KR102609283B1 - A cerebral organoids with improved neural activity and a method for producing the same - Google Patents
A cerebral organoids with improved neural activity and a method for producing the same Download PDFInfo
- Publication number
- KR102609283B1 KR102609283B1 KR1020230096922A KR20230096922A KR102609283B1 KR 102609283 B1 KR102609283 B1 KR 102609283B1 KR 1020230096922 A KR1020230096922 A KR 1020230096922A KR 20230096922 A KR20230096922 A KR 20230096922A KR 102609283 B1 KR102609283 B1 KR 102609283B1
- Authority
- KR
- South Korea
- Prior art keywords
- cerebral
- organoids
- cerebral organoids
- mtor
- day
- Prior art date
Links
- 210000002220 organoid Anatomy 0.000 title claims abstract description 147
- 230000002490 cerebral effect Effects 0.000 title claims abstract description 128
- 230000001537 neural effect Effects 0.000 title claims abstract description 61
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 19
- 102000013530 TOR Serine-Threonine Kinases Human genes 0.000 claims abstract description 67
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 claims abstract description 67
- 239000000556 agonist Substances 0.000 claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 33
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims abstract description 26
- 230000004069 differentiation Effects 0.000 claims abstract description 25
- 229940079593 drug Drugs 0.000 claims abstract description 24
- 239000003814 drug Substances 0.000 claims abstract description 24
- 230000014509 gene expression Effects 0.000 claims description 42
- 108090000623 proteins and genes Proteins 0.000 claims description 29
- 230000001965 increasing effect Effects 0.000 claims description 28
- 239000002609 medium Substances 0.000 claims description 21
- 102000004169 proteins and genes Human genes 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 10
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound 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](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 10
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 8
- 229930182566 Gentamicin Natural products 0.000 claims description 6
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 6
- 229930182555 Penicillin Natural products 0.000 claims description 6
- 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 claims description 6
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 claims description 6
- 108010082117 matrigel Proteins 0.000 claims description 6
- 229940049954 penicillin Drugs 0.000 claims 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 claims description 5
- 102100022983 B-cell lymphoma/leukemia 11B Human genes 0.000 claims description 5
- 102100032883 DNA-binding protein SATB2 Human genes 0.000 claims description 5
- 101000903697 Homo sapiens B-cell lymphoma/leukemia 11B Proteins 0.000 claims description 5
- 101000655236 Homo sapiens DNA-binding protein SATB2 Proteins 0.000 claims description 5
- 101001092197 Homo sapiens RNA binding protein fox-1 homolog 3 Proteins 0.000 claims description 5
- 102100035530 RNA binding protein fox-1 homolog 3 Human genes 0.000 claims description 5
- 229960005322 streptomycin Drugs 0.000 claims description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 4
- 239000012583 B-27 Supplement Substances 0.000 claims description 4
- 101000976075 Homo sapiens Insulin Proteins 0.000 claims description 4
- 101000727472 Homo sapiens Reticulon-4 Proteins 0.000 claims description 4
- 239000012580 N-2 Supplement Substances 0.000 claims description 4
- 101150079937 NEUROD1 gene Proteins 0.000 claims description 4
- 108700020297 NeuroD Proteins 0.000 claims description 4
- 102100032063 Neurogenic differentiation factor 1 Human genes 0.000 claims description 4
- 102100029831 Reticulon-4 Human genes 0.000 claims description 4
- 102100021796 Sonic hedgehog protein Human genes 0.000 claims description 4
- 101710113849 Sonic hedgehog protein Proteins 0.000 claims description 4
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 claims description 4
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 4
- 102100024506 Bone morphogenetic protein 2 Human genes 0.000 claims description 3
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 claims description 3
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 claims description 3
- 102000017926 CHRM2 Human genes 0.000 claims description 3
- 102100036462 Delta-like protein 1 Human genes 0.000 claims description 3
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 3
- 108090000368 Fibroblast growth factor 8 Proteins 0.000 claims description 3
- 102100022103 Histone-lysine N-methyltransferase 2A Human genes 0.000 claims description 3
- 101000762366 Homo sapiens Bone morphogenetic protein 2 Proteins 0.000 claims description 3
- 101000928537 Homo sapiens Delta-like protein 1 Proteins 0.000 claims description 3
- 101001045846 Homo sapiens Histone-lysine N-methyltransferase 2A Proteins 0.000 claims description 3
- 101000928929 Homo sapiens Muscarinic acetylcholine receptor M2 Proteins 0.000 claims description 3
- 101000720704 Homo sapiens Neuronal migration protein doublecortin Proteins 0.000 claims description 3
- 101001094741 Homo sapiens POU domain, class 4, transcription factor 1 Proteins 0.000 claims description 3
- 101000650694 Homo sapiens Roundabout homolog 1 Proteins 0.000 claims description 3
- 208000029726 Neurodevelopmental disease Diseases 0.000 claims description 3
- 102100025929 Neuronal migration protein doublecortin Human genes 0.000 claims description 3
- 102000001759 Notch1 Receptor Human genes 0.000 claims description 3
- 108010029755 Notch1 Receptor Proteins 0.000 claims description 3
- 102100035395 POU domain, class 4, transcription factor 1 Human genes 0.000 claims description 3
- 102100039277 Pleiotrophin Human genes 0.000 claims description 3
- 101710098940 Pro-epidermal growth factor Proteins 0.000 claims description 3
- 102100027702 Roundabout homolog 1 Human genes 0.000 claims description 3
- 239000003797 essential amino acid Substances 0.000 claims description 3
- 102100033793 ALK tyrosine kinase receptor Human genes 0.000 claims description 2
- 102100022142 Achaete-scute homolog 1 Human genes 0.000 claims description 2
- 102100022545 Bone morphogenetic protein 8B Human genes 0.000 claims description 2
- 102100038451 CDK5 regulatory subunit-associated protein 2 Human genes 0.000 claims description 2
- 102000034615 Glial cell line-derived neurotrophic factor Human genes 0.000 claims description 2
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 claims description 2
- 102100034221 Growth-regulated alpha protein Human genes 0.000 claims description 2
- 101000779641 Homo sapiens ALK tyrosine kinase receptor Proteins 0.000 claims description 2
- 101000901099 Homo sapiens Achaete-scute homolog 1 Proteins 0.000 claims description 2
- 101000899368 Homo sapiens Bone morphogenetic protein 8B Proteins 0.000 claims description 2
- 101000882873 Homo sapiens CDK5 regulatory subunit-associated protein 2 Proteins 0.000 claims description 2
- 101001069921 Homo sapiens Growth-regulated alpha protein Proteins 0.000 claims description 2
- 101001033279 Homo sapiens Interleukin-3 Proteins 0.000 claims description 2
- 101000603763 Homo sapiens Neurogenin-1 Proteins 0.000 claims description 2
- 101000613490 Homo sapiens Paired box protein Pax-3 Proteins 0.000 claims description 2
- 101001135199 Homo sapiens Partitioning defective 3 homolog Proteins 0.000 claims description 2
- 101000651890 Homo sapiens Slit homolog 2 protein Proteins 0.000 claims description 2
- 101000651893 Homo sapiens Slit homolog 3 protein Proteins 0.000 claims description 2
- 101000800639 Homo sapiens Teneurin-1 Proteins 0.000 claims description 2
- 102100039064 Interleukin-3 Human genes 0.000 claims description 2
- 108010018650 MEF2 Transcription Factors Proteins 0.000 claims description 2
- 102100039229 Myocyte-specific enhancer factor 2C Human genes 0.000 claims description 2
- 102000048238 Neuregulin-1 Human genes 0.000 claims description 2
- 108090000556 Neuregulin-1 Proteins 0.000 claims description 2
- 102100038550 Neurogenin-1 Human genes 0.000 claims description 2
- 102100040891 Paired box protein Pax-3 Human genes 0.000 claims description 2
- 102100033496 Partitioning defective 3 homolog Human genes 0.000 claims description 2
- 102100027340 Slit homolog 2 protein Human genes 0.000 claims description 2
- 102100033213 Teneurin-1 Human genes 0.000 claims description 2
- 229960005070 ascorbic acid Drugs 0.000 claims description 2
- 235000010323 ascorbic acid Nutrition 0.000 claims description 2
- 239000011668 ascorbic acid Substances 0.000 claims description 2
- 238000007877 drug screening Methods 0.000 claims description 2
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 claims 4
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 claims 4
- 235000019155 vitamin A Nutrition 0.000 claims 4
- 239000011719 vitamin A Substances 0.000 claims 4
- 229940045997 vitamin a Drugs 0.000 claims 4
- 101000979001 Homo sapiens Methionine aminopeptidase 2 Proteins 0.000 claims 2
- 101000969087 Homo sapiens Microtubule-associated protein 2 Proteins 0.000 claims 2
- 102100023174 Methionine aminopeptidase 2 Human genes 0.000 claims 2
- 239000013589 supplement Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 25
- 230000008569 process Effects 0.000 abstract description 14
- 239000005557 antagonist Substances 0.000 abstract description 10
- 238000011161 development Methods 0.000 abstract description 10
- 210000005036 nerve Anatomy 0.000 abstract description 5
- 210000004556 brain Anatomy 0.000 abstract description 4
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 abstract description 2
- 206010070863 Toxicity to various agents Diseases 0.000 abstract description 2
- 230000003278 mimic effect Effects 0.000 abstract 1
- 230000019491 signal transduction Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 29
- HKVAMNSJSFKALM-GKUWKFKPSA-N Everolimus Chemical compound C1C[C@@H](OCCO)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 HKVAMNSJSFKALM-GKUWKFKPSA-N 0.000 description 27
- 229960005167 everolimus Drugs 0.000 description 27
- 230000004083 survival effect Effects 0.000 description 21
- 230000011664 signaling Effects 0.000 description 16
- 230000003247 decreasing effect Effects 0.000 description 13
- 210000000056 organ Anatomy 0.000 description 13
- 230000018109 developmental process Effects 0.000 description 10
- 230000006870 function Effects 0.000 description 8
- 210000000130 stem cell Anatomy 0.000 description 8
- 108010020004 Microtubule-Associated Proteins Proteins 0.000 description 7
- 102000009664 Microtubule-Associated Proteins Human genes 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 230000008859 change Effects 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 description 5
- 102100024270 Transcription factor SOX-2 Human genes 0.000 description 5
- 238000010304 firing Methods 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 230000000638 stimulation Effects 0.000 description 5
- 239000002033 PVDF binder Substances 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000003590 rho kinase inhibitor Substances 0.000 description 4
- 210000001082 somatic cell Anatomy 0.000 description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- 229960002518 gentamicin Drugs 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000008035 nerve activity Effects 0.000 description 3
- 230000003988 neural development Effects 0.000 description 3
- 210000002569 neuron Anatomy 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 101150037123 APOE gene Proteins 0.000 description 2
- 102100029470 Apolipoprotein E Human genes 0.000 description 2
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical compound C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 description 2
- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 description 2
- 201000007547 Dravet syndrome Diseases 0.000 description 2
- 108700039887 Essential Genes Proteins 0.000 description 2
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 229930182816 L-glutamine Natural products 0.000 description 2
- 102000008135 Mechanistic Target of Rapamycin Complex 1 Human genes 0.000 description 2
- 108010035196 Mechanistic Target of Rapamycin Complex 1 Proteins 0.000 description 2
- 102000009308 Mechanistic Target of Rapamycin Complex 2 Human genes 0.000 description 2
- 108010034057 Mechanistic Target of Rapamycin Complex 2 Proteins 0.000 description 2
- 208000036572 Myoclonic epilepsy Diseases 0.000 description 2
- -1 NDP Proteins 0.000 description 2
- 102100032421 Protein S100-A6 Human genes 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 208000006289 Rett Syndrome Diseases 0.000 description 2
- 206010073677 Severe myoclonic epilepsy of infancy Diseases 0.000 description 2
- 102100039037 Vascular endothelial growth factor A Human genes 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 208000029560 autism spectrum disease Diseases 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000011536 extraction buffer Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000002547 new drug Substances 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000004114 suspension culture Methods 0.000 description 2
- CDOVNWNANFFLFJ-UHFFFAOYSA-N 4-[6-[4-(1-piperazinyl)phenyl]-3-pyrazolo[1,5-a]pyrimidinyl]quinoline Chemical compound C1CNCCN1C1=CC=C(C2=CN3N=CC(=C3N=C2)C=2C3=CC=CC=C3N=CC=2)C=C1 CDOVNWNANFFLFJ-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102100026865 Cyclin-dependent kinase 5 activator 1 Human genes 0.000 description 1
- 102100030013 Endoribonuclease Human genes 0.000 description 1
- 101710199605 Endoribonuclease Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010044099 Ephrin-B1 Proteins 0.000 description 1
- 102000006397 Ephrin-B1 Human genes 0.000 description 1
- 102100022466 Eukaryotic translation initiation factor 4E-binding protein 1 Human genes 0.000 description 1
- 108050000946 Eukaryotic translation initiation factor 4E-binding protein 1 Proteins 0.000 description 1
- 102100026561 Filamin-A Human genes 0.000 description 1
- 101710150822 G protein-regulated inducer of neurite outgrowth 1 Proteins 0.000 description 1
- 102100031132 Glucose-6-phosphate isomerase Human genes 0.000 description 1
- 108010070600 Glucose-6-phosphate isomerase Proteins 0.000 description 1
- 102100022645 Glutamate receptor ionotropic, NMDA 1 Human genes 0.000 description 1
- 102100038885 Histone acetyltransferase p300 Human genes 0.000 description 1
- 101000913549 Homo sapiens Filamin-A Proteins 0.000 description 1
- 101000882390 Homo sapiens Histone acetyltransferase p300 Proteins 0.000 description 1
- 101000979216 Homo sapiens Necdin Proteins 0.000 description 1
- 101000572989 Homo sapiens POU domain, class 3, transcription factor 3 Proteins 0.000 description 1
- 101000633511 Homo sapiens Photoreceptor-specific nuclear receptor Proteins 0.000 description 1
- 101001064282 Homo sapiens Platelet-activating factor acetylhydrolase IB subunit beta Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 102100023210 Necdin Human genes 0.000 description 1
- 108090000770 Neuropilin-2 Proteins 0.000 description 1
- 102000001756 Notch2 Receptor Human genes 0.000 description 1
- 108010029751 Notch2 Receptor Proteins 0.000 description 1
- 102100026456 POU domain, class 3, transcription factor 3 Human genes 0.000 description 1
- 102100029533 Photoreceptor-specific nuclear receptor Human genes 0.000 description 1
- 102100030655 Platelet-activating factor acetylhydrolase IB subunit beta Human genes 0.000 description 1
- 229920012266 Poly(ether sulfone) PES Polymers 0.000 description 1
- 102100034433 Protein kinase C-binding protein NELL2 Human genes 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 102100022122 Ras-related C3 botulinum toxin substrate 1 Human genes 0.000 description 1
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 1
- 101710113029 Serine/threonine-protein kinase Proteins 0.000 description 1
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- KLGQSVMIPOVQAX-UHFFFAOYSA-N XAV939 Chemical compound N=1C=2CCSCC=2C(O)=NC=1C1=CC=C(C(F)(F)F)C=C1 KLGQSVMIPOVQAX-UHFFFAOYSA-N 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000004931 aggregating effect Effects 0.000 description 1
- 230000004900 autophagic degradation Effects 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 238000012054 celltiter-glo Methods 0.000 description 1
- 210000004720 cerebrum Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000032459 dedifferentiation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 108010064131 neuronal Cdk5 activator (p25-p35) Proteins 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 108010062302 rac1 GTP Binding Protein Proteins 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000007651 self-proliferation Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 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 1
- 230000025366 tissue development Effects 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229940121396 wnt pathway inhibitor Drugs 0.000 description 1
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
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
-
- 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
-
- 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
- G01N33/483—Physical analysis of biological material
- G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
- G01N33/4836—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures using multielectrode arrays
-
- 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
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Optics & Photonics (AREA)
- Neurology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Neurosurgery (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
본 발명은 mTOR 작용제를 처치하여 신경 활성이 증진된 대뇌 오가노이드를 제조하는 방법에 관한 것으로, 유도만능줄기세포(iPSC)를 분화시켜 대뇌 오가노이드를 제조하는 과정 중에 mTOR 작용제 및 길항제를 처치하여 mTOR 신호전달계가 대뇌의 성장 및 분화에 미치는 영향을 확인하였고, 이를 이용하여 mTOR 작용제를 이용하면 대뇌 오가노이드의 초기 성장 속도를 향상시킬 수 있고 대뇌 오가노이드의 발달(분화) 및 신경 활성 기능을 증진시킬 수 있다는 것을 확인하였다. 이를 이용하면 대뇌를 보다 잘 모방하는 대뇌 오가노이드를 제조할 수 있고 제조된 대뇌 오가노이드는 약물의 독성 또는 약물이 신경에 미치는 영향을 평가하는 것에 유용하게 활용될 수 있을 것이다.The present invention relates to a method of producing cerebral organoids with enhanced neural activity by treating mTOR agonists. During the process of producing cerebral organoids by differentiating induced pluripotent stem cells (iPSCs), mTOR agonists and antagonists are treated to produce mTOR. The effect of the signal transduction system on cerebral growth and differentiation was confirmed, and using this, mTOR agonists can improve the initial growth rate of cerebral organoids and enhance the development (differentiation) and neural activity function of cerebral organoids. It was confirmed that it was possible. Using this, it is possible to manufacture cerebral organoids that better mimic the brain, and the manufactured cerebral organoids can be usefully used to evaluate drug toxicity or the effects of drugs on nerves.
Description
본 발명은 대뇌 오가노이드 및 이의 제조 방법, 더 구체적으로 신경 활성이 증진된 대뇌 오가노이드 및 이의 제조 방법에 관한 것이다.The present invention relates to cerebral organoids and methods for producing the same, and more specifically, to cerebral organoids with enhanced neural activity and methods for producing the same.
기존의 동물 모델을 사용한 연구 결과들은 인간 관련 질환이나 신약 개발 등의 적용에 많은 한계들을 가졌으나, 인간줄기세포 배양 기술이 개발되면서 배아 및 조직 발달, 각종 질병 원인 및 기전 등에 관한 연구들이 획기적으로 발전하고 있다. 줄기세포는 크게 배아줄기세포(embryonic stem cell, ESC)와 유도만능줄기세포(induced pluripotent stem cell, iPSC)로 구분되며, 이들 줄기세포는 공통적으로 자가증식이 가능하고, 특정 환경에서 각종 세포로 분화되는 다분화능을 갖는 특징이 있다.Research results using existing animal models had many limitations in application to human-related diseases or new drug development, but with the development of human stem cell culture technology, research on embryonic and tissue development, causes and mechanisms of various diseases, etc. has advanced dramatically. I'm doing it. Stem cells are largely divided into embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). These stem cells are commonly capable of self-proliferation and differentiate into various cells in a specific environment. It has the characteristic of having multipotency.
성인의 피부세포 등의 체세포에 역분화를 일으키는 특정 유전자를 도입하여 제조되는 유도만능줄기세포의 개발로 인해 배아줄기세포가 가지는 윤리적 문제점이 해결되었고, 환자 본인의 체세포를 이용함으로써 환자 맞춤형 질병모델, 신약 스크리닝, 약물 독성 테스트 등이 가능해졌다.The development of induced pluripotent stem cells, which are manufactured by introducing a specific gene that causes dedifferentiation into somatic cells such as adult skin cells, has solved the ethical problems with embryonic stem cells, and by using the patient's own somatic cells, a customized disease model, New drug screening and drug toxicity testing have become possible.
최근에는, 줄기세포의 내재적 자기조직화 성질을 이용하여 부분적으로 생체 내(in vivo) 장기 모사를 할 수 있는 기능적인 삼차원 구조체인, 오가노이드의 개발로 이어졌다. 특히, 신체의 장기 중에서도 태아 단계 및 출생 초기에 발달이 완성되는 뇌의 경우, 뇌에 직접적인 접근이 거의 불가능하여 연구를 하기 위해 현실적으로 많은 제약이 따르는 문제가 있다. 이러한 문제를 해결하기 위해 줄기세포를 신경세포가 포함된 뇌의 구성세포로 분화시키는 기술로 발달시키기 위한 연구가 진행 중이다(J Biomed Sci, 28:30, 2021).Recently, the intrinsic self-organizing properties of stem cells have been exploited, leading to the development of organoids, functional three-dimensional structures that can partially simulate organs in vivo . In particular, among the organs of the body, in the case of the brain, whose development is completed in the fetal stage and early after birth, direct access to the brain is almost impossible, which poses many practical limitations for research. To solve this problem, research is underway to develop technology to differentiate stem cells into brain cells containing neurons (J Biomed Sci, 28:30, 2021).
오가노이드(organoid)는 줄기세포나 장기 기원세포로부터 분리한 세포를 3차원적으로 배양하거나 응집 또는 재조합하여 만든 조직 또는 기관의 형태와 기능 모두를 재현하는 작은 배양체를 의미한다. 이러한 오가노이드는 기관 또는 조직을 구성하는 여러 특이적 세포 집단들을 포함하고 있고, 실제 조직 또는 기관과 유사한 형태 및 구조적 조직화가 이루어져 있으며, 각 기관이 가지는 특수한 기능을 재현할 수 있다. 오가노이드는 공통적인 일련의 과정에 의해 형성되는데, 기능이 같은 세포들끼리 뭉쳐 적절한 위치로 배치되며, 세포들의 구획이 분리된 후에는 더욱 세부적인 분화가 일어난다. 오가노이드는 실제 장기와 유사한 형태로 분자 신호 조절 등과 같은 동물 모델에서는 구현하기 힘든 연구를 시험관 내(in vitro)에서 구현할 수 있어 기초 연구에 매우 유용함은 물론 인간의 발생과정, 질환 모델 확립, 의약품 유효성 평가 스크리닝, 세포치료제 개발 등 다양한 분야에 매우 유용하게 사용될 수 있는 기술이다 (한국 등록 특허 제10-2228400호; J Biomed Sci, 28:30, 2021).Organoid refers to a small culture that reproduces both the form and function of a tissue or organ created by three-dimensionally cultivating, aggregating, or recombining cells isolated from stem cells or organ origin cells. These organoids contain several specific cell populations that make up an organ or tissue, have a form and structural organization similar to an actual tissue or organ, and can reproduce the special functions of each organ. Organoids are formed through a series of common processes. Cells with the same function are grouped together and placed in an appropriate location. After the cell compartments are separated, more detailed differentiation occurs. Organoids are similar to actual organs and can carry out research in vitro that is difficult to implement in animal models, such as regulating molecular signals, making them very useful for basic research, as well as human developmental processes, establishing disease models, and drug effectiveness. It is a technology that can be very useful in various fields such as evaluation screening and cell therapy development (Korea Registered Patent No. 10-2228400; J Biomed Sci, 28:30, 2021).
mTOR(mammalian target of rapamycin)은 세린/트레오닌 단백질 인산화효소이고, 세포의 증식, 분화, 생존 및 자가 포식 조절에 관여하는 것으로 알려져 있다 (Cell, 168(6):960-976, 2017). 대뇌의 발달에 mTOR 신호 전달계가 미치는 영향은 mTOR 신호 전달계의 상위 기작을 조절하는 단백질인 Rab39b를 결손시킨 돌연변이 마우스(mouse) 및 오가노이드를 이용하여 간접적으로 관찰된 바가 있다. Rab39b를 결손시키면 PI3K-Akt-mTOR로 이어지는 신호 전달계의 활성 수준이 증가하고 그에 따라 대뇌의 성장 및 발달이 향상된다는 결과를 확인할 수 있다(Genes & Development, 34:580-597, 2020).mTOR (mammalian target of rapamycin) is a serine/threonine protein kinase and is known to be involved in regulating cell proliferation, differentiation, survival, and autophagy (Cell, 168(6):960-976, 2017). The influence of the mTOR signaling system on cerebral development has been indirectly observed using mutant mice and organoids lacking Rab39b, a protein that regulates the upper mechanisms of the mTOR signaling system. The results show that when Rab39b is deleted, the activity level of the signaling system leading to PI3K-Akt-mTOR increases, thereby improving cerebral growth and development (Genes & Development, 34:580-597, 2020).
그러나, 현재까지 mTOR 신호 전달계를 향상시키는 작용제(agonist)를 이용하여 대뇌 오가노이드를 제조한 사례는 없을 뿐 아니라, 이러한 작용제를 이용함으로써 신경 활성이 증진된 대뇌 오가노이드를 얻을 수 있음을 밝힌 사례는 전무하다.However, to date, there have been no cases of manufacturing cerebral organoids using agonists that enhance the mTOR signaling system, and there has been no case showing that cerebral organoids with enhanced neural activity can be obtained by using such agonists. There are none.
본 발명의 목적은 mTOR 작용제를 이용하여 신경 활성이 증진된 대뇌 오가노이드를 제조하는 방법과 이러한 제조방법을 통해 얻은 대뇌 오가노이드를 제공하는 것이다.The purpose of the present invention is to provide a method for producing cerebral organoids with enhanced neural activity using an mTOR agonist and a cerebral organoid obtained through this production method.
상기 목적을 달성하기 위하여, In order to achieve the above purpose,
본 발명은 (a) 유도만능줄기세포를 매트리겔(Matrigel)이 코팅된 조건에서 부착 배양하는 단계; (b) 배양된 유도만능줄기세포의 콜로니(colony)를 분리하는 단계; (c) 분리된 콜로니를 부유 배양하는 단계; 및 (d) 배양 중인 단계 (c)의 콜로니에 mTOR 작용제를 처치하는 단계;를 포함하는 대뇌 오가노이드 제조 방법을 제공한다.The present invention includes the steps of (a) attaching and culturing induced pluripotent stem cells under Matrigel-coated conditions; (b) isolating colonies of cultured induced pluripotent stem cells; (c) suspending culture of the separated colonies; and (d) treating the colony of step (c) being cultured with an mTOR agonist.
또한, 본 발명은 상기 제조 방법으로 제조된 대뇌 오가노이드를 제공한다.Additionally, the present invention provides cerebral organoids produced by the above production method.
또한, 본 발명은 (a) 본 발명의 제조방법으로 제조된 대뇌 오가노이드에 약물을 처리하는 단계; (b) 상기 대뇌 오가노이드의 신경 활성을 측정하는 단계; 및 (c) 약물 처리 전 대조군의 신경 활성도와 단계 (b)의 신경 활성을 비교하는 단계;를 포함하는 신경 발달 장애를 치료 또는 개선하기 위한 약물 스크리닝 방법을 제공한다. 상기 신경 발달 장애는 자폐스펙트럼 장애, 드라베 증후군(Dravet syndrome) 또는 레트 증후군(Rett syndrome)을 포함할 수 있다.In addition, the present invention includes the steps of (a) treating cerebral organoids prepared by the production method of the present invention with a drug; (b) measuring neural activity of the cerebral organoid; and (c) comparing the neural activity of the control group before drug treatment with the neural activity of step (b). The neurodevelopmental disorder may include autism spectrum disorder, Dravet syndrome, or Rett syndrome.
본 발명은 mTOR 작용제를 이용하여 신경 활성이 증진된 대뇌 오가노이드를 제조하는 방법에 관한 것으로, 대뇌 오가노이드를 제조하는 과정에서 mTOR 작용제를 처치하면, 대뇌 오가노이드의 성장 속도 및 생존율이 향상되는 것을 확인하였고, 신경 관련 유전자의 발현이 증가하는 것을 확인하였으며, 신경의 활성도가 증가하는 것을 확인하였다. 실제 장기를 모사하기 위해 제조되는 오가노이드의 특성과 신경 활성이 중요한 뇌라는 특수한 장기의 특성을 고려할 때, 본 발명의 제조 방법을 통해 생체 내 대뇌를 보다 잘 모방(모사)한 대뇌 오가노이드를 제조할 수 있다.The present invention relates to a method of producing cerebral organoids with enhanced neural activity using an mTOR agonist, and shows that treatment with an mTOR agonist during the process of producing cerebral organoids improves the growth rate and survival rate of the cerebral organoids. It was confirmed that the expression of nerve-related genes increased, and that nerve activity increased. Considering the characteristics of organoids manufactured to simulate actual organs and the characteristics of a special organ called the brain, where neural activity is important, a cerebral organoid that better mimics the in vivo cerebrum is manufactured through the manufacturing method of the present invention. can do.
도 1은 유도만능줄기세포(induced pluripotent stem cell, iPSC)를 대뇌 오가노이드로 분화시키는 과정 중에 mTOR 작용제(agonist)인 MHY1485(MHY) 또는 mTOR 길항제(antagonist)인 에버롤리무스(everolimus, EVER)를 처치하였을 때, 대뇌 오가노이드 내에서의 mTOR 신호전달계 활성 수준이 변화하는지 확인하기 위해 웨스턴블롯(western blot)을 수행한 결과이다.
도 2는 대뇌 오가노이드 제조 과정 중에 대뇌 오가노이드의 크기를 관찰한 결과이다. 유도만능줄기세포의 콜로니를 부유 배양하면서 대뇌 오가노이드로 분화시키는 과정에서, 부유 배양 10일차부터 MHY1485 또는 에버롤리무스를 처치하였고, 약물을 처치하지 않고 배양한 대뇌 오가노이드를 대조군(C)으로 이용하였다. 도 2a는 배양 10일차, 43일차, 70일차의 대뇌 오가노이드 사진이고, 도 2b는 배양 0일차부터 120일차까지의 대뇌 오가노이드의 지름 변화를 기록한 그래프이다. 도 2c는 저농도(10 nM 또는 100 nM)의 약물을 처치하였을 때의 대뇌 오가노이드의 지름 변화를 기록한 그래프이다.
도 3은 배양 10일차(a), 43일차(b), 70일차(c), 120일차(d), 각 시점에서의 대뇌 오가노이드 지름(μm)을 기록한 그래프이다.
도 4는 배양 10일차(a), 43일차(b), 70일차(c), 120일차(d), 각 시점에서의 대뇌 오가노이드의 생존율을 기록한 그래프이고, 도 4e는 저농도(10 nM 또는 100 nM)의 약물을 처치하였을 때 배양 43일차의 오가노이드 생존율을 비교한 그래프이다.
도 5는 대뇌 오가노이드 제조 과정에서 배양 10일차부터 각 약물을 처치하였을 때 신경 발달과 관련된 유전자의 발현 변화를 확인한 결과를 보여준다. 도 5a는 배양 43일차의 MHY1485 처치 대뇌 오가노이드에서 나타난 유전자 발현의 결과이고, 도 5b는 에버롤리무스 처치 대뇌 오가노이드에서 나타난 유전자 발현의 결과이다. 도 5c 및 도 5d는 배양 120일차까지 각 약물을 처치하였을 때의 유전자 발현의 결과이다. 유전자 발현 변화는 하우스키핑(housekeeping) 유전자의 발현 수준과 대비하여 나타내었다.
도 6은 대뇌 오가노이드의 용해물을 이용하여 웨스턴블롯을 수행한 결과로, 각 약물 처치에 따른 SOX2, MAP2, NeuN, CTIP2, SATB2, GAPDH의 발현량 차이를 나타낸 것이다. 도 6a는 배양 43일차의 대뇌 오가노이드를 이용한 결과이고, 도 6b는 배양 120일차의 대뇌 오가노이드를 이용한 결과이다.
도 7은 다중전극어레이(multielectrode array, MEA)와 이를 이용하여 신경 활성을 측정한 결과를 나타낸 것이다. 도 7a는 다중전극어레이의 개략도이고, 도 7b는 배양 기간 지속에 따른 mean firing rate(Hz)의 변화를 나타내는 그래프이다. 배양 120일차의 대뇌 오가노이드의 신경 활성은 평균 발화 속도(mean firing rate, MFR)(c), 네트워크 돌발 주파수(network burst frequency)(d), 전극 돌발 크기(electrode burst size)(e), 및 돌발 수(the number of bursts)(f)를 이용하여 나타난다.Figure 1 shows the use of MHY1485 (MHY), an mTOR agonist, or everolimus (EVER), an mTOR antagonist, during the process of differentiating induced pluripotent stem cells (iPSC) into cerebral organoids. This is the result of western blot to confirm whether the level of mTOR signaling system activity in cerebral organoids changes when treated.
Figure 2 shows the results of observing the size of cerebral organoids during the cerebral organoid manufacturing process. In the process of differentiating induced pluripotent stem cell colonies into cerebral organoids through suspension culture, MHY1485 or everolimus was treated from the 10th day of suspension culture, and cerebral organoids cultured without drug treatment were used as a control group (C). did. Figure 2a is a photograph of cerebral organoids on days 10, 43, and 70 of culture, and Figure 2b is a graph recording the change in diameter of cerebral organoids from day 0 to day 120 of culture. Figure 2c is a graph recording the change in diameter of cerebral organoids when treated with a low concentration (10 nM or 100 nM) of the drug.
Figure 3 is a graph recording the cerebral organoid diameter (μm) at each time point, on the 10th day (a), the 43rd day (b), the 70th day (c), and the 120th day (d) of culture.
Figure 4 is a graph recording the survival rate of cerebral organoids at each time point, day 10 (a), day 43 (b), day 70 (c), and day 120 (d) of culture, and Figure 4e is a graph recording the survival rate of cerebral organoids at low concentration (10 nM or This is a graph comparing the organoid survival rate on the 43rd day of culture when treated with 100 nM) of the drug.
Figure 5 shows the results of confirming changes in the expression of genes related to neural development when each drug was treated from the 10th day of culture during the manufacturing of cerebral organoids. Figure 5a shows the results of gene expression in cerebral organoids treated with MHY1485 on day 43 of culture, and Figure 5b shows the results of gene expression in cerebral organoids treated with everolimus. Figures 5c and 5d show the results of gene expression when each drug was treated until the 120th day of culture. Gene expression changes were expressed in comparison with the expression levels of housekeeping genes.
Figure 6 shows the results of Western blot using lysates of cerebral organoids, showing differences in expression levels of SOX2, MAP2, NeuN, CTIP2, SATB2, and GAPDH according to each drug treatment. Figure 6a shows the results using cerebral organoids on day 43 of culture, and Figure 6b shows the results using cerebral organoids on day 120 of culture.
Figure 7 shows a multielectrode array (MEA) and the results of measuring neural activity using it. Figure 7a is a schematic diagram of a multi-electrode array, and Figure 7b is a graph showing the change in mean firing rate (Hz) as the culture period continues. Neuronal activity of cerebral organoids on day 120 of culture was measured by mean firing rate (MFR) (c), network burst frequency (d), electrode burst size (e), and It is expressed using the number of bursts (f).
이하, 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 (a) 유도만능줄기세포를 매트리겔(Matrigel)이 코팅된 조건에서 부착 배양하는 단계; (b) 배양된 유도만능줄기세포의 콜로니(colony)를 분리하는 단계; (c) 분리된 콜로니를 부유 배양하는 단계; 및 (d) 배양 중인 단계 (c)의 콜로니에 mTOR 작용제를 처치하는 단계;를 포함하는 대뇌 오가노이드 제조 방법을 제공한다.The present invention includes the steps of (a) attaching and culturing induced pluripotent stem cells under Matrigel-coated conditions; (b) isolating colonies of cultured induced pluripotent stem cells; (c) suspending culture of the separated colonies; and (d) treating the colony of step (c) being cultured with an mTOR agonist.
상기 유도만능줄기세포(induced pluripotent stem cell, iPSC)는 체세포 또는 이미 분화된 세포를 처리하여 만능 분화성을 갖게 된 세포를 의미한다. 여기서 처리하는 방법은 화합물, 유전적 변환 또는 특정 조건으로 배양하는 방법 등을 포함하나 이에 한정되는 것은 아니다. The induced pluripotent stem cell (iPSC) refers to a cell that has pluripotent differentiation by processing somatic cells or already differentiated cells. Treatment methods herein include, but are not limited to, compounds, genetic transformation, or culturing under specific conditions.
상기 유도만능줄기세포는 체세포 또는 이미 분화된 세포를 처리하여 직접 제조한 것일 수도 있으나, 상업적으로 판매하는 유도만능줄기세포를 구매하여 사용할 수 있다. 일례로, IMR-90-1, IMR-90-2, IMR-90-3, IMR-90-4, 또는 SIGi001-A-1을 이용할 수 있고, IMR-90-4를 이용하는 것이 바람직하다.The induced pluripotent stem cells may be manufactured directly by processing somatic cells or already differentiated cells, but commercially sold induced pluripotent stem cells can be purchased and used. For example, IMR-90-1, IMR-90-2, IMR-90-3, IMR-90-4, or SIGi001-A-1 can be used, and IMR-90-4 is preferably used.
상기 단계 (b)는 부착 배양된 세포를 분리하기 위해 통상적으로 이용되는 물리적 및/또는 화학적 방법이 이용될 수 있고 공지된 줄기세포 배양 방법에서 이용되는 방법이면 특별히 제한되지 않는다. 하지만 세포 해리용 용액(cell dissociation buffer)을 처리하여 방치한 후, 세포 스크래퍼(scrapper)를 이용하여 물리적으로 긁어내는 방법, 즉, 물리적 및 화학적 방법을 동시에 사용하는 것이 바람직하다.The step (b) is not particularly limited as long as physical and/or chemical methods commonly used to separate adherent cultured cells can be used and methods used in known stem cell culture methods. However, it is preferable to process the cell dissociation buffer, leave it, and then physically scrape it off using a cell scraper, that is, use both physical and chemical methods at the same time.
상기 mTOR는 mammalian target of rapamycin의 약어로, FRAP1(FK506 binding protein 12-rapamycin-associated protein 1)으로도 불리는 인산화 효소의 일종이다. mTOR는 다른 단백질과 결합되어 단백질 결합체인 mTORC1(mTOR complex 1)과 mTORC2(mTOR complex 2)의 핵심 성분으로 작용한다.The mTOR is an abbreviation for mammalian target of rapamycin, and is a type of phosphorylation enzyme also called FRAP1 (FK506 binding protein 12-rapamycin-associated protein 1). mTOR binds to other proteins and acts as a key component of the protein complexes mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2).
상기 mTOR 작용제는 mTOR 신호 전달계의 활성을 증진시키는 약물로서, mTORC1 작용제 및/또는 mTORC2 작용제일 수 있다. 일례로, MHY1485, NV-5297, 및 NV-5138로 이루어진 군으로부터 선택된 1종 이상인 것일 수 있고, 바람직하게는 MHY1485일 수 있다.The mTOR agonist is a drug that enhances the activity of the mTOR signaling system and may be an mTORC1 agonist and/or an mTORC2 agonist. For example, it may be one or more selected from the group consisting of MHY1485, NV-5297, and NV-5138, and preferably MHY1485.
상기 mTOR 작용제의 유효 처치 농도는 500 nM 내지 2 μΜ일 수 있고, 바람직하게는 750 nM 내지 1.5 μM일 수 있으며, 더욱 바람직하게는 1 μM일 수 있다.The effective treatment concentration of the mTOR agonist may be 500 nM to 2 μΜ, preferably 750 nM to 1.5 μM, and more preferably 1 μM.
상기 mTOR 작용제를 처치하는 기간은 단계 (c)를 시작한 이후 적어도 10일, 적어도 15일, 적어도 20일, 적어도 25일, 적어도 30일일 수 있고, 최대 180일일 수 있다.The period of treatment with the mTOR agonist may be at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, and up to 180 days after starting step (c).
상기 오가노이드는 생체(인체) 장기와 유사한 구조, 세포의 구성 및 기능을 보유한 3차원 구조체를 의미한다. The organoid refers to a three-dimensional structure that has a structure, cell composition, and function similar to a living (human body) organ.
또한, 본 발명은 앞서 기재한 제조 방법을 이용하여 제조된 대뇌 오가노이드를 제공한다.Additionally, the present invention provides cerebral organoids prepared using the manufacturing method described above.
또한, 본 발명은 (a) 앞서 기재한 제조 방법을 이용하여 제조된 대뇌 오가노이드에 약물을 처리하기 전의 신경 활성도를 측정하는 단계; (b) 약물을 처리한 후의 상기 대뇌 오가노이드의 신경 활성도를 측정하는 단계; 및 (c) 단계 (a)의 신경 활성도와 단계 (b)의 신경 활성도를 비교하는 단계를 포함하는 신경 발달 장애를 치료 또는 개선하기 위한 약물 스크리닝 방법을 제공한다. 상기 신경 발달 장애는 자폐스펙트럼 장애, 드라베 증후군(Dravet syndrome) 또는 레트 증후군(Rett syndrome)를 포함할 수 있다.In addition, the present invention includes the steps of (a) measuring neural activity before treating the cerebral organoid prepared using the above-described production method with a drug; (b) measuring the neural activity of the cerebral organoid after treatment with the drug; and (c) comparing the neural activity of step (a) with the neural activity of step (b). The neurodevelopmental disorder may include autism spectrum disorder, Dravet syndrome, or Rett syndrome.
상기 신경 활성을 측정하는 방법은 신경 관련 오가노이드의 기능적 평가를 위한 공지된 기술이라면 특별히 제한되지 않으나, 칼슘 이미징(Calcium imaging), 패치 클램프(patch clamp) 및 다중전극어레이(multielectrode array, MEA)로 이루어지는 군으로부터 선택된 적어도 하나인 것이 바람직하고, 다중전극어레이로 측정하는 것이 더욱 바람직하다.The method of measuring the neural activity is not particularly limited as long as it is a known technique for functional evaluation of nerve-related organoids, but may include calcium imaging, patch clamp, and multielectrode array (MEA). It is preferable that it is at least one selected from the group consisting of, and it is more preferable to measure with a multi-electrode array.
본 발명의 구체적인 실시예에서, 대뇌 오가노이드를 제조하기 위해 유도만능줄기세포를 배양 및 분화시키는 과정을 수행하였고, 그 과정에서 배양 10일차부터 mTOR 신호전달계를 향상시키는 작용제인 MHY1485 또는 mTOR 신호전달계를 억제하는 길항제인 에버롤리무스(everolimus)를 배지에 추가하여 배양하였다. mTOR 작용제인 MHY1485 및 길항제인 에버롤리무스는 대뇌 오가노이드의 mTOR 신호 전달계를 증가시키거나(MHY1485) 감소시키는(에버롤리무스) 효과를 보였다(도 1). 약물을 배지에 추가하여 배양한 결과, mTOR 작용제인 MHY1485를 1 μM의 농도로 처치하면 대뇌 오가노이드의 크기가 증가하고 에버롤리무스를 1 μM의 농도로 처치하면 크기가 감소하는 경향이 있었다(도 2). 세부적으로 배양 지속 기간에 따라 오가노이드의 성장 정도를 비교해보면 약물을 처치하기 시작하는 배양 10일차의 대뇌 오가노이드의 크기는 그룹에 관계없이 차이가 없었다(도 3a). 약물을 처치하고 약 33일이 지난 배양 43일차에는 MHY1485를 처치한 오가노이드의 지름이 10,000 μm 수준으로 약물을 처치하지 않은 대조군에 비해 지름이 약 2배에 달하는 것을 확인할 수 있고 에버롤리무스를 처치하면 대조군에 비해 지름이 작은 것을 확인할 수 있었다(도 3b). 하지만 배양 70일차 또는 120일차의 대뇌 오가노이드를 비교하면 MHY1485를 처치한 경우에는 대조군과 큰 차이가 없었으나, 에버롤리무스를 처치한 경우에는 대조군에 비해 대뇌 오가노이드의 크기가 작은 경향이 있었다(도 3c 및 3d). 즉, mTOR 신호전달계에 의해 대뇌 오가노이드의 성장을 조절할 수 있으나, 배양 기간이 길어지면 대뇌 오가노이드의 성장이 포화되기 때문에 MHY1485의 영향은 배양 초기 단계에 더 크게 영향을 미치는 것을 확인할 수 있다. 대뇌 오가노이드의 성장뿐만 아니라 MHY1485 및 에버롤리무스가 대뇌 오가노이드의 생존율에도 영향을 미치는지 관찰하였다. 대뇌 오가노이드의 크기와 마찬가지로 약물을 처치하기 시작하는 배양 10일차에는 대뇌 오가노이드의 생존율은 그룹 간의 차이가 없었다(도 4a). 하지만 MHY1485를 처치한 대뇌 오가노이드의 경우, 배양 43일차 및 70일차의 생존율이 대조군과 비교하여 증가한 것을 확인할 수 있었으나, 배양 120일차에는 큰 차이가 없었다(도 4b, 4c, 및 4d). 배양 기간이 길어지면 MHY1485의 영향이 감소하지만 70일차까지 생존율이 증가시킬 수 있었다. In a specific example of the present invention, the process of culturing and differentiating induced pluripotent stem cells was performed to produce cerebral organoids, and in the process, MHY1485, an agent that improves the mTOR signaling system, or the mTOR signaling system was added from the 10th day of culture. Everolimus, an inhibitory antagonist, was added to the medium and cultured. MHY1485, an mTOR agonist, and everolimus, an antagonist, showed the effect of increasing (MHY1485) or decreasing (everolimus) the mTOR signaling system in cerebral organoids (Figure 1). As a result of culture by adding the drug to the medium, the size of cerebral organoids tended to increase when MHY1485, an mTOR agonist, was treated at a concentration of 1 μM, and the size tended to decrease when treated with everolimus at a concentration of 1 μM (Figure 2). In detail, comparing the growth of organoids according to the duration of culture, there was no difference in the size of cerebral organoids regardless of the group on the 10th day of culture when drug treatment began (Figure 3a). On the 43rd day of culture, about 33 days after treatment with the drug, the diameter of the organoid treated with MHY1485 was approximately 10,000 μm, which was about twice the diameter of the control group that was not treated with the drug, and everolimus was treated. It was confirmed that the diameter was smaller than that of the control group (Figure 3b). However, when comparing the cerebral organoids on the 70th or 120th day of culture, there was no significant difference from the control group when treated with MHY1485, but the size of the cerebral organoids tended to be smaller when treated with everolimus compared to the control group ( Figures 3c and 3d). In other words, the growth of cerebral organoids can be controlled by the mTOR signaling system, but as the culture period becomes longer, the growth of cerebral organoids becomes saturated, so it can be seen that the effect of MHY1485 has a greater effect on the initial stage of culture. In addition to the growth of cerebral organoids, we observed whether MHY1485 and everolimus affected the survival rate of cerebral organoids. As with the size of the cerebral organoids, there was no difference in the survival rate of the cerebral organoids between groups on the 10th day of culture when drug treatment began (Figure 4a). However, in the case of cerebral organoids treated with MHY1485, the survival rate on days 43 and 70 of culture was confirmed to be increased compared to the control group, but there was no significant difference on day 120 of culture (Figures 4b, 4c, and 4d). As the culture period increases, the effect of MHY1485 decreases, but survival rate can be increased up to day 70.
오가노이드의 생존 및 성장뿐 아니라 실질적으로 기능에 영향을 미칠 수 있는 분화 상태의 변화가 있는지 확인하기 위해 신경 마커의 발현 수준을 비교하였다. 그 결과, 배양 10일차부터 43일차까지 MHY1485를 처치하면 신경 마커 유전자 중 8가지의 발현이 증가하고 3가지의 발현만 감소하지만, 에버롤리무스를 처치하면 3가지의 신경 마커만 발현이 증가하고 27가지의 신경 마커의 발현은 감소하였다(도 5a 및 5b). 또한, 배양 10일차부터 120일차까지 MHY1485를 처치하면 21가지의 신경 마커의 발현이 증가하고 3가지 유전자의 발현만 감소하였으나, 에버롤리무스를 처치하면 2가지 신경 마커의 발현만 증가하고 4가지 신경 마커의 발현은 감소하였다(도 5c 및 5d). 유전자의 발현뿐만 아니라 MHY1485를 처치한 43일차의 대뇌 오가노이드는 미분화 세포의 마커인 SOX2의 발현이 감소하고 신경 분화 마커인 MAP2 및 NeuN의 발현이 증가하였고, MHY1485를 처치한 120일차의 대뇌 오가노이드는 CTIP2 및 SATB2의 발현이 증가하였다. 즉, MHY1485를 처치하면 대뇌 오가노이드의 성장뿐만 아니라 발달(분화) 과정도 촉진시킬 수 있고, 분화 과정에 미치는 영향은 배양 기간이 증가할수록 커지는 것을 확인할 수 있었다.Expression levels of neural markers were compared to determine whether there were changes in differentiation status that could substantially affect organoid survival and growth, as well as function. As a result, when MHY1485 was treated from day 10 to day 43 of culture, the expression of 8 neural marker genes increased and the expression of only 3 decreased, but when everolimus was treated, the expression of only 3 neural marker genes increased 27 Expression of neuronal markers in the branches was decreased (Figures 5A and 5B). In addition, when MHY1485 was treated from the 10th day to the 120th day of culture, the expression of 21 neural markers increased and the expression of only 3 genes decreased, but when everolimus was treated, the expression of only 2 neural markers increased and the expression of 4 neural markers increased. The expression of the marker decreased (Figures 5c and 5d). In addition to gene expression, cerebral organoids on day 43 after treatment with MHY1485 showed decreased expression of SOX2, a marker for undifferentiated cells, and increased expression of MAP2 and NeuN, markers of neural differentiation, and cerebral organoids on day 120 after treatment with MHY1485. showed increased expression of CTIP2 and SATB2. In other words, it was confirmed that treatment with MHY1485 can promote not only the growth of cerebral organoids but also the development (differentiation) process, and the effect on the differentiation process increases as the culture period increases.
최종적으로 오가노이드는 실제 장기의 기능을 모사하여야 하기 때문에 대뇌 오가노이드의 신경 활성 정도를 다중전극어레이를 이용하여 측정하였다(도 7a). 대뇌 오가노이드의 신경 활성 기능은 배양 70일차까지는 측정되지 않았고 70일 이후부터 전기 자극에 대한 반응을 보이기 시작하였다(도 7b). 대뇌 오가노이드를 제조하면서 MHY1485를 처치한 그룹의 경우, 평균 발화 속도(mean firing rate), 네트워크 돌발 주파수(network burst frequency), 전극 돌발 크기(electrode burst size), 돌발 수(the number of burst)와 같은 전기 자극에 대한 물리적 반응이 약 20% 가량 증가하는 것을 관찰하였다(도 7c-7f). Ultimately, because organoids must simulate the functions of actual organs, the degree of neural activity of cerebral organoids was measured using a multielectrode array (Figure 7a). The neural activity function of the cerebral organoids was not measured until the 70th day of culture, and they began to respond to electrical stimulation after 70 days (Figure 7b). For the group treated with MHY1485 while producing cerebral organoids, the mean firing rate, network burst frequency, electrode burst size, number of bursts and It was observed that the physical response to the same electrical stimulation increased by approximately 20% (Figures 7c-7f).
즉, 유도만능줄기세포를 이용하여 대뇌 오가노이드로 분화되는 과정에서 mTOR 신호 전달계의 활성은 대뇌 오가노이드의 성장 및 생존율을 높이고 신경 분화 및 신경 활성을 증진시키는 역할을 한다는 것을 확인할 수 있었다. 따라서 mTOR 신호전달계 작용제를 이용하여 대뇌 오가노이드의 성장 및 생존율을 향상시켜 제조된 대뇌 오가노이드의 수득율을 높일 수 있고 신경 발달 정도가 높고 신경 활성도가 높은 실제 장기와 보다 유사한 대뇌 오가노이드를 제조할 수 있다.In other words, it was confirmed that in the process of differentiation into cerebral organoids using induced pluripotent stem cells, the activity of the mTOR signaling system increases the growth and survival rate of cerebral organoids and plays a role in enhancing neural differentiation and neural activity. Therefore, by using mTOR signaling system agonists, the growth and survival rate of cerebral organoids can be improved, thereby increasing the yield of manufactured cerebral organoids, and it is possible to manufacture cerebral organoids that are more similar to actual organs with a high degree of neural development and high neural activity. there is.
이하에서는, 구체적인 실시예를 통하여 본 발명을 더욱 상세하게 설명한다.Below, the present invention will be described in more detail through specific examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해서 한정되는 것은 아니다.However, the following examples only illustrate the present invention, and the content of the present invention is not limited by the following examples.
<실시예 1> 대뇌 오가노이드의 제조<Example 1> Preparation of cerebral organoids
매트리겔(Matrigel)이 코팅된 6-웰(well) 배양 플레이트를 제조하기 위해 DMEM/F12 배지에 매트리겔 100 μl, 1%의 페니실린(penicillin), 0.2%의 겐타마이신(gentamicin)을 혼합하여 배양 플레이트에 넣은 후 37 ℃, 5% CO2 세포 배양기에서 30분간 코팅하였다. 매트리겔이 코팅된 배양 플레이트에 mTeSRTM1 배지를 이용하여 유도만능줄기세포(induced pluripotent stem cell, iPSC)인 IMR-90-4 세포를 부착 배양하였다. 세포는 37℃, 5% CO2 조건에서 배양되었고 형성된 iPSC 콜로니를 분리할 때는 세포 분리용 배지(Gentle cell dissociation media) 1 mL을 6분 간 처리한 후, 스크래퍼(scraper)를 이용하여 살살 긁어 내었다. iPSC 콜로니를 분리시키는 방법에는 여러 가지 방법이 있으나, 세포 분리용 배지를 처리한 후 스크래퍼를 이용하여 세포를 긁어내어 떨어뜨리는 방법을 이용할 때 오가노이드의 생존율을 향상시킬 수 있었다. To prepare a 6-well culture plate coated with Matrigel, 100 μl of Matrigel, 1% penicillin, and 0.2% gentamicin were mixed and cultured in DMEM/F12 medium. After putting it into the plate, it was coated in a cell incubator at 37°C and 5% CO 2 for 30 minutes. IMR-90-4 cells, which are induced pluripotent stem cells (iPSC), were attached and cultured on a Matrigel-coated culture plate using mTeSR TM 1 medium. Cells were cultured at 37°C and 5% CO 2 conditions. When separating iPSC colonies, they were treated with 1 mL of Gentle cell dissociation media for 6 minutes and then gently scraped off using a scraper. . There are several methods for isolating iPSC colonies, but the survival rate of organoids was improved when using a method of scraping off the cells using a scraper after treating the cell isolation medium.
분리된 콜로니의 세포 수를 측정하여 U자형 바닥 저부착(U-bottom ultra-low-attachment) 96-웰 배양 플레이트의 각 웰에 20,000개의 세포를 분주하여 부유 배양하였다. 이 때 이용된 배양배지는 DMEM/F12에 50 μM Rho 인산화효소 저해제(Y-27632) 및 5% 열-불활성화 FBS를 추가하여 이용하였다. 그 후, 아래의 표 1에 기재된 조성의 신경 유도 배지(neural induction media, NIM)를 이용하여 세포를 배양하였고, 신경 유도 배지는 배양 10일차까지 2일에 한 번씩 교체하였다. 배양 2일차에는 NIM 배지에 열-불활성화 FBS 첨가없이 50 μM Rho 인산화효소 저해제(Y-27632)를 첨가하였고, 4일차에는 Rho 인산화효소 저해제를 더 이상 첨가하지 않았다. 전술한 과정을 거쳐 IMR-90-4 오가노이드를 5% CO2, 37℃ 조건에서 0일차부터 10일차까지 정치 배양하였다.The number of cells in the isolated colonies was measured, and 20,000 cells were dispensed into each well of a U-bottom ultra-low-attachment 96-well culture plate and cultured in suspension. The culture medium used at this time was DMEM/F12 with 50 μM Rho kinase inhibitor (Y-27632) and 5% heat-inactivated FBS added. Afterwards, the cells were cultured using neural induction media (NIM) with the composition shown in Table 1 below, and the neural induction media was replaced every two days until the 10th day of culture. On the second day of culture, 50 μM Rho kinase inhibitor (Y-27632) was added to the NIM medium without heat-inactivated FBS, and on the fourth day, the Rho kinase inhibitor was no longer added. Through the above-described process, IMR-90-4 organoids were cultured statically from day 0 to day 10 under 5% CO 2 and 37°C conditions.
이후, 오가노이드를 96-웰 배양 플레이트에서 평평한 저흡착 6-웰 배양 플레이트로 옮겼으며, 이때 각 웰마다 8개의 오가노이드를 넣었다. 10일차부터 18일차까지는 표 2에 기재된 신경 분화 배지 Ⅰ(neural differentiation media-Ⅰ, NDM-Ⅰ)를 이용하여 배양하였고, 배양 플레이트를 80 rpm/분의 속도로 회전시키면서 배양을 진행하였고, 배지를 2일에 한 번씩 교체하였다. Afterwards, the organoids were transferred from the 96-well culture plate to a flat, low-adsorption 6-well culture plate, with 8 organoids placed in each well. From day 10 to day 18, the culture was performed using neural differentiation media-Ⅰ (NDM-Ⅰ) shown in Table 2, and the culture was performed while rotating the culture plate at a speed of 80 rpm/min, and the medium was It was replaced once every two days.
18일차부터 120일까지는 표 2에 기재된 신경 분화 배지 Ⅱ(NDM-Ⅱ)를 이용하여 배양하였고, 배양 배지를 4일에 한 번씩 교체하였다. 모든 오가노이드용 배지는 0.22 μm 폴리에테르술폰(polyethersulfone, PES) 진공 필터를 이용하여 거른 후 사용하였다.From day 18 to day 120, the cells were cultured using Neural Differentiation Medium II (NDM-II) listed in Table 2, and the culture medium was changed once every 4 days. All medium for organoids were filtered using a 0.22 μm polyethersulfone (PES) vacuum filter before use.
배양 배지에 페니실린/스트렙토마이신(penicillin/streptomycin) 뿐만 아니라 겐타마이신(gentamycin)을 함께 처리하였고, 배양 배지를 0.22 μm 필터를 이용하여 여과한 다음 사용함으로써 장기 배양에 따른 배양 배지의 오염 위험을 최소화시켰다.The culture medium was treated with gentamycin as well as penicillin/streptomycin, and the culture medium was filtered using a 0.22 μm filter before use to minimize the risk of contamination of the culture medium due to long-term culture. .
<실시예 2> 대뇌 오가노이드의 성장에 미치는 mTOR 작용제의 영향 확인<Example 2> Confirmation of the effect of mTOR agonist on the growth of cerebral organoids
대뇌 오가노이드 제조시 mTOR 작용제가 대뇌 오가노이드의 성장에 영향을 확인하기 위해서 mTOR 작용제(agonist)인 MHY1485와 mTOR 길항제(antagonist)인 에버롤리무스(everolimus)를 처치하여 영향을 확인하고자 하였다. In order to determine the effect of mTOR agonists on the growth of cerebral organoids during the preparation of cerebral organoids, MHY1485, an mTOR agonist, and everolimus, an mTOR antagonist, were administered to determine the effect.
실시예 1에 기재된 방법으로 대뇌 오가노이드를 제조하면서 배양 10일차부터 MHY1485 또는 에버롤리무스를 각각 1 또는 5 μM의 농도로 배지를 주기적으로 교체하면서 110일 간 처치하였다.Cerebral organoids were prepared by the method described in Example 1, and starting from the 10th day of culture, MHY1485 or everolimus were treated at a concentration of 1 or 5 μM, respectively, for 110 days while periodically changing the medium.
각 약물의 처리에 의해 mTOR 신호전달계 활성 수준에 변화가 생기는지 확인하기 위해 웨스턴 블롯을 수행하였다.Western blot was performed to determine whether the level of mTOR signaling system activity changed due to treatment with each drug.
웨스턴 블롯을 위해 대뇌 오가노이드에 차갑게 준비한 전체-세포 추출용 버퍼(whole-cell extract buffer, pH 7.4)를 추가하여 세포 용해물을 수득하였다. 단백질의 농도를 BCA 단백질 정량 키트를 이용하여 측정하고 동량의 단백질을 SDS-PAGE로 내린 후, PVDF(polyvinylidene fluoride, 플루오르화 폴리비닐리덴)에 전사(transfer)하여 이용하였다. 각 PVDF 멤브레인은 블로킹(blocking) 후 1차 항체로서 mTOR 단백질과 mTOR 신호전달계의 하위 단백질인 S6K, p4EBP1 및 각각의 인산화 형태에 대한 항체를 이용하였고 각 1차 항체에 대응하는 2차 항체(HRP-융합 항체)를 순차적으로 넣어 혼성화시켰다. 단백질 밴드는 케미닥(Chemidoc™MP)을 이용하여 확인하였고 GAPDH를 대조군으로 이용하였다.For Western blotting, cell lysates were obtained by adding cold prepared whole-cell extraction buffer (pH 7.4) to the cerebral organoids. The protein concentration was measured using a BCA protein quantification kit, and an equal amount of protein was subjected to SDS-PAGE and then transferred to PVDF (polyvinylidene fluoride) for use. After blocking, each PVDF membrane was treated with antibodies against the mTOR protein and the sub-proteins of the mTOR signaling system, S6K, p4EBP1, and their respective phosphorylated forms, as primary antibodies, and secondary antibodies (HRP-) corresponding to each primary antibody were used. fusion antibodies) were sequentially added and hybridized. Protein bands were confirmed using Chemidoc™MP, and GAPDH was used as a control.
mTOR 작용제인 MHY1485를 처치함에 따라 대뇌 오가노이드 내의 mTOR 단백질의 활성이 증가하여 하위 단백질인 4E-BP1 및 S6K의 인산화 형태가 증가하였고 mTOR 길항제인 에버롤리무스를 처치한 대뇌 오가노이드의 경우, 반대로 4E-BP1 및 S6K의 인산화 형태가 감소하여 MHY1485 및 에버롤리무스가 의도한대로 정상적으로 작용하고 있음을 알 수 있다(도 1).As the mTOR agonist MHY1485 was treated, the activity of mTOR protein in cerebral organoids increased, resulting in an increase in the phosphorylated forms of downstream proteins 4E-BP1 and S6K. In the case of cerebral organoids treated with the mTOR antagonist everolimus, on the contrary, 4E -The phosphorylated forms of BP1 and S6K were reduced, indicating that MHY1485 and everolimus were functioning normally as intended (Figure 1).
대뇌 오가노이드 제조 과정에서 mTOR 작용제 또는 길항제를 처치하면서 세포의 크기(지름)을 측정한 결과는 도 1에서 확인할 수 있다. 전반적으로 세포의 크기가 MHY1485 처치 조건에서 세포의 크기가 커진 것을 확인할 수 있다(도 2a 및 2b). 세부적으로는 약물을 처치하기 시작하는 배양 10일차에는 각 대뇌 오가노이드 그룹 간의 크기 차이가 없었다(도 3a). 배양 43일차에 대뇌 오가노이드의 크기 및 생존율을 확인한 결과, 10일 내지 43일 동안 1 μM의 mTOR 작용제 MHY1485를 처치하였을 때 그렇지 않은 경우보다 평균 지름이 1.89배 증가하였고(도 3b) 배양 70일차 이후에는 평균 지름의 차이를 확인할 수 없었다(도 3c 및 3d). 배양 70일차 이후에는 세포의 성장이 충분히 이루어졌기 때문에 차이가 관찰되지 않는 것으로 사료된다.The results of measuring the size (diameter) of cells while treating them with mTOR agonists or antagonists during the production of cerebral organoids can be seen in Figure 1. Overall, it can be seen that the cell size increased under MHY1485 treatment conditions (Figures 2a and 2b). In detail, there was no difference in size between each cerebral organoid group on the 10th day of culture when drug treatment began (Figure 3a). As a result of checking the size and survival rate of cerebral organoids on the 43rd day of culture, when treated with 1 μM of the mTOR agonist MHY1485 for 10 to 43 days, the average diameter increased 1.89 times compared to the untreated case (Figure 3b), and after the 70th day of culture No difference in average diameter could be confirmed (Figures 3c and 3d). It is believed that no differences are observed after the 70th day of culture because the cells have grown sufficiently.
하지만 저농도 조건인 10 nM 또는 100 nM 조건으로 처치하였을 때는 대뇌 오가노이드의 성장(크기)에 대한 영항을 확인할 수 없었고(도 2c), 고농도 조건인 5 μM로 MHY1485 또는 에버롤리무스를 처치하면 대뇌 오가노이드가 사망하는 것을 관찰할 수 있었다(도 2b).However, when treated at a low concentration of 10 nM or 100 nM, no effect on the growth (size) of cerebral organoids could be confirmed (Figure 2c), and when treated with MHY1485 or everolimus at a high concentration of 5 μM, cerebral organoids were not observed. Noid death could be observed (Figure 2b).
즉, 대뇌 오가노이드를 제조하는 과정 중에 mTOR 신호전달계의 활성을 높이는 mTOR 작용제를 적정 농도로 처치하면, 배양 초기 단계에서의 대뇌 오가노이드가 성장하는 속도를 향상시킬 수 있음을 알 수 있다.In other words, it can be seen that if an mTOR agonist that increases the activity of the mTOR signaling system is administered at an appropriate concentration during the process of manufacturing cerebral organoids, the growth rate of cerebral organoids in the early stage of culture can be improved.
<실시예 3> 대뇌 오가노이드의 생존율에 미치는 mTOR 작용제의 영향 확인<Example 3> Confirmation of the effect of mTOR agonist on the survival rate of cerebral organoids
대뇌 오가노이드 제조시 mTOR 작용제가 대뇌 오가노이드의 성장에 영향을 확인하기 위해서 mTOR 작용제인 MHY1485와 mTOR 길항제인 에버롤리무스(everolimus)를 처치하여 영향을 확인하고자 하였다. In order to determine the effect of mTOR agonists on the growth of cerebral organoids during the preparation of cerebral organoids, MHY1485, an mTOR agonist, and everolimus, an mTOR antagonist, were administered to determine the effect.
실시예 1에 기재된 방법으로 대뇌 오가노이드를 제조하면서 배양 10일차부터 MHY1485 또는 에버롤리무스를 각각 1 μM의 농도로 배지를 주기적으로 교체하면서 110일 간 처치하였다.Cerebral organoids were prepared by the method described in Example 1, and starting from the 10th day of culture, MHY1485 or everolimus were treated at a concentration of 1 μM each for 110 days while periodically changing the medium.
배양 10일차, 43일차, 70일차, 120일차의 대뇌 오가노이드의 생존율을 확인하기 위해 셀타이터-글로 3D(CellTiter-Glo® 3D)를 세포 및 배지와 동일한 용량으로 세포 및 배지에 추가하여 5분간 섞어주고 25분간 실온에서 방치한 후, 스펙트라맥스 M5(SpectraMax M5)를 이용하여 발광을 측정하였고, 대뇌 오가노이드의 크기는 현미경(Nikon Eclipse Ti2)을 이용하여 측정하였다.To check the survival rate of cerebral organoids on days 10, 43, 70, and 120 of culture, CellTiter-Glo ® 3D was added to the cells and medium in the same volume as the cells and medium for 5 minutes. After mixing and leaving at room temperature for 25 minutes, luminescence was measured using SpectraMax M5, and the size of cerebral organoids was measured using a microscope (Nikon Eclipse Ti2).
mTOR 작용제인 MHY1485를 1 μM의 농도로 처치하면 약물을 처치하지 않은 대뇌 오가노이드와 비교하여 43일차 대뇌 오가노이드의 생존율이 59.8% 증가하였고, 70일차 대뇌 오가노이드의 생존율 또한 유의미한 수준으로 높은 것을 확인할 수 있다(도 4b 및 4c). 하지만 120일차까지 배양하면 차이를 확인할 수 없었다.When treated with MHY1485, an mTOR agonist, at a concentration of 1 μM, the survival rate of cerebral organoids at day 43 increased by 59.8% compared to cerebral organoids not treated with the drug, and the survival rate of cerebral organoids at day 70 was also significantly higher. (Figures 4b and 4c). However, no difference could be confirmed when cultured until day 120.
mTOR 길항제인 1 μM의 에버롤리무스를 처치하였을 때는 대조군과 대비하여 생존율의 변화를 확인할 수 없었다(도 4).When treated with 1 μM everolimus, an mTOR antagonist, no change in survival rate was confirmed compared to the control group (Figure 4).
하지만 오가노이드의 크기 결과와 동일하게 더 낮은 농도인 10 nM 및 100 nM로 약물을 처치하였을 때도 대뇌 오가노이드의 생존율의 변화를 확인할 수 없었다(도 4e).However, consistent with the organoid size results, no change in the survival rate of cerebral organoids could be confirmed even when the drug was treated at lower concentrations of 10 nM and 100 nM (Figure 4e).
따라서 1 μM의 mTOR 작용제(MHY1485)를 이용하면 대뇌 오가노이드의 생존율을 높여 더 많은 대뇌 오가노이드를 제조할 수 있을 것으로 유추할 수 있다.Therefore, it can be inferred that using 1 μM of the mTOR agonist (MHY1485) can increase the survival rate of cerebral organoids and produce more cerebral organoids.
<실시예 4> 대뇌 오가노이드의 신경 마커 발현에 미치는 mTOR 작용제의 영향 확인<Example 4> Confirmation of the effect of mTOR agonist on the expression of neural markers in cerebral organoids
실시예 2에 기재한 것과 같이 대뇌 오가노이드를 제작하면서 mTOR 작용제인 MHY1485 또는 mTOR 길항제인 에버롤리무스를 처치하였고, 신경 분화에 미치는 영향을 확인하고자 qPCR (정량 PCR, quantitative PCR)을 수행하였다. qPCR을 수행하기 위해서 대뇌 오가노이드로부터 RNeasy Mini kit를 이용하여 RNA를 추출하였다. RT2 First Strand kit를 이용하여 추출한 RNA로부터 cDNA를 제조하였고, 인간 CD RT2 Profiler PCR array 및 SYBR Green chemistry를 이용하여 cDNA를 분석함으로써 신경의 발달과 관련된 유전자의 발현을 확인하였다. 각 유전자의 발현 수준은 5개의 하우스키핑 유전자(housekeeping gene)의 발현 수준과 비교하여 분석하였다. As described in Example 2, cerebral organoids were prepared while being treated with the mTOR agonist MHY1485 or the mTOR antagonist everolimus, and qPCR (quantitative PCR) was performed to confirm the effect on neural differentiation. To perform qPCR, RNA was extracted from cerebral organoids using the RNeasy Mini kit. cDNA was prepared from RNA extracted using the RT2 First Strand kit, and the expression of genes related to neural development was confirmed by analyzing the cDNA using the human CD RT2 Profiler PCR array and SYBR Green chemistry. The expression level of each gene was analyzed by comparing it with the expression level of five housekeeping genes.
배양 43일차에 1 μM의 MHY1485를 처치한 대뇌 오가노이드의 경우, 8가지 신경 마커 BMP2, BMP8B, CHRM2, CXCL1, GDNF, IL3, TENM1, SHH)의 발현이 증가하였고 3가지 신경 마커(NDN, NEUROD1, NOTCH2)의 발현이 감소하였다(도 5a). 하지만 1 μM의 에버롤리무스를 동일한 기간 동안 처치한 경우에는 3가지 신경 마커(BMP2, CHRM2, TH)의 발현만 증가하였고 27가지 신경 마커(CDK5R1, DCX, DLL1, EFNB1, EGF, EP300, FLNA, GPI, GRIN1, MAP2, KMT2A, NDN, NEUROD1, NOTCH1, NOTCH2, NR2E3, NRP2, PAFAH1B1, POU3F3, POU4F1, PTN, RAC1, ROBO1, RTN4, SOX2, STAT3, VEGFA)의 발현이 감소하였다(도 5b). In the case of cerebral organoids treated with 1 μM MHY1485 on the 43rd day of culture, the expression of eight neuronal markers BMP2, BMP8B, CHRM2, CXCL1, GDNF, IL3, TENM1, and SHH increased, and three neuronal markers (NDN, NEUROD1) , NOTCH2) expression was decreased (Figure 5a). However, when 1 μM everolimus was treated for the same period, the expression of only 3 neuronal markers (BMP2, CHRM2, TH) increased and 27 neuronal markers (CDK5R1, DCX, DLL1, EFNB1, EGF, EP300, FLNA, The expression of GPI, GRIN1, MAP2, KMT2A, NDN, NEUROD1, NOTCH1, NOTCH2, NR2E3, NRP2, PAFAH1B1, POU3F3, POU4F1, PTN, RAC1, ROBO1, RTN4, SOX2, STAT3, VEGFA) was decreased (Figure 5b).
배양 120일차의 대뇌 오가노이드 또한 1 μM의 MHY1485를 처치한 경우에는 21가지의 신경 관련 유전자(ALK, ASCL1, CDK5RAP2, DCX, DLL1, EGF, MAP2, MEF2C, KMT2A, NEUROD1, NEUROG1, NOTCH1, NRG1, PARD3, PAX3, POU4F1, PTN, ROBO1, RTN4, SHH, SLIT2)의 발현이 증가하고 3가지 유전자(APOE, NDP, S100A6)의 발현만 감소하였으나(도 5c), 1 μM의 에버롤리무스를 처치한 경우에는 2가지 신경 관련 유전자(NOTCH1, RTN4)의 발현만 증가하였고 4가지 유전자(APOE, NDP, S100A6, VEGFA)의 발현은 감소하였다(도 5d).Cerebral organoids on day 120 of culture were also treated with 1 μM MHY1485, and 21 neuron-related genes (ALK, ASCL1, CDK5RAP2, DCX, DLL1, EGF, MAP2, MEF2C, KMT2A, NEUROD1, NEUROG1, NOTCH1, NRG1, The expression of PARD3, PAX3, POU4F1, PTN, ROBO1, RTN4, SHH, SLIT2) increased and the expression of only three genes (APOE, NDP, S100A6) decreased (Figure 5c), but treatment with 1 μM everolimus In this case, the expression of only two nerve-related genes (NOTCH1, RTN4) increased, and the expression of four genes (APOE, NDP, S100A6, VEGFA) decreased (Figure 5d).
유전자 수준의 차이가 실제로 단백질 수준에서도 반영이 되는지 확인하기 위해서 웨스턴 블롯을 수행하였다. Western blot was performed to confirm whether differences at the gene level were actually reflected at the protein level.
웨스턴 블롯을 위해 대뇌 오가노이드에 차갑게 준비한 전체-세포 추출용 버퍼(whole-cell extract buffer, pH 7.4)를 추가하여 세포 용해물을 수득하였다. 단백질의 농도를 BCA 단백질 정량 키트를 이용하여 측정하고 동량의 단백질을 SDS-PAGE로 내린 후, PVDF(polyvinylidene fluoride, 플루오르화 폴리비닐리덴)에 전사(transfer)하여 이용하였다. 각 PVDF 멤브레인은 블로킹(blocking) 후 1차 항체와 각각의 2차 항체(HRP-융합 항체)를 순차적으로 넣어 혼성화시켰다. 단백질 밴드는 케미닥(Chemidoc™MP)을 이용하여 확인하였고 GAPDH 단백질을 대조군으로 이용하였다.For Western blotting, cell lysates were obtained by adding cold prepared whole-cell extraction buffer (pH 7.4) to the cerebral organoids. The protein concentration was measured using a BCA protein quantification kit, and an equal amount of protein was subjected to SDS-PAGE and then transferred to PVDF (polyvinylidene fluoride) for use. Each PVDF membrane was hybridized by sequentially adding the primary antibody and each secondary antibody (HRP-fusion antibody) after blocking. Protein bands were confirmed using Chemidoc™MP, and GAPDH protein was used as a control.
그 결과, 배양 43일차 1 μM의 MHY1485 처치 대뇌 오가노이드에서는 신경 마커인 MAP2(microtubule-associated protein 2) 및 NeuN의 발현이 증가하는 것을 확인할 수 있고 줄기세포의 다분화능 마커이자 신경이 분화됨에 따라 감소하는 단백질인 SOX2의 양이 감소한 것을 확인할 수 있다. 하지만 1 μM의 에버롤리무스 처치 대뇌 오가노이드에서는 SOX2, MAP2 및 NeuN의 발현이 변화하지 않는 것을 확인하였다(도 6a).As a result, in cerebral organoids treated with 1 μM MHY1485 on the 43rd day of culture, the expression of MAP2 (microtubule-associated protein 2) and NeuN, which are neural markers, was confirmed to increase, and it is a pluripotency marker for stem cells and decreases as neurons differentiate. It can be seen that the amount of SOX2, a protein that does this, has decreased. However, it was confirmed that the expression of SOX2, MAP2, and NeuN did not change in cerebral organoids treated with 1 μM everolimus (Figure 6a).
배양 120일차의 대뇌 오가노이드는 1 μM의 MHY1485를 처치하면 CTIP2의 발현은 22% 정도 증가하였고SATB2의 발현은 57% 정도 증가하였으나, 1 μM의 에버롤리무스를 처치하면 CTIP2 및 SATB2의 발현은 감소하였다(도 6b).In cerebral organoids on day 120 of culture, when treated with 1 μM MHY1485, the expression of CTIP2 increased by about 22% and the expression of SATB2 increased by about 57%, but when treated with 1 μM everolimus, the expression of CTIP2 and SATB2 decreased. (Figure 6b).
MHY1485를 처치함으로써 대뇌 오가노이드의 신경 분화가 더 잘 이루어지는 것을 확인할 수 있다.It can be confirmed that neural differentiation of cerebral organoids is better achieved by treating MHY1485.
<실시예 5> 대뇌 오가노이드 신경 활성에 미치는 영향<Example 5> Effect on cerebral organoid nerve activity
mTOR 작용제인 MHY1485를 처치함으로써 대뇌 오가노이드의 성장 및 분화가 향상되는 것을 확인하였기에 실제로 신경의 활성에도 영향을 미치는지 확인하고자 다중전극어레이(multi-electrode array, MEA)를 수행하였다(도 7a). Since it was confirmed that treatment with the mTOR agonist MHY1485 improved the growth and differentiation of cerebral organoids, a multi-electrode array (MEA) was performed to confirm whether it actually affected neural activity (FIG. 7a).
MEA 플레이트를 0.01% 폴리-에틸렌이민(poly-ethyleneimine, PEI)으로 코팅한 후, 배양된 대뇌 오가노이드를 MEA 플레이트의 전극 위에 놓고 37 ℃, 5% CO2 조건에서 4시간 동안 배양하였다. 그 후, 배양 배지를 제거하여 전극과 오가노이드가 밀착하도록 한 후 전기 자극에 대한 반응을 관찰하였다. 평균 발화 속도(Mean firing rate, MFR) 결과는 신경의 활성도를 나타낸다.After coating the MEA plate with 0.01% poly-ethyleneimine (PEI), the cultured cerebral organoids were placed on the electrodes of the MEA plate and cultured for 4 hours at 37°C and 5% CO2 conditions. Afterwards, the culture medium was removed to ensure close contact between the electrode and the organoid, and then the response to electrical stimulation was observed. Mean firing rate (MFR) results indicate nerve activity.
관찰 결과, 대뇌 오가노이드는 배양 70일차에는 신경 활성을 보이지 않았고 70일 이후부터 전기 자극에 대한 물리적 반응이 보이기 시작하였다(MFR: 0.032)(도 7b)As a result of observation, cerebral organoids did not show neural activity on the 70th day of culture, and began to show physical responses to electrical stimulation after 70 days (MFR: 0.032) (Figure 7b)
대뇌 오가노이드를 제조하는 과정 중에 1 μM의 MHY1485를 처치하면, 평균 발화 속도(MFR), 네트워크 돌발 주파수(network burst frequency), 전극 돌발 크기(electrode burst size), 돌발 수(the number of bursts)와 같은 전기 자극에 대한 물리적 반응이 각각 18.9%, 65.7%, 70.4%, 및 43.2% 가량 증가하는 것을 관찰하였다(도 7c-7f).When treated with 1 μM MHY1485 during the preparation of cerebral organoids, the mean firing rate (MFR), network burst frequency, electrode burst size, the number of bursts and It was observed that the physical response to the same electrical stimulation increased by 18.9%, 65.7%, 70.4%, and 43.2%, respectively (FIGS. 7c-7f).
하지만 에버롤리무스를 처치하면 아무 것도 처치하지 않은 대조군에 비해 MFR의 경우 9.2%, network burst frequency의 경우 21.6%, electrode burst size의 경우, 31.5%, 및 the number of bursts의 경우, 1.1% 정도 감소하였다(도 7c-7f).However, treatment with everolimus decreased MFR by 9.2%, network burst frequency by 21.6%, electrode burst size by 31.5%, and the number of bursts by 1.1% compared to the untreated control group. (Figures 7c-7f).
즉, MHY1485를 처치하여 대뇌 오가노이드를 제조하면 대뇌 오가노이드의 신경 활성 반응이 상대적으로 향상되는 것을 알 수 있고, 이를 통해 정상적인 기능을 갖는 대뇌 오가노이드로 분화하였음을 알 수 있다.In other words, when cerebral organoids are prepared by treating them with MHY1485, the neural activity response of the cerebral organoids is relatively improved, which shows that they have differentiated into cerebral organoids with normal functions.
본 실시예를 통해 유도만능줄기세포가 대뇌 오가노이드로 분화되는 과정은 mTOR 신호전달계의 활성에 따라 증진될 수 있다는 것을 확인할 수 있고, 대뇌 오가노이드를 제조할 때 mTOR 작용제를 첨가하는 과정을 통해 대뇌 오가노이드의 성장을 촉진하고 신경의 분화를 향상시키며, 신경 활성을 증진시킬 수 있음을 확인할 수 있다.Through this example, it can be confirmed that the process of differentiation of induced pluripotent stem cells into cerebral organoids can be enhanced depending on the activity of the mTOR signaling system, and that the process of adding an mTOR agonist when producing cerebral organoids It can be confirmed that it can promote the growth of organoids, improve neural differentiation, and enhance neural activity.
Claims (12)
(a) 유도만능줄기세포를 매트리겔(Matrigel)이 코팅된 조건에서 배양하는 단계;
(b) 2차원적으로 배양된 유도만능줄기세포의 콜로니(colony)를 분리하는 단계;
(c) 분리된 콜로니를 3차원 배양하는 단계;
(d) 단계 (c)의 콜로니를 48% DMEM/F12, 48% 신경기질 A 배지(neurobasal A media), 50 μM 베타-머캅토에탄올, 1% N2 보충제(supplement), 2% 비타민 A 배제 B27 보충제(B27 supplement without vitamin A), 1% 글루타맥스(Glutamax), 0.025% 인간 인슐린 용액(human insulin solution) 1% 페니실린/스트렙토마이신(penicillin/streptomycin), 및 0.25% 겐타마이신(gentamycin)을 포함하는 신경 분화 배지 Ⅰ에서 10일차부터 18일차까지 배양하는 단계로서, 상기 신경 분화 배지 Ⅰ에 mTOR 작용제로서의 MHY1485를 첨가하여 배양하는 단계; 및
(e) 단계 (d)의 콜로니를 48% DMEM/F12, 48% 신경기질 A 배지(neurobasal A media), 50 μM 베타-머캅토에탄올, 1% N2 보충제(supplement), 2% 비타민 A 배제 B27 보충제(B27 supplement without vitamin A), 1% 글루타맥스(Glutamax), 0.5% MEM-비필수 아미노산(Minimum Essential Media-Non-Essential Amino Acids, MEM-NEAA), 0.025% 인간 인슐린 용액(human insulin solution), 1% 페니실린/스트렙토마이신(penicillin/streptomycin), 및 0.25% 겐타마이신(gentamycin), 20 ng/ml BDNF, 200 μM cAMP, 및 200 μM 아스코르브산(ascorbic acid)를 포함하는 신경 분화 배지 Ⅱ에서 18일차부터 120일차까지 배양하는 단계로서, 상기 신경 분화 배지 Ⅱ에 mTOR 작용제로서의 MHY1485를 첨가하여 배양하는 단계;
를 포함하는, 대뇌 오가노이드 제조 방법.
A method for producing cerebral organoids with enhanced growth and enhanced differentiation ability, comprising:
(a) culturing induced pluripotent stem cells in Matrigel-coated conditions;
(b) isolating colonies of two-dimensionally cultured induced pluripotent stem cells;
(c) culturing the isolated colonies in three dimensions;
(d) Colonies from step (c) were cultured in 48% DMEM/F12, 48% neurobasal A media, 50 μM beta-mercaptoethanol, 1% N2 supplement, 2% vitamin A excluded B27. Contains B27 supplement without vitamin A, 1% Glutamax, 0.025% human insulin solution, 1% penicillin/streptomycin, and 0.25% gentamycin. A step of culturing from day 10 to day 18 in neural differentiation medium I, adding MHY1485 as an mTOR agonist to the neural differentiation medium I and culturing; and
(e) Colonies from step (d) were cultured in 48% DMEM/F12, 48% neurobasal A media, 50 μM beta-mercaptoethanol, 1% N2 supplement, 2% vitamin A excluded B27. Supplement (B27 supplement without vitamin A), 1% Glutamax, 0.5% MEM-Non-Essential Amino Acids (MEM-NEAA), 0.025% human insulin solution ), 1% penicillin/streptomycin, and 0.25% gentamycin, 20 ng/ml BDNF, 200 μM cAMP, and 200 μM ascorbic acid in neural differentiation medium II. Culturing from day 18 to day 120, adding MHY1485 as an mTOR agonist to the neural differentiation medium II and culturing;
Method for producing cerebral organoids, comprising:
상기 단계 (a)의 유도만능줄기세포는 IMR-90-1, IMR-90-2, IMR-90-3, IMR-90-4, 또는 SIGi001-A-1인 것인, 대뇌 오가노이드 제조 방법.
According to paragraph 1,
The induced pluripotent stem cells in step (a) are IMR-90-1, IMR-90-2, IMR-90-3, IMR-90-4, or SIGi001-A-1, a method for producing cerebral organoids. .
상기 단계 (d) 및 (e)에서 mTOR 작용제는 500 nM 내지 2 μM의 농도로 첨가되는 것인, 대뇌 오가노이드 제조 방법.
According to paragraph 1,
In steps (d) and (e), the mTOR agonist is added at a concentration of 500 nM to 2 μM.
상기 단계 (d) 및 (e)에서 mTOR 작용제는 1 μM의 농도로 첨가되는 것인, 대뇌 오가노이드 제조 방법.
According to paragraph 1,
In steps (d) and (e), the mTOR agonist is added at a concentration of 1 μM.
상기 단계 (d) 이후 30일 내지 35일차의 대뇌 오가노이드는 신경 마커인 BMP2, BMP8B, CHRM2, CXCL1, GDNF, IL3, TENM1, 및 SHH로 이루어진 군으로부터 선택되는 하나 이상의 유전자의 발현이 증가한 것인, 대뇌 오가노이드 제조 방법.
According to paragraph 1,
Cerebral organoids 30 to 35 days after step (d) have increased expression of one or more genes selected from the group consisting of neural markers BMP2, BMP8B, CHRM2, CXCL1, GDNF, IL3, TENM1, and SHH. , Method for producing cerebral organoids.
상기 단계 (d) 이후 100 내지 110일차의 대뇌 오가노이드는 신경 마커인 ALK, ASCL1, CDK5RAP2, DCX, DLL1, EGF, MAP2, MEF2C, KMT2A, NEUROD1, NEUROG1, NOTCH1, NRG1, PARD3, PAX3, POU4F1, PTN, ROBO1, RTN4, SHH, 및 SLIT2로 이루어진 군으로부터 선택되는 하나 이상의 유전자의 발현이 증가한 것인, 대뇌 오가노이드 제조 방법.
According to paragraph 1,
Cerebral organoids at 100 to 110 days after step (d) contain neural markers ALK, ASCL1, CDK5RAP2, DCX, DLL1, EGF, MAP2, MEF2C, KMT2A, NEUROD1, NEUROG1, NOTCH1, NRG1, PARD3, PAX3, POU4F1, A method of producing cerebral organoids, wherein the expression of one or more genes selected from the group consisting of PTN, ROBO1, RTN4, SHH, and SLIT2 is increased.
상기 대뇌 오가노이드는 신경 마커인 MAP2, NeuN, CTIP2, 및 SATB2으로 이루어진 군으로부터 선택되는 하나 이상의 단백질의 발현이 증가한 것인, 대뇌 오가노이드 제조 방법.
According to paragraph 1,
A method of producing a cerebral organoid, wherein the cerebral organoid has increased expression of one or more proteins selected from the group consisting of neural markers MAP2, NeuN, CTIP2, and SATB2.
A cerebral organoid prepared by the method of claim 1.
(b) 약물을 처리한 후의 상기 대뇌 오가노이드의 신경 활성도를 측정하는 단계; 및
(c) 단계 (a)의 신경 활성도와 단계 (b)의 신경 활성도를 비교하는 단계;
를 포함하는 신경 발달 장애를 개선 또는 치료하기 위한 약물 스크리닝 방법.
(a) measuring neural activity before treating the cerebral organoid prepared by the method of claim 1 with a drug;
(b) measuring the neural activity of the cerebral organoid after treatment with the drug; and
(c) comparing the neural activity of step (a) with the neural activity of step (b);
A drug screening method for improving or treating neurodevelopmental disorders including.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020230096922A KR102609283B1 (en) | 2023-07-25 | 2023-07-25 | A cerebral organoids with improved neural activity and a method for producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020230096922A KR102609283B1 (en) | 2023-07-25 | 2023-07-25 | A cerebral organoids with improved neural activity and a method for producing the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR102609283B1 true KR102609283B1 (en) | 2023-12-01 |
Family
ID=89124315
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020230096922A KR102609283B1 (en) | 2023-07-25 | 2023-07-25 | A cerebral organoids with improved neural activity and a method for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR102609283B1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117778313A (en) * | 2024-02-23 | 2024-03-29 | 成都云测医学生物技术有限公司 | Differentiation method and application of mesenchymal stem cells obtained from brain organoids |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20220050670A (en) * | 2020-10-16 | 2022-04-25 | 서강대학교산학협력단 | Mini-brain structure and Preparation method thereof |
| KR20220067388A (en) * | 2020-11-17 | 2022-05-24 | 서강대학교산학협력단 | Cerebral organoid-motor neuron spheroid structure and manufacturing method thereof |
| KR20220068135A (en) * | 2020-11-18 | 2022-05-25 | 서울대학교산학협력단 | Method for preparing brain organoid and method for screening neurodegenerative disease treatment agent using the brain organoid |
| KR20230076437A (en) * | 2021-11-24 | 2023-05-31 | 순천향대학교 산학협력단 | Advancement of the retinal organoid early differentiation process through regulation of the mTOR signaling pathway |
| KR20230100458A (en) * | 2021-12-28 | 2023-07-05 | 주식회사 오간팩토리 | Method of providing information for diagnosing of cerebral-related diseases and drug screening using three-dimensional cerebral cell aggregates |
-
2023
- 2023-07-25 KR KR1020230096922A patent/KR102609283B1/en active IP Right Grant
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20220050670A (en) * | 2020-10-16 | 2022-04-25 | 서강대학교산학협력단 | Mini-brain structure and Preparation method thereof |
| KR20220067388A (en) * | 2020-11-17 | 2022-05-24 | 서강대학교산학협력단 | Cerebral organoid-motor neuron spheroid structure and manufacturing method thereof |
| KR20220068135A (en) * | 2020-11-18 | 2022-05-25 | 서울대학교산학협력단 | Method for preparing brain organoid and method for screening neurodegenerative disease treatment agent using the brain organoid |
| KR20230076437A (en) * | 2021-11-24 | 2023-05-31 | 순천향대학교 산학협력단 | Advancement of the retinal organoid early differentiation process through regulation of the mTOR signaling pathway |
| KR20230100458A (en) * | 2021-12-28 | 2023-07-05 | 주식회사 오간팩토리 | Method of providing information for diagnosing of cerebral-related diseases and drug screening using three-dimensional cerebral cell aggregates |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117778313A (en) * | 2024-02-23 | 2024-03-29 | 成都云测医学生物技术有限公司 | Differentiation method and application of mesenchymal stem cells obtained from brain organoids |
| CN117778313B (en) * | 2024-02-23 | 2024-05-24 | 成都云测医学生物技术有限公司 | Differentiation method and application of mesenchymal stem cells obtained from brain organoids |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7023820B2 (en) | Cortical interneurons and other neuronal cells generated by directing the differentiation of pluripotent cells and pluripotent cells | |
| JP6835335B2 (en) | Method for producing telencephalon or its precursor tissue | |
| US10857185B2 (en) | Compositions and methods for reprogramming non-neuronal cells into neuron-like cells | |
| CN104894060B (en) | Inducing somatic transdifferentiation is the method and its application of neural stem cell | |
| KR102609283B1 (en) | A cerebral organoids with improved neural activity and a method for producing the same | |
| KR101870240B1 (en) | Differentiation method of adipose-derived mesenchymal stem cells into neural stem cells, neural cells, and GABAergic neural cells | |
| JP2019502407A (en) | Nerve organoid composition and method of use | |
| JP7169468B2 (en) | Improved retinal organoids and methods of making same | |
| US10724000B2 (en) | Small molecule based conversion of somatic cells into neural crest cells | |
| WO2022051847A9 (en) | Methods for generating neural progenitor cells with a spinal cord identity | |
| US20230287343A1 (en) | Method for generating a three-dimensional neuromuscular organoid in vitro | |
| Xie et al. | Reproducible and efficient generation of functionally active neurons from human hiPSCs for preclinical disease modeling | |
| US11781108B2 (en) | Method for producing nervous system cells | |
| EP4379045A1 (en) | Method for producing highly proliferative cell, and highly proliferative cell and use thereof | |
| CN114867849B (en) | Method for production of induced dopaminergic neuron progenitor cells using direct reprogramming | |
| TWI613446B (en) | Neurogenesis screening method and system using adipose tissue derived stem cells | |
| Kwak et al. | Guidelines for manufacturing and application of organoids: brain | |
| Ruiz-Lozano et al. | Stem cells as in vitro models of disease | |
| Glass et al. | Brain organoids: models of cell type diversity, connectivity, and disease phenotypes | |
| CN114540278B (en) | Microvascular in-vitro culture method and culture solution | |
| US20240052317A1 (en) | Methods for Differentiating Stromal Cells or Pericytes | |
| EP4397750A1 (en) | Method for producing inner ear stria vascularis marginal cells, chemical agent assessment method, and cell culture for assessing chemical agent | |
| US20240150710A1 (en) | Assembled three-dimensional cultures of human neurons and glia and their use | |
| Smits | Generation of midbrain organoids as a model to study Parkinson's disease | |
| Lein et al. | Autonomic and Enteric Neurons: Cell Culture☆ |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant |