 Glaucoma Diseases 0.000 description 1
- 206010019909 Hernia Diseases 0.000 description 1
- 101000762366 Homo sapiens Bone morphogenetic protein 2 Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 208000034461 Progressive cone dystrophy Diseases 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 208000002367 Retinal Perforations Diseases 0.000 description 1
- 206010038848 Retinal detachment Diseases 0.000 description 1
- 206010038897 Retinal tear Diseases 0.000 description 1
- 208000007014 Retinitis pigmentosa Diseases 0.000 description 1
- 241000283984 Rodentia Species 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
- 229930006000 Sucrose Natural products 0.000 description 1
- 208000005400 Synovial Cyst Diseases 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical compound IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 description 1
- 108091000117 Tyrosine 3-Monooxygenase Proteins 0.000 description 1
- 102000048218 Tyrosine 3-monooxygenases Human genes 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 206010064930 age-related macular degeneration Diseases 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 210000000411 amacrine cell Anatomy 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 210000004227 basal ganglia Anatomy 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 208000006752 brain edema Diseases 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000021617 central nervous system development Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000001638 cerebellum Anatomy 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 210000004720 cerebrum Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229960003260 chlorhexidine Drugs 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 201000008615 cone dystrophy Diseases 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 229930182912 cyclosporin Natural products 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011257 definitive treatment Methods 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 108010007093 dispase Proteins 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 239000003349 gelling agent Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 210000004565 granule cell Anatomy 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- BCQZXOMGPXTTIC-UHFFFAOYSA-N halothane Chemical compound FC(F)(F)C(Cl)Br BCQZXOMGPXTTIC-UHFFFAOYSA-N 0.000 description 1
- 229960003132 halothane Drugs 0.000 description 1
- 210000001320 hippocampus Anatomy 0.000 description 1
- 210000002287 horizontal cell Anatomy 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 230000001146 hypoxic effect Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002684 laminectomy Methods 0.000 description 1
- 230000003137 locomotive effect Effects 0.000 description 1
- 208000002780 macular degeneration Diseases 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 210000001259 mesencephalon Anatomy 0.000 description 1
- 230000027939 micturition Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000002161 motor neuron Anatomy 0.000 description 1
- 230000006771 negative regulation of oligodendrocyte apoptotic process Effects 0.000 description 1
- 210000000933 neural crest Anatomy 0.000 description 1
- 230000000324 neuroprotective effect Effects 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229960000988 nystatin Drugs 0.000 description 1
- VQOXZBDYSJBXMA-NQTDYLQESA-N nystatin A1 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/CC/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)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 VQOXZBDYSJBXMA-NQTDYLQESA-N 0.000 description 1
- 230000028266 oligodendrocyte apoptotic process Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000009984 peri-natal effect Effects 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 201000005936 periventricular leukomalacia Diseases 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 210000000608 photoreceptor cell Anatomy 0.000 description 1
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 210000000449 purkinje cell Anatomy 0.000 description 1
- 210000001609 raphe nuclei Anatomy 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000004264 retinal detachment Effects 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 210000004116 schwann cell Anatomy 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 101150088976 shh gene Proteins 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 206010062261 spinal cord neoplasm Diseases 0.000 description 1
- 238000013223 sprague-dawley female rat Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000011476 stem cell transplantation Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000000946 synaptic effect Effects 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 210000004357 third molar Anatomy 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 229940034208 thyroxine Drugs 0.000 description 1
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 1
- 229940035722 triiodothyronine Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Materials For Medical Uses (AREA)
Description
[1]歯髄幹細胞を含むことを特徴とする、神経疾患治療用組成物。
[2]歯髄幹細胞が乳歯由来であることを特徴とする、[1]に記載の神経疾患治療用組成物。
[3]神経疾患が脊髄損傷、末梢神経麻痺等の外傷性疾患、筋萎縮性側索硬化症、アルツハイマー病、パーキンソン病、進行性核上清麻痺、ハンチントン病、多系統萎縮症、脊髄小脳変性症等の神経変性疾患、脳虚血、脳内出血等に伴う脳梗塞による神経細胞の変性・脱落、及び神経細胞の障害を伴う網膜疾患からなる群より選択される疾患ないし病態である、[1]又は[2]に記載の神経疾患治療用組成物。
[4]神経疾患が脊髄損傷である、[1]又は[2]に記載の神経疾患治療用組成物。
[5]歯髄幹細胞が、採取後に分化誘導処理をしていない未分化型歯髄幹細胞であることを特徴とする、[1]又は[2]に記載の神経疾患治療用組成物。
[6]歯髄幹細胞が、ネスチン陽性、ダブルコルチン陽性、β-IIIチューブリン陽性、NeuN陽性、GFAP陽性、CNPase陽性であり、且つ脳由来神経成長因子の産生能を有する、[5]に記載の神経疾患治療用組成物。
[7]歯髄幹細胞が、特定の細胞系譜へと分化誘導した分化誘導型歯髄幹細胞であることを特徴とする、[1]又は[2]に記載の神経疾患治療用組成物。
[8][1]〜[7]のいずれか一項に記載の神経疾患治療用組成物を、神経疾患患者に投与することを特徴とする、神経疾患の治療法。
自然に脱落した乳歯(又は抜歯した乳歯、或いは永久歯)をクロロヘキシジンまたはイソジン溶液で消毒した後、歯冠部を分割し歯科用リーマーにて歯髄組織を回収する。
採取した歯髄組織を基本培地(10%ウシ血清・抗生物質含有ダルベッコ変法イーグル培地)に懸濁し、2mg/mlのコラゲナーゼ及びディスパーゼで37℃、1時間処理する。5分間の遠心操作(5000回転/分)により酵素処理後の歯髄細胞を回収する。セルストレーナーによる細胞選別はSHEDやDPSCの神経幹細胞分画の回収効率を低下させるので原則、使用しない。
細胞を4cc基本培地で再懸濁し、直径6cmの付着性細胞培養用ディッシュに播種する。5%CO2、37℃に調整したインキュベータにて3日間培養した後、コロニーを形成した接着性細胞を0.05%トリプシン・EDTAにて5分間、37℃で処理する。ディッシュから剥離した歯髄細胞を直径10cmの付着性細胞培養用ディッシュに播種し拡大培養を行う。例えば、肉眼で観察してサブコンフルエント(培養容器の表面の約70%を細胞が占める状態)又はコンフルエントに達したときに細胞を培養容器から剥離して回収し、再度、培養液を満たした培養容器に播種する。継代培養を繰り返し行ってもよい。例えば継代培養を1〜8回行い、必要な細胞数(例えば約1×107個/ml)まで増殖させる。尚、培養容器からの細胞の剥離は、トリプシン処理など常法で実施することができる。以上の培養の後、細胞を回収して保存することにしてもよい(保存条件は例えば-198℃)。様々なドナーから回収した細胞を歯髄幹細胞バンクの形態で保存することにしてもよい。
次に、細胞を回収する。トリプシン処理等で培養容器から細胞を剥離した後、遠心処理を施すことによって細胞を回収することができる。このようにして回収した細胞を用いて本発明の組成物を調製する。
(1)生体吸収性材料
有機系生体吸収性材料としてヒアルロン酸、コラーゲン、フィブリノーゲン(例えばボルヒール(登録商標))等を使用することができる。
ゲル化材料は、生体親和性が高いものを用いることが好ましく、ヒアルロン酸、コラーゲン又はフィブリン糊等を用いることができる。ヒアルロン酸、コラーゲンとしては種々のものを選択して用いることができるが、本発明の組成物の適用目的(適用組織)に適したものを採用することが好ましい。用いるコラーゲンは可溶性(酸可溶性コラーゲン、アルカリ可溶性コラーゲン、酵素可溶性コラーゲン等)であることが好ましい。
本発明の組成物は、水系の溶媒を含むものであってもよい。水系の溶媒としては、滅菌水、生理食塩水、リン酸塩溶液等の緩衝液等を用いることができる。尚、調製した細胞を生理食塩水やPBS(リン酸緩衝生理食塩水)に懸濁して本発明の組成物とし(他の成分を含有しない)、患部に適用することもできる。
本発明の組成物は、上記の成分の他、抗生物質、安定化剤、保存剤、pH調整剤等を含んでいても良い。また、成長因子を含ませることもできる。
本発明の組成物は、自家移植又は同種移植による神経疾患の治療に利用される。歯髄幹細胞をそのまま投与してもよいが、組織生着効率を高めるために、予めスフィア(sphere)を形成させた上で投与しても良い。本発明の組成物の投与は、直接投与でも間接投与でもよい。例えば、直接投与では神経損傷部位に本発明の組成物が注入、埋入、填入、又は塗布等によって移植される。適度な流動性を有するゲル状に調製すれば、填入、注入、又は塗布等、簡便な手技で適用することができる。また、ゲル状であれば注射針等を用いて適用部位に容易に填入でき(患部を開放することなく適用することも可能である)。間接投与では、例えば、静脈注射や髄腔内投与によって血液・脳脊髄液の循環に乗せて細胞を患部に送達する。
脱落乳歯及び抜去した乳歯・永久歯から歯髄幹細胞(SHED、DPSC)を採取し拡大培養を行った。SHED及びDPSCの70%以上の細胞が、神経幹細胞マーカーのネスチン及びダブルコルチン、分化した神経細胞マーカーのβ-IIIチューブリン及びNeuN、アストロサイトマーカーのGFAP、オリゴデンドロサイトマーカーのCNPaseなど、全ての神経系譜マーカーを共発現する細胞集団であることをFACS及び蛍光免疫染色法で確認した(図1)。
拡大培養したSHED及びDPSC(5継代目)は口腔粘膜上皮細胞と比較して、SHEDで12倍量、DPSCで3倍量のNT-3 (Neurotrophin-3)遺伝子を発現していた(図2)。また、SHEDで60倍量、DPSCで30倍量のBDNF(Brain Derived Neurotrophic Factor)遺伝子を発現することが明らかとなった(図2)。
(1)脊髄損傷モデル作製と歯髄細胞移植
Halothaneを用いた8週齢雌性Sprague-Dawleyラットを麻酔した。第9〜10胸椎を椎弓切除後、脊髄を外科メスにて切断し脊髄完全切断モデルラットとした。
歯髄細胞の移植による下肢運動機能の改善効果は、排尿後のラットの下肢運動を5分間観察し、Basso-Beattie-Bresnahan Locomotor Rating Scaleに準じて評価した。図3に示すように、培地のみ注入したコントロール群で下肢運動機能が回復したラットはいなかった(0.9±0.89, n=10)。一方、SHED又はDPSCを移植したラットでは下肢運動機能が有意に改善した(SHED:6.57±0.54, n=12。DPSC: 6±1.89, n=10)。
脊髄切断部位を超えた神経線維の伸張は、切断した脊髄の末梢側へのセロトニン(serotonin, 5-hydroxytryptamine, 5-HT)輸送能を、抗セロトニン抗体を用いて検出することによって評価した。セロトニンはモノアミン神経伝達物質で視床下部や大脳基底核、延髄の縫線核などに高濃度に分布している。脊髄切断面を超えて上位中枢神経(脳および脳幹)からの神経線維が伸張していれば、切断面より遠位側でセロトニン陽性神経線維が確認できる。再生した神経軸索の検出には抗Neurofilament-M抗体を用いた。パラホルムアルデヒドによる灌流固定後、脊髄を採取し再固定を行った。30%シュークロース液に浸透させた後、コンパウンドに包埋し凍結切片を作製した。図3Bに示すように、培地のみを注入したラットでは切断末梢側にセロトニンは確認されなかった(n=5)。一方、SHED移植群では切断部位を超えて、末梢側脊髄にセロトニンが脳から輸送されていた(n=10)。SHEDを移植した切断部位では抗Neurofilament-M抗体で染色される再生神経線維が多く検出されたが、培地を注入したコントロール群では陽性神経線維がほとんど観察されなかった(図3C)。
移植したSHEDの脊髄切断部位周囲における分化について、抗ヒト細胞核染色抗体、成熟型神経細胞マーカー(抗NeuN抗体)、成熟型アストロサイト特異的抗体(抗GFAP抗体)、成熟型オリゴデンドロサイト特異的抗体(抗MBP抗体)による蛍光免疫組織染色で評価した。移植した多くの未分化型SHEDはオリゴデンドロサイトに分化していた。ごくわずかなSHEDが神経細胞へ分化するが、アストロサイトに分化したSHEDは検出されなかった(図4)。
本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。
Claims (3)
- ネスチン陽性、ダブルコルチン陽性、β-IIIチューブリン陽性、NeuN陽性、GFAP陽性、CNPase陽性であり、且つ脳由来神経成長因子の産生能を有する歯髄幹細胞を含むことを特徴とする、脊髄損傷治療用組成物。
- 歯髄幹細胞が乳歯由来であることを特徴とする、請求項1に記載の脊髄損傷治療用組成物。
- 歯髄幹細胞が、採取後に分化誘導処理をしていない未分化型歯髄幹細胞であることを特徴とする、請求項1又は2に記載の脊髄損傷治療用組成物。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2010092585A JP5608927B2 (ja) | 2010-04-13 | 2010-04-13 | 歯髄幹細胞を用いた神経疾患治療用組成物 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2010092585A JP5608927B2 (ja) | 2010-04-13 | 2010-04-13 | 歯髄幹細胞を用いた神経疾患治療用組成物 |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2011219432A JP2011219432A (ja) | 2011-11-04 |
JP5608927B2 true JP5608927B2 (ja) | 2014-10-22 |
Family
ID=45036866
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2010092585A Active JP5608927B2 (ja) | 2010-04-13 | 2010-04-13 | 歯髄幹細胞を用いた神経疾患治療用組成物 |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP5608927B2 (ja) |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014185470A1 (ja) * | 2013-05-14 | 2014-11-20 | 株式会社再生医療推進機構 | 神経損傷の治療用移植材の製造方法 |
JP2016008198A (ja) * | 2014-06-24 | 2016-01-18 | 国立大学法人名古屋大学 | 間質性膀胱炎の治療 |
KR101895648B1 (ko) * | 2014-11-06 | 2018-09-05 | 단국대학교 천안캠퍼스 산학협력단 | 치수줄기세포를 포함하는 신경질환 치료용 약학 조성물 및 이의 제조방법 |
IL264825B (en) * | 2016-08-14 | 2022-08-01 | Univ Ramot | Exosomes from mesenchymal cells for the treatment of neurological diseases |
CN108624560B (zh) * | 2018-06-01 | 2022-04-08 | 南京艾尔普再生医学科技有限公司 | 一种分化培养基及少突胶质前体细胞的制备方法 |
JPWO2020027163A1 (ja) | 2018-07-31 | 2021-09-24 | Jcrファーマ株式会社 | 歯髄由来細胞の製造方法 |
EP4098267A4 (en) | 2020-01-30 | 2024-03-06 | JCR Pharmaceuticals Co., Ltd. | MEDICINAL COMPOSITION COMPRISING CELLS DERIVED FROM DENTAL PULP |
CN111973631A (zh) * | 2020-08-21 | 2020-11-24 | 卡替(上海)生物技术有限公司 | 牙髓间充质干细胞在制备阿尔茨海默病治疗药物中的用途 |
CN117618653B (zh) * | 2023-12-07 | 2024-06-04 | 北京大学口腔医学院 | 一种用于面神经缺损修复的3d打印神经再生导管及其制备方法 |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009072527A1 (ja) * | 2007-12-05 | 2009-06-11 | National University Corporation Nagoya University | 歯髄幹細胞を用いた自家又は同種移植用組成物及びその用途 |
ITRM20080342A1 (it) * | 2008-06-26 | 2009-12-27 | Univ Degli Studi Udine | Cellule di polpa dentale midollo-simili, metodi per isolamento ed uso. |
JP5793724B2 (ja) * | 2008-08-22 | 2015-10-14 | 公益財団法人ヒューマンサイエンス振興財団 | 脳梗塞治療材 |
JP2010268715A (ja) * | 2009-05-20 | 2010-12-02 | Nagoya Univ | 乳歯歯髄幹細胞に特徴的な遺伝子発現群の利用 |
KR20130008594A (ko) * | 2010-03-26 | 2013-01-22 | 고쿠리츠 다이가쿠 호우징 나고야 다이가쿠 | 손상부 치료용 조성물 |
-
2010
- 2010-04-13 JP JP2010092585A patent/JP5608927B2/ja active Active
Also Published As
Publication number | Publication date |
---|---|
JP2011219432A (ja) | 2011-11-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5608927B2 (ja) | 歯髄幹細胞を用いた神経疾患治療用組成物 | |
Qi et al. | Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells repair critical-sized bone defects through enhanced angiogenesis and osteogenesis in osteoporotic rats | |
Zhou et al. | A comparison of the use of adipose-derived and bone marrow-derived stem cells for peripheral nerve regeneration in vitro and in vivo | |
Shimizu et al. | Peripheral nerve regeneration by the in vitro differentiated-human bone marrow stromal cells with Schwann cell property | |
Chen et al. | Transplantation of bone marrow stromal cells for peripheral nerve repair | |
DE60132429T2 (de) | Pluripotente aus von fettgewebe stammenden stromazellen erzeugte stammzellen und deren verwendung | |
Choi et al. | Transplantation of cultured bone marrow stromal cells to improve peripheral nerve regeneration | |
CA2600653C (en) | Pluripotent stem cell derived from cardiac tissue | |
US20080026462A1 (en) | Meningeal-derived stem cells | |
US9157070B2 (en) | Methods and combination comprising eukaryotic cells and recombinant spider silk protein | |
CN107709545A (zh) | 从干细胞生成肌肉谱系细胞 | |
Wei et al. | An improved method for isolating Schwann cells from postnatal rat sciatic nerves | |
JP2012031127A (ja) | 臍帯由来間葉系幹細胞を含む組成物 | |
KR101686315B1 (ko) | 편도 유래 중간엽 줄기세포로부터 슈반 세포의 분화 방법 | |
WO2013146992A1 (ja) | 歯髄由来の多能性幹細胞の製造方法 | |
KR20200012991A (ko) | 개과동물 양막-유래 다분화능 줄기세포 | |
JP6993026B2 (ja) | 再生治療用組成物 | |
EP2063902A1 (en) | Therapeutic cell medicine comprising skin tissue derived stem cell | |
Yang et al. | Dorsal root ganglion neurons induce transdifferentiation of mesenchymal stem cells along a Schwann cell lineage | |
KR20070080561A (ko) | 중추 또는 말초 신경계 손상 치료용 조성물 | |
JP2017119646A (ja) | 性ホルモン分泌促進剤及び生殖細胞増殖促進剤 | |
EP2845898B1 (en) | Method for culturing neural crest stem cells, and use thereof | |
US20230113275A1 (en) | Expandable cell populations from brain biopsies of living subjects | |
US20230285353A1 (en) | A chemical cocktail driving expansion of myogenic stem cells | |
Zhang et al. | Cerebral function of bone marrow multipotent adult progenitor cells after transplantation in Parkinson's disease rat models |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20130326 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20140402 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20140528 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20140613 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20140725 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20140812 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20140813 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 5608927 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313113 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313113 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |