JP7169468B2 - 改良された網膜オルガノイドおよびその製造方法 - Google Patents
改良された網膜オルガノイドおよびその製造方法 Download PDFInfo
- Publication number
- JP7169468B2 JP7169468B2 JP2021573323A JP2021573323A JP7169468B2 JP 7169468 B2 JP7169468 B2 JP 7169468B2 JP 2021573323 A JP2021573323 A JP 2021573323A JP 2021573323 A JP2021573323 A JP 2021573323A JP 7169468 B2 JP7169468 B2 JP 7169468B2
- Authority
- JP
- Japan
- Prior art keywords
- retinal
- cells
- organoids
- microglial
- obtaining
- 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
- 210000002220 organoid Anatomy 0.000 title claims description 170
- 230000002207 retinal effect Effects 0.000 title claims description 130
- 238000000034 method Methods 0.000 title claims description 60
- 210000004027 cell Anatomy 0.000 claims description 166
- 230000002025 microglial effect Effects 0.000 claims description 98
- 210000001519 tissue Anatomy 0.000 claims description 48
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims description 25
- 238000012258 culturing Methods 0.000 claims description 22
- 210000001778 pluripotent stem cell Anatomy 0.000 claims description 22
- 210000000130 stem cell Anatomy 0.000 claims description 19
- 210000000274 microglia Anatomy 0.000 claims description 13
- 210000000608 photoreceptor cell Anatomy 0.000 claims description 12
- 210000001525 retina Anatomy 0.000 claims description 11
- 210000003583 retinal pigment epithelium Anatomy 0.000 claims description 10
- 108090000835 CX3C Chemokine Receptor 1 Proteins 0.000 claims description 8
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 claims description 8
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 claims description 8
- 210000002287 horizontal cell Anatomy 0.000 claims description 5
- 210000003994 retinal ganglion cell Anatomy 0.000 claims description 5
- 210000000411 amacrine cell Anatomy 0.000 claims description 4
- 102100039196 CX3C chemokine receptor 1 Human genes 0.000 claims description 3
- 101000757378 Homo sapiens Transcription factor AP-2-alpha Proteins 0.000 claims description 3
- 102000018210 Recoverin Human genes 0.000 claims description 3
- 108010076570 Recoverin Proteins 0.000 claims description 3
- 102100022972 Transcription factor AP-2-alpha Human genes 0.000 claims description 3
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims description 2
- 210000004498 neuroglial cell Anatomy 0.000 claims 1
- 239000002609 medium Substances 0.000 description 19
- 208000002780 macular degeneration Diseases 0.000 description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 12
- 239000003814 drug Substances 0.000 description 12
- 230000014509 gene expression Effects 0.000 description 12
- 208000017442 Retinal disease Diseases 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 11
- 230000000694 effects Effects 0.000 description 10
- 206010064930 age-related macular degeneration Diseases 0.000 description 9
- 230000008859 change Effects 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 9
- 239000003795 chemical substances by application Substances 0.000 description 8
- 230000004069 differentiation Effects 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 102000004298 CX3C Chemokine Receptor 1 Human genes 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 208000007014 Retinitis pigmentosa Diseases 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 210000000056 organ Anatomy 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 6
- 230000035899 viability Effects 0.000 description 6
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 5
- 108091010877 Allograft inflammatory factor 1 Proteins 0.000 description 5
- 102100040121 Allograft inflammatory factor 1 Human genes 0.000 description 5
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 5
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 5
- 238000003501 co-culture Methods 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 206010012689 Diabetic retinopathy Diseases 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 102100033499 Interleukin-34 Human genes 0.000 description 4
- 101710181549 Interleukin-34 Proteins 0.000 description 4
- 208000027073 Stargardt disease Diseases 0.000 description 4
- 208000014769 Usher Syndromes Diseases 0.000 description 4
- 208000035475 disorder Diseases 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 229940000406 drug candidate Drugs 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 208000017532 inherited retinal dystrophy Diseases 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 239000012679 serum free medium Substances 0.000 description 4
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 3
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 3
- 206010019899 Hereditary retinal dystrophy Diseases 0.000 description 3
- 208000026350 Inborn Genetic disease Diseases 0.000 description 3
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 description 3
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 3
- 210000005156 Müller Glia Anatomy 0.000 description 3
- 206010038933 Retinopathy of prematurity Diseases 0.000 description 3
- 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 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000003169 central nervous system Anatomy 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 208000016361 genetic disease Diseases 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 208000015122 neurodegenerative disease Diseases 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 238000011536 re-plating Methods 0.000 description 3
- 229930002330 retinoic acid Natural products 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 229960003080 taurine Drugs 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 229960001727 tretinoin Drugs 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 108091023037 Aptamer Proteins 0.000 description 2
- 201000004569 Blindness Diseases 0.000 description 2
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 101000762379 Homo sapiens Bone morphogenetic protein 4 Proteins 0.000 description 2
- 101000746022 Homo sapiens CX3C chemokine receptor 1 Proteins 0.000 description 2
- 102000000646 Interleukin-3 Human genes 0.000 description 2
- 108010002386 Interleukin-3 Proteins 0.000 description 2
- 239000007760 Iscove's Modified Dulbecco's Medium Substances 0.000 description 2
- 201000007737 Retinal degeneration Diseases 0.000 description 2
- 102000036693 Thrombopoietin Human genes 0.000 description 2
- 108010041111 Thrombopoietin Proteins 0.000 description 2
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000006735 deficit Effects 0.000 description 2
- 238000007876 drug discovery Methods 0.000 description 2
- 238000007877 drug screening Methods 0.000 description 2
- 210000003981 ectoderm Anatomy 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 238000007901 in situ hybridization Methods 0.000 description 2
- 208000027866 inflammatory disease Diseases 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 229940076264 interleukin-3 Drugs 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 230000003278 mimic effect Effects 0.000 description 2
- 210000000653 nervous system Anatomy 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 108091008695 photoreceptors Proteins 0.000 description 2
- SIOXPEMLGUPBBT-UHFFFAOYSA-N picolinic acid Chemical compound OC(=O)C1=CC=CC=N1 SIOXPEMLGUPBBT-UHFFFAOYSA-N 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 230000004258 retinal degeneration Effects 0.000 description 2
- 210000001082 somatic cell Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 238000002723 toxicity assay Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- 238000012604 3D cell culture Methods 0.000 description 1
- BWRRWBIBNBVHQF-UHFFFAOYSA-N 4-(3-pyridin-2-yl-1,2,4-oxadiazol-5-yl)butanoic acid Chemical compound O1C(CCCC(=O)O)=NC(C=2N=CC=CC=2)=N1 BWRRWBIBNBVHQF-UHFFFAOYSA-N 0.000 description 1
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=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-KKGHZKTASA-N 0.000 description 1
- 241000293679 Boraria media Species 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- AQGNHMOJWBZFQQ-UHFFFAOYSA-N CT 99021 Chemical compound CC1=CNC(C=2C(=NC(NCCNC=3N=CC(=CC=3)C#N)=NC=2)C=2C(=CC(Cl)=CC=2)Cl)=N1 AQGNHMOJWBZFQQ-UHFFFAOYSA-N 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- ZEOWTGPWHLSLOG-UHFFFAOYSA-N Cc1ccc(cc1-c1ccc2c(n[nH]c2c1)-c1cnn(c1)C1CC1)C(=O)Nc1cccc(c1)C(F)(F)F Chemical compound Cc1ccc(cc1-c1ccc2c(n[nH]c2c1)-c1cnn(c1)C1CC1)C(=O)Nc1cccc(c1)C(F)(F)F ZEOWTGPWHLSLOG-UHFFFAOYSA-N 0.000 description 1
- 108091027757 Deoxyribozyme Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102100031968 Ephrin type-B receptor 2 Human genes 0.000 description 1
- 102100023593 Fibroblast growth factor receptor 1 Human genes 0.000 description 1
- 101710182386 Fibroblast growth factor receptor 1 Proteins 0.000 description 1
- 102100020715 Fms-related tyrosine kinase 3 ligand protein Human genes 0.000 description 1
- 101710162577 Fms-related tyrosine kinase 3 ligand protein Proteins 0.000 description 1
- 208000010412 Glaucoma Diseases 0.000 description 1
- 102000002254 Glycogen Synthase Kinase 3 Human genes 0.000 description 1
- 108010014905 Glycogen Synthase Kinase 3 Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101000851007 Homo sapiens Vascular endothelial growth factor receptor 2 Proteins 0.000 description 1
- 208000032578 Inherited retinal disease Diseases 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 238000003231 Lowry assay Methods 0.000 description 1
- 238000009013 Lowry's assay Methods 0.000 description 1
- 101100335081 Mus musculus Flt3 gene Proteins 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 208000032860 Partial vision loss Diseases 0.000 description 1
- 108010051742 Platelet-Derived Growth Factor beta Receptor Proteins 0.000 description 1
- 102100026547 Platelet-derived growth factor receptor beta Human genes 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 102100020718 Receptor-type tyrosine-protein kinase FLT3 Human genes 0.000 description 1
- 208000032430 Retinal dystrophy Diseases 0.000 description 1
- 206010057430 Retinal injury Diseases 0.000 description 1
- 206010038923 Retinopathy Diseases 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 150000001413 amino acids Chemical class 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
- 229960003942 amphotericin b Drugs 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- -1 antibodies Proteins 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 230000003366 colagenolytic effect Effects 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 210000001900 endoderm Anatomy 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 108010003374 fms-Like Tyrosine Kinase 3 Proteins 0.000 description 1
- 201000006321 fundus dystrophy Diseases 0.000 description 1
- 230000005021 gait Effects 0.000 description 1
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 210000001654 germ layer Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000003365 immunocytochemistry Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229940073577 lithium chloride Drugs 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 239000012577 media supplement Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 208000004141 microcephaly Diseases 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- PJUIMOJAAPLTRJ-UHFFFAOYSA-N monothioglycerol Chemical compound OCC(O)CS PJUIMOJAAPLTRJ-UHFFFAOYSA-N 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 230000005305 organ development Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000008756 pathogenetic mechanism Effects 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229940081066 picolinic acid Drugs 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000028396 retina morphogenesis in camera-type eye Effects 0.000 description 1
- 230000004491 retinal development Effects 0.000 description 1
- 210000001116 retinal neuron Anatomy 0.000 description 1
- 230000004286 retinal pathology Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000012134 supernatant fraction Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000025366 tissue development Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 230000004393 visual impairment Effects 0.000 description 1
- 238000001262 western blot Methods 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
- C12N5/0618—Cells of the nervous system
- C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
-
- 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
- C12N5/062—Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
-
- 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
-
- 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
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- 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
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
-
- 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
- C12N2501/135—Platelet-derived growth factor [PDGF]
-
- 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
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- 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
- C12N2501/165—Vascular endothelial growth factor [VEGF]
-
- 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
- C12N2501/20—Cytokines; Chemokines
- C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
-
- 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
- C12N2501/20—Cytokines; Chemokines
- C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
-
- 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
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
-
- 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
- C12N2501/40—Regulators of development
- C12N2501/415—Wnt; Frizzeled
-
- 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
- C12N2501/70—Enzymes
- C12N2501/72—Transferases [EC 2.]
- C12N2501/727—Kinases (EC 2.7.)
-
- 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
- C12N2501/998—Proteins not provided for elsewhere
-
- 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
- C12N2501/999—Small molecules not provided for elsewhere
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
- C12N2502/083—Coculture with; Conditioned medium produced by cells of the nervous system sensory transducers
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
- C12N2502/086—Coculture with; Conditioned medium produced by cells of the nervous system glial 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent 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
- C12N2513/00—3D culture
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Neurology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Neurosurgery (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Acoustics & Sound (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Ophthalmology & Optometry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Toxicology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
ミクログリア細胞またはミクログリア前駆細胞と網膜オルガノイドを、ミクログリア細胞がオルガノイドに組み込まれる条件下で共培養する工程、
を含む、網膜組織または網膜オルガノイドを得るための方法が提供される。
ミクログリア細胞および/またはミクログリア前駆細胞の集団を得る工程と、
多能性幹細胞由来の網膜オルガノイドを得る工程と、
ミクログリア細胞と網膜オルガノイドを、ミクログリア細胞がオルガノイドに組み込まれる条件下で共培養する工程と、
を含む。
いくつかの実施形態において、重要な工程は、多能性であるか、または多能性になるように誘導され得る幹細胞を得ることである。いくつかの実施形態において、例示的なiPS細胞株は、iPS-SB-Ad4;iPS-SB-Ad3;iPS-DF19-9;iPS-DF4-3;iPS-DF6-9;iPS(Foreskin);およびiPS(IMR90)を含むが、これらに限定されない。
1.SB-Ad3細胞株由来のもののような人工多能性幹細胞(iPSC)を、カスタムmTeSR1媒体における密度15x103細胞/cm2のマトリゲル被覆プレート(カスタムmTeSR1は、増殖因子:塩化リチウム、GABA、パイコリン酸、bFGF、TGFβ1、BMP4を含まないTeSR1である;ステムセルテクノロジーから入手可能)+10μM Rockインヒビターに24時間平板培養
2.カスタムmTeSR1を毎日供給
3.個々のコロニーがみえたら(2~4日)、分化誘導:
a.BMP4を80ng/ml含むmTeSRカスタム培地
4.毎日培地を交換
5.4日目に、培地を以下に変える:
a.StemPro-34無血清培地(SFM)(2mM GutaMAX-I、Life Technologiesを含む)
b.25ng/ml bFGF(塩基性線維芽細胞増殖因子)
c.100ng/ml SCF(幹細胞因子)
d.80ng/ml VEGF(血管内皮増殖因子)
6.6日目に、培地を以下に変える:
a.StemPro-34SFM無血清培地(2mM GlutaMAX-I、Life Technologiesを含む)
b.50ng/ml SCF(幹細胞因子)
c.50ng/ml IL-3(インターロイキン3)
d.5ng/ml トロンボポイエチン(TPO)
e.50ng/ml マクロファージコロニー刺激因子(M-CSF)
f.50ng/ml fms様チロシンキナーゼ3(Flt3)リガンド
7.10日目に、上清画分をペレット化し、新しい培地に再懸濁し、ディッシュに戻す。
8.14日目に、ペレット浮遊細胞を以下に再懸濁する:
a.StemPro-34
b.50ng/ml M-CSF
c.50ng/ml Flt3リガンド
d.25ng/ml 顆粒球マクロファージコロニー刺激因子(GM-CSF)
e.細胞をディッシュに戻して再度平板培養する。
9.CD14および/またはCX3CR1陽性細胞が40%を超えるまで4日ごとにステップ#8を繰り返し、この時点で、それらは、ポイント8に従って増殖させ続けるか、または組織培養処理プレート上でミクログリア成熟のために再度平板培養することができる。
10.CD14+および/またはCX3CR1+細胞を再度平板培養した後、培地をミクログリア培地に変更する:
a.2mMのGlutaMAX-Iを添加したRPMI-1640増殖培地
b.10ng/mlのGM-CSF
c.100ng/mlインターロイキン34(IL-34)
11.その後3~4日ごとに培地を交換する
注:ペニシリン-ストレプトマイシン(pen/strep)は、細胞培養のすべての段階で添加され、mTeSR1培地はヒトESおよびiPS細胞のためのフィーダーフリーの維持培地である。
1.-2日目:
a.iPSC細胞をPBSで洗浄する。これらは、SB-Ad4細胞株、すなわち、ミクログリア様細胞を得るために使用されるものとは異なる細胞株からの細胞であり得るが、同じiPSC株を使用することもできる。
b.accutase(商標)(室温)を3分間加える。Accutase(商標)は、タンパク質分解酵素およびコラーゲン分解酵素の細胞分離溶液である。
c.mTeSR1+10uM Rho関連、コイルドコイル含有プロテインキナーゼ(ROCK)阻害剤で希釈する
d.1000rpmで3分間遠心分離
e.10ml mTeSR1+10uM ROCKiで再懸濁し、カウント
f.100ul mTeSR1+10uM ROCKi中でリピジュア(lipidure)でプレコートした96ウェルプレートのウェル当たり7000細胞をプレートする
g.48時間触れない
2.0日目:
a.200ulの分化培地を加え、その後培地を100ulに半交換することにより2日ごとに供給する
b.培地組成物
i.41%イスコブ改変ダルベッコ培地(IMDM)
ii.41% HAMのF12栄養混合物
iii.15% KnockOut(商標)Serum replacement(KOSR)、すなわち、多能性幹細胞の成長を支持する、より明確なFBSを含まない培地サプリメント
iv.1%グルタマックス(Glutamax)
v.1%化学的に定義された脂質濃縮物
vi.1%ペン(Pen)/ストレップ(Strep)
vii.225uM 1-チオグリセロール
3.6日目:
a.2.25nM BMP4を添加
b.1/2を3日毎に新しい培地に交換
4.18日目:
a.培地を反転(reversal)培地に変更し、2日ごとにフィード(栄養を供給)(feed)する
b.DMEM/F12(グルタマックス含有)
c.1% N2
d.4uM CHIR99021(GSK-3酵素の阻害剤)
e.2.5uM SU5402(MEK/ERK経路阻害薬、VEGFR2、FGFR1およびPDGFRBを阻害)
f.1%ペン/ストレップ
5.24日目:
a.培地を維持培地に変更する
b.DMEM/F12(グルタマックス含有)
c.5% FBS
d.1% N2
e.0.25uMレチノイン酸
f.0.1mMタウリン
g.1%ペン/ストレップ
h.0.25ug/ml Fungizone(商標)(アムホテリシンB)
i.週3回、2ヶ月間、培地の色が変わらない範囲でフィードし、その後、週2回
1.上澄み由来のCD14+/CX3XR1+ミクログリア前駆体(MDPの工程10)を、ミクログリア培地中の組織培養プレート上に平板培養し、7日間成熟させる
2.8日目に、細胞を解離させ、次の増殖培地中でオルガノイド当たり5000細胞の密度で網膜様オルガノイド(RLOD法を介して得られる)でウェル中で再平板培養する:
a.DMEM/F12(グルタマックス含有)
b.5% FBS
c.1% N2
d.0.25uMレチノイン酸
e.0.1mMタウリン
f.1%ペン/ストレップ
g.10ng/mlのGM-CSF
h.100ng/ml IL-34
これを共培養と呼ぶ。
3.共培養された細胞は、オルガノイド内に組み込まれたまま14日間放置され、週に2回フィードされる。
4.14日後、ミクログリア細胞を層状3D網膜様構造に取り込む改良された網膜オルガノイドが存在する。改良されたオルガノイドの形成を示す画像を図1に示す。
1.上澄み由来のCD14+/CX3XR1+ミクログリア前駆体(MDPの工程10)を、ミクログリア培地中の組織培養プレート上に平板培養し、7日間成熟させる
2.8日目に、細胞を解離させ、次の増殖培地中でオルガノイド当たり5000細胞の密度で網膜様オルガノイド(RLOD法を介して得られる)でウェル中で再平板培養する:
a.DMEM/F12(グルタマックス含有)
b.5% FBS
c.1% N2
d.0.25uMレチノイン酸
e.0.1mMタウリン
f.1%ペン/ストレップ
g.10ng/ml GM-CSF
h.100ng/ml IL-34
これを共培養と呼ぶ。
3.共培養細胞を55日間オルガノイド内に集積させ、週に2回フィードする。
5.55日後に、ミクログリア細胞を層状3D網膜様構造に取り込む改良された網膜オルガノイドが存在する。改良されたオルガノイドの形成を示す画像を図4に示す。
Claims (22)
- ミクログリア細胞またはミクログリア前駆細胞と網膜オルガノイドとを、ミクログリア細胞がオルガノイドに組み込まれる条件下で共培養する工程を含む、網膜組織または網膜オルガノイドを得るための方法。
- ミクログリア細胞および/またはミクログリア前駆細胞の集団を得る工程と、
多能性幹細胞由来網膜オルガノイドを得る工程と、
前記ミクログリア細胞と前記網膜オルガノイドを、前記ミクログリア細胞が前記オルガノイドに組み込まれる条件下で共培養する工程と、
を含む、請求項1に記載の網膜組織または網膜オルガノイドを得るための方法。 - 得られたミクログリアまたはミクログリア前駆細胞の集団が、CD14および/またはCX3CR1に対して陽性である>40%の細胞を有する、請求項2に記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞の集団を得る工程が、多能性幹細胞を造血前駆細胞に分化させ、次いでミクログリア前駆細胞またはミクログリア様細胞に分化させることを含む、請求項1~3のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- ミクログリア細胞またはミクログリア前駆細胞の集団を得るとき、それらが人工多能性幹細胞由来ミクログリア細胞である、請求項1~4のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記多能性幹細胞由来網膜オルガノイドが、天然に存在する網膜に類似する層状構造を有し、ミクログリア細胞、任意の網膜色素上皮(RPE)層、並びに、光受容体細胞(PRC)、アマクリン細胞、ミュラーグリア細胞、水平細胞、双極細胞および網膜神経節細胞から選択される複数の細胞タイプを含む、請求項1~5のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 多能性幹細胞由来網膜オルガノイドがAP-2α、HuC/D、Prox1、リカバリンおよび/またはCRXに対して陽性である、請求項1~6のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞と前記網膜オルガノイドとを共培養する前に、前記ミクログリア細胞を3日以上成熟させたままにする、請求項1~7のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞と前記網膜オルガノイドとを共培養する前に、前記ミクログリア細胞を5日以上成熟させたままにする、請求項1~7のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞と前記網膜オルガノイドとを共培養する前に、前記ミクログリア細胞を7日間成熟させたままにする、請求項1~7のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞と前記網膜オルガノイドとを共培養するとき、オルガノイドが250日齢未満である、請求項1~10のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞と前記網膜オルガノイドとを共培養するとき、オルガノイドが200日齢未満である、請求項1~10のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞と前記網膜オルガノイドとを共培養するとき、オルガノイドが150日齢未満である、請求項1~10のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞と前記網膜オルガノイドとを共培養するとき、オルガノイドが100日齢である、請求項1~10のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞と前記網膜オルガノイドとを共培養するとき、前記ミクログリア細胞を、オルガノイドあたり<10000細胞の密度で平板培養する、請求項1~14のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞と前記網膜オルガノイドとを共培養するとき、前記ミクログリア細胞を、オルガノイドあたり<8000細胞の密度で平板培養する、請求項1~14のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞と前記網膜オルガノイドとを共培養するとき、前記ミクログリア細胞を、オルガノイドあたり5000細胞の密度で平板培養する、請求項1~14のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞と前記網膜オルガノイドとを共培養するとき、7日を超えて共培養する、前記1~17のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞と前記網膜オルガノイドとを共培養するとき、10日を超えて共培養する、前記1~17のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞と前記網膜オルガノイドとを共培養するとき、14日以上共培養する、前記1~17のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞と前記網膜オルガノイドとを共培養するとき、14日共培養する、前記1~17のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
- 前記ミクログリア細胞と前記網膜オルガノイドとを共培養するとき、55日を超えて共培養する、前記1~17のいずれかに記載の網膜組織または網膜オルガノイドを得るための方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB1908224.7 | 2019-06-10 | ||
GB1908224.7A GB2584664B (en) | 2019-06-10 | 2019-06-10 | Improved retinal organoids and methods of making the same |
PCT/GB2020/051387 WO2020249935A1 (en) | 2019-06-10 | 2020-06-08 | Improved retinal organoids and methods of making the same |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2022527676A JP2022527676A (ja) | 2022-06-02 |
JP7169468B2 true JP7169468B2 (ja) | 2022-11-10 |
Family
ID=67107905
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2021573323A Active JP7169468B2 (ja) | 2019-06-10 | 2020-06-08 | 改良された網膜オルガノイドおよびその製造方法 |
Country Status (6)
Country | Link |
---|---|
US (1) | US20220267722A1 (ja) |
EP (1) | EP3980528A1 (ja) |
JP (1) | JP7169468B2 (ja) |
CA (1) | CA3140619C (ja) |
GB (1) | GB2584664B (ja) |
WO (1) | WO2020249935A1 (ja) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113632765B (zh) * | 2021-03-31 | 2023-01-03 | 中山大学中山眼科中心 | 视网膜新生血管疾病动物模型、构建方法及其应用 |
CN114752565B (zh) * | 2022-04-18 | 2024-02-27 | 北京市眼科研究所 | 带有免疫细胞的视网膜类器官及其构建方法 |
EP4379046A1 (en) | 2022-11-30 | 2024-06-05 | Universidade Nova De Lisboa | A 3d cellular model of early diabetic retinopathy |
WO2024116114A1 (en) | 2022-11-30 | 2024-06-06 | Universidade Nova De Lisboa | A 3d cellular model of early diabetic retinopathy |
CN116121173B (zh) * | 2023-03-14 | 2023-09-01 | 广州湾区生物基因科技有限公司 | 一种眼组织类器官及其衍生细胞系、其制备方法和应用 |
CN117050945B (zh) * | 2023-10-11 | 2024-01-26 | 北京市眼科研究所 | 体外培养类玻璃体的方法 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2019502407A (ja) | 2016-01-14 | 2019-01-31 | オハイオ ステイト イノベイション ファウンデーション | 神経オルガノイド組成物および使用方法 |
Family Cites Families (27)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030161817A1 (en) * | 2001-03-28 | 2003-08-28 | Young Henry E. | Pluripotent embryonic-like stem cells, compositions, methods and uses thereof |
DE60041821D1 (de) * | 1999-09-24 | 2009-04-30 | Cybios Llc | Pluripotent embryonale stammzellen-ähnliche zellen, zusammensetzungen und ihre vervendungen |
US7795026B2 (en) * | 2000-01-21 | 2010-09-14 | The Johns Hopkins University School Of Medicine | Methods for obtaining human embryoid body-derived cells |
US11241460B2 (en) * | 2013-03-15 | 2022-02-08 | Astellas Institute For Regenerative Medicine | Photoreceptors and photoreceptor progenitors produced from pluripotent stem cells |
CN105683366B (zh) * | 2013-08-23 | 2020-11-06 | 住友化学株式会社 | 用于制备视网膜组织和视网膜相关细胞的方法 |
GB201402692D0 (en) * | 2014-02-16 | 2014-04-02 | Univ Newcastle | Synthetic retina |
JP6682446B2 (ja) * | 2014-10-24 | 2020-04-15 | 大日本住友製薬株式会社 | 網膜組織の製造方法 |
IL251855B2 (en) * | 2014-10-24 | 2023-09-01 | Sumitomo Pharma Co Ltd | A method for producing nerve tissue |
US20190002835A1 (en) * | 2015-12-31 | 2019-01-03 | President And Fellows Of Harvard College | Methods for generating neural tissue and uses thereof |
EP3442543A4 (en) * | 2016-02-11 | 2020-01-22 | The Johns Hopkins University | COMPOSITIONS AND METHODS FOR NEUROGENESIS |
AU2017228466B2 (en) * | 2016-03-03 | 2023-05-25 | New York Stem Cell Foundation, Inc. | Microglia derived from pluripotent stem cells and methods of making and using the same |
WO2017176810A1 (en) * | 2016-04-04 | 2017-10-12 | Biotime, Inc. | Pluripotent stem cell-derived 3d retinal tissue and uses thereof |
CA3021828A1 (en) * | 2016-04-22 | 2017-10-26 | Sumitomo Dainippon Pharma Co., Ltd. | Method for producing retinal tissue |
CA3044509A1 (en) * | 2016-11-25 | 2018-05-31 | Riken | Cell population for transplantation and method for producing same |
JP2020513794A (ja) * | 2017-02-28 | 2020-05-21 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | 多能性幹細胞および造血始原細胞からのヒトミクログリア様細胞の分化と使用 |
US20210139844A1 (en) * | 2017-06-15 | 2021-05-13 | University Of North Texas Health Science Center | Reprogramming fibroblasts to retinal cells |
CA3070212A1 (en) * | 2017-07-20 | 2019-01-24 | Riken | Method for preserving neural tissue |
WO2019028088A1 (en) * | 2017-07-31 | 2019-02-07 | Biotime, Inc. | COMPOSITIONS AND METHODS FOR RESTORING OR PREVENTING LOSS OF VISION CAUSED BY TRAUMATIC DISEASE OR INJURY |
US20190127684A1 (en) * | 2017-11-01 | 2019-05-02 | Yale University | Composition and methods for culturing retinal progenitor cells |
US11002727B2 (en) * | 2017-12-18 | 2021-05-11 | University Of Maryland, Baltimore County | Screen printing tissue models |
GB201800546D0 (en) * | 2018-01-12 | 2018-02-28 | Ucl Business Plc | Treatment |
WO2019217630A1 (en) * | 2018-05-09 | 2019-11-14 | The Regents Of The University Of Colorado, A Body Corporate | Stem cell-derived cell cultures, stem cell-derived three-dimensional tissue products, and methods of making and using the same |
MX2021000614A (es) * | 2018-07-17 | 2021-07-02 | Univ California | Células diferenciadas de células pluripotentes inmunodiseñadas. |
WO2020102260A1 (en) * | 2018-11-13 | 2020-05-22 | Prellis Biologics, Inc. | Compositions and methods for printing three-dimensional structures corresponding to biological material |
WO2020204827A1 (en) * | 2019-03-29 | 2020-10-08 | Agency For Science, Technology And Research | Microglia-sufficient brain organoids |
CN114401990A (zh) * | 2019-06-04 | 2022-04-26 | 维西欧制药公司 | 用于调节髓系细胞炎性表型的抗psgl-1组合物和方法及其用途 |
EP4259649A1 (en) * | 2020-12-10 | 2023-10-18 | The Trustees of Columbia University in the City of New York | Dual expression vector for gene augmentation for crumbs complex homologue 1 (crb1) mutations |
-
2019
- 2019-06-10 GB GB1908224.7A patent/GB2584664B/en active Active
-
2020
- 2020-06-08 WO PCT/GB2020/051387 patent/WO2020249935A1/en unknown
- 2020-06-08 JP JP2021573323A patent/JP7169468B2/ja active Active
- 2020-06-08 EP EP20731183.8A patent/EP3980528A1/en active Pending
- 2020-06-08 CA CA3140619A patent/CA3140619C/en active Active
- 2020-06-08 US US17/617,908 patent/US20220267722A1/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2019502407A (ja) | 2016-01-14 | 2019-01-31 | オハイオ ステイト イノベイション ファウンデーション | 神経オルガノイド組成物および使用方法 |
Non-Patent Citations (2)
Title |
---|
IOVS, 2018, Vol. 59, No.6, pp.2586-2603 |
Neuron, 2017, VOl. 94, pp.278-293 |
Also Published As
Publication number | Publication date |
---|---|
GB201908224D0 (en) | 2019-07-24 |
EP3980528A1 (en) | 2022-04-13 |
GB2584664A (en) | 2020-12-16 |
GB2584664B (en) | 2023-05-24 |
WO2020249935A1 (en) | 2020-12-17 |
US20220267722A1 (en) | 2022-08-25 |
JP2022527676A (ja) | 2022-06-02 |
CA3140619C (en) | 2023-02-28 |
CA3140619A1 (en) | 2020-12-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7169468B2 (ja) | 改良された網膜オルガノイドおよびその製造方法 | |
Oldak et al. | Complete human day 14 post-implantation embryo models from naive ES cells | |
US11345890B2 (en) | Neural organoid composition and methods of use | |
JP6367824B2 (ja) | 三次元不均一分化組織培養 | |
JP6756610B2 (ja) | 多分化能細胞および多能性細胞の分化を方向付けることによって発生させる皮質介在ニューロンおよびその他のニューロン細胞 | |
CN106103702B (zh) | 制备端脑或其前体组织的方法 | |
KR20200010279A (ko) | 중간 중배엽 세포로부터 신장 전구 세포로의 분화 유도 방법 및 다능성 줄기세포로부터 신장 전구 세포로의 분화 유도 방법 | |
KR102026418B1 (ko) | In vitro에서 성숙된 인간 장관 오가노이드의 제조 방법 및 이의 용도 | |
JP2002522069A (ja) | 移植可能なヒトニューロン幹細胞 | |
JP2005500847A (ja) | 分化誘導薬の同定のためのスクリーニング検定及び細胞治療用の分化細胞の調製 | |
CN106574243B (zh) | 表达腺病毒e4orf1的神经细胞及其制备方法和应用 | |
EP3609511A1 (en) | Personalized 3d neural culture system for generating human oligodendrocytes and studying myelination in vitro | |
TW200411058A (en) | Process for producing nerve cells | |
KR102146274B1 (ko) | 장관 오가노이드의 제조 방법 및 이의 용도 | |
US20210332329A1 (en) | Novel renal progenitor cell marker and method for concentrating renal progenitor cells using same | |
Markert et al. | Transcriptional comparison of adult human primary Retinal Pigment Epithelium, human pluripotent stem cell-derived Retinal Pigment Epithelium, and ARPE19 cells | |
CN110121555B (zh) | 包含工程化内皮细胞的血脑屏障 | |
WO2015099206A1 (ko) | 파브리 병의 유도-만능 줄기세포 모델 및 이의 용도 | |
KR20190127041A (ko) | 간 오가노이드 및 그의 제조 방법 | |
KR102050223B1 (ko) | 배아줄기세포로부터 소장 오가노이드의 제조방법 | |
WO2023166111A1 (en) | Method for the generation of outer radial glial (org) cells | |
Motohashi et al. | Induction of melanocytes from embryonic stem cells and their therapeutic potential | |
US20150037885A1 (en) | Method of producing human retinal pigmented epithelial cells | |
EP3995570A1 (en) | Composition for promoting proliferation of stem cells, containing, as active ingredient, cp1p or pharmaceutically acceptable salt thereof | |
Choe et al. | A brain metastasis model for breast cancer using human embryonic stem cell-derived cerebral organoids |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220131 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20220131 |
|
A871 | Explanation of circumstances concerning accelerated examination |
Free format text: JAPANESE INTERMEDIATE CODE: A871 Effective date: 20220131 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20220621 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220803 |
|
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: 20221011 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20221028 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 7169468 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |