CN112553160B - Method and culture medium for chemically inducing cortical neurons - Google Patents
Method and culture medium for chemically inducing cortical neurons Download PDFInfo
- Publication number
- CN112553160B CN112553160B CN202011562592.8A CN202011562592A CN112553160B CN 112553160 B CN112553160 B CN 112553160B CN 202011562592 A CN202011562592 A CN 202011562592A CN 112553160 B CN112553160 B CN 112553160B
- Authority
- CN
- China
- Prior art keywords
- stem cells
- culture medium
- serum
- cortical
- neural stem
- 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
- 210000003618 cortical neuron Anatomy 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 28
- 239000001963 growth medium Substances 0.000 title claims abstract description 22
- 230000001939 inductive effect Effects 0.000 title claims abstract description 22
- 241000282414 Homo sapiens Species 0.000 claims abstract description 41
- 230000006698 induction Effects 0.000 claims abstract description 38
- 230000004069 differentiation Effects 0.000 claims abstract description 19
- 210000002966 serum Anatomy 0.000 claims abstract description 17
- 150000003384 small molecules Chemical class 0.000 claims abstract description 8
- 229940121773 Secretase inhibitor Drugs 0.000 claims abstract description 5
- 210000001178 neural stem cell Anatomy 0.000 claims description 41
- 210000004027 cell Anatomy 0.000 claims description 36
- 239000003112 inhibitor Substances 0.000 claims description 17
- 210000001778 pluripotent stem cell Anatomy 0.000 claims description 17
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 claims description 10
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims description 10
- 229930003231 vitamin Natural products 0.000 claims description 9
- 235000013343 vitamin Nutrition 0.000 claims description 9
- 239000011782 vitamin Substances 0.000 claims description 9
- 229940088594 vitamin Drugs 0.000 claims description 9
- KIDHWZJUCRJVML-UHFFFAOYSA-N putrescine Chemical compound NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 claims description 8
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims description 6
- 102000004877 Insulin Human genes 0.000 claims description 5
- 108090001061 Insulin Proteins 0.000 claims description 5
- 102000008730 Nestin Human genes 0.000 claims description 5
- 108010088225 Nestin Proteins 0.000 claims description 5
- 229940125396 insulin Drugs 0.000 claims description 5
- 210000005055 nestin Anatomy 0.000 claims description 5
- 229960003387 progesterone Drugs 0.000 claims description 5
- 239000000186 progesterone Substances 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 239000005700 Putrescine Substances 0.000 claims description 4
- 108010071390 Serum Albumin Proteins 0.000 claims description 4
- 102000007562 Serum Albumin Human genes 0.000 claims description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical group [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 4
- 102000004338 Transferrin Human genes 0.000 claims description 4
- 229930003779 Vitamin B12 Natural products 0.000 claims description 4
- -1 ZO-1 Proteins 0.000 claims description 4
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 claims description 4
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 4
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims description 4
- 229930195729 fatty acid Natural products 0.000 claims description 4
- 239000000194 fatty acid Substances 0.000 claims description 4
- 150000004665 fatty acids Chemical class 0.000 claims description 4
- 230000001537 neural effect Effects 0.000 claims description 4
- 229960001471 sodium selenite Drugs 0.000 claims description 4
- 235000015921 sodium selenite Nutrition 0.000 claims description 4
- 239000011781 sodium selenite Substances 0.000 claims description 4
- 239000012581 transferrin Substances 0.000 claims description 4
- 235000019163 vitamin B12 Nutrition 0.000 claims description 4
- 239000011715 vitamin B12 Substances 0.000 claims description 4
- 101710150822 G protein-regulated inducer of neurite outgrowth 1 Proteins 0.000 claims description 3
- 101000766307 Gallus gallus Ovotransferrin Proteins 0.000 claims description 3
- 102100022645 Glutamate receptor ionotropic, NMDA 1 Human genes 0.000 claims description 3
- 102100022193 Glutamate receptor ionotropic, delta-1 Human genes 0.000 claims description 3
- 101000873546 Homo sapiens Glutamate decarboxylase 1 Proteins 0.000 claims description 3
- 101000900493 Homo sapiens Glutamate receptor ionotropic, delta-1 Proteins 0.000 claims description 3
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 claims description 3
- 108091006162 SLC17A6 Proteins 0.000 claims description 3
- 108091006283 SLC17A7 Proteins 0.000 claims description 3
- 102100024270 Transcription factor SOX-2 Human genes 0.000 claims description 3
- 102100038039 Vesicular glutamate transporter 1 Human genes 0.000 claims description 3
- 102100038036 Vesicular glutamate transporter 2 Human genes 0.000 claims description 3
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 239000002356 single layer Substances 0.000 claims description 3
- 108010032788 PAX6 Transcription Factor Proteins 0.000 claims description 2
- 102100037506 Paired box protein Pax-6 Human genes 0.000 claims description 2
- 108010031318 Vitronectin Proteins 0.000 claims description 2
- 102100035140 Vitronectin Human genes 0.000 claims description 2
- 210000002469 basement membrane Anatomy 0.000 claims description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 2
- 150000003722 vitamin derivatives Chemical class 0.000 claims 3
- 238000004115 adherent culture Methods 0.000 claims 1
- 210000002569 neuron Anatomy 0.000 abstract description 13
- 239000003814 drug Substances 0.000 abstract description 8
- 208000015122 neurodegenerative disease Diseases 0.000 abstract description 8
- 239000000126 substance Substances 0.000 abstract description 8
- 241001465754 Metazoa Species 0.000 abstract description 7
- 230000004770 neurodegeneration Effects 0.000 abstract description 7
- 238000000338 in vitro Methods 0.000 abstract description 6
- 238000004113 cell culture Methods 0.000 abstract description 5
- 238000012216 screening Methods 0.000 abstract description 5
- 238000011282 treatment Methods 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 208000012902 Nervous system disease Diseases 0.000 abstract description 3
- 230000005847 immunogenicity Effects 0.000 abstract description 2
- 230000000192 social effect Effects 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 16
- 239000002609 medium Substances 0.000 description 15
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 14
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 14
- 239000003550 marker Substances 0.000 description 9
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 8
- DWJXYEABWRJFSP-XOBRGWDASA-N DAPT Chemical compound N([C@@H](C)C(=O)N[C@H](C(=O)OC(C)(C)C)C=1C=CC=CC=1)C(=O)CC1=CC(F)=CC(F)=C1 DWJXYEABWRJFSP-XOBRGWDASA-N 0.000 description 8
- 238000011977 dual antiplatelet therapy Methods 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 239000000872 buffer Substances 0.000 description 7
- 239000007853 buffer solution Substances 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 6
- 101000979001 Homo sapiens Methionine aminopeptidase 2 Proteins 0.000 description 6
- 101000969087 Homo sapiens Microtubule-associated protein 2 Proteins 0.000 description 6
- 102100023174 Methionine aminopeptidase 2 Human genes 0.000 description 6
- 239000007640 basal medium Substances 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 230000008672 reprogramming Effects 0.000 description 6
- 210000000130 stem cell Anatomy 0.000 description 6
- 108010085895 Laminin Proteins 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 210000005036 nerve Anatomy 0.000 description 5
- 210000003061 neural cell Anatomy 0.000 description 5
- 230000006907 apoptotic process Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000007877 drug screening Methods 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108010039918 Polylysine Proteins 0.000 description 3
- 238000001190 Q-PCR Methods 0.000 description 3
- 238000002123 RNA extraction Methods 0.000 description 3
- 229930003268 Vitamin C Natural products 0.000 description 3
- 229930003427 Vitamin E Natural products 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 230000024245 cell differentiation Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 239000002131 composite material Substances 0.000 description 3
- 230000001054 cortical effect Effects 0.000 description 3
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 3
- 238000003125 immunofluorescent labeling Methods 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 239000010410 layer Substances 0.000 description 3
- 210000000653 nervous system Anatomy 0.000 description 3
- 238000007747 plating Methods 0.000 description 3
- 229920000656 polylysine Polymers 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000004017 serum-free culture medium Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000002103 transcriptional effect Effects 0.000 description 3
- 235000019154 vitamin C Nutrition 0.000 description 3
- 239000011718 vitamin C Substances 0.000 description 3
- 235000019165 vitamin E Nutrition 0.000 description 3
- 239000011709 vitamin E Substances 0.000 description 3
- 229940046009 vitamin E Drugs 0.000 description 3
- 208000024827 Alzheimer disease Diseases 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 2
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 208000018737 Parkinson disease Diseases 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 239000000853 adhesive Substances 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000002771 cell marker Substances 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 210000003981 ectoderm Anatomy 0.000 description 2
- 230000000408 embryogenic effect Effects 0.000 description 2
- 210000001900 endoderm Anatomy 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000003716 mesoderm Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000004766 neurogenesis Effects 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 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
- 102100034111 Activin receptor type-1 Human genes 0.000 description 1
- 239000012583 B-27 Supplement Substances 0.000 description 1
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 description 1
- 102100025423 Bone morphogenetic protein receptor type-1A Human genes 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102100035902 Glutamate decarboxylase 1 Human genes 0.000 description 1
- 102100030652 Glutamate receptor 1 Human genes 0.000 description 1
- 101000799140 Homo sapiens Activin receptor type-1 Proteins 0.000 description 1
- 101000762379 Homo sapiens Bone morphogenetic protein 4 Proteins 0.000 description 1
- 101000934638 Homo sapiens Bone morphogenetic protein receptor type-1A Proteins 0.000 description 1
- 101001010445 Homo sapiens Glutamate receptor 1 Proteins 0.000 description 1
- 101001139134 Homo sapiens Krueppel-like factor 4 Proteins 0.000 description 1
- 101000766306 Homo sapiens Serotransferrin Proteins 0.000 description 1
- 102100020677 Krueppel-like factor 4 Human genes 0.000 description 1
- 239000002211 L-ascorbic acid Substances 0.000 description 1
- 235000000069 L-ascorbic acid Nutrition 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 239000012580 N-2 Supplement Substances 0.000 description 1
- 101710126211 POU domain, class 5, transcription factor 1 Proteins 0.000 description 1
- 102100035423 POU domain, class 5, transcription factor 1 Human genes 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000023715 cellular developmental process Effects 0.000 description 1
- 208000015114 central nervous system disease Diseases 0.000 description 1
- 210000003710 cerebral cortex Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000008143 early embryonic development Effects 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 230000008451 emotion Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 206010015037 epilepsy Diseases 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 210000001654 germ layer Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 210000004884 grey matter Anatomy 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 210000004126 nerve fiber Anatomy 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000000474 nursing effect Effects 0.000 description 1
- 210000004248 oligodendroglia Anatomy 0.000 description 1
- 230000005305 organ development Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000008447 perception Effects 0.000 description 1
- 108010055896 polyornithine Proteins 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 description 1
- GPTFURBXHJWNHR-UHFFFAOYSA-N protopine Chemical compound C1=C2C(=O)CC3=CC=C4OCOC4=C3CN(C)CCC2=CC2=C1OCO2 GPTFURBXHJWNHR-UHFFFAOYSA-N 0.000 description 1
- 239000013014 purified material Substances 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 210000000697 sensory organ Anatomy 0.000 description 1
- 210000001626 skin fibroblast Anatomy 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 208000020431 spinal cord injury Diseases 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000011269 treatment regimen Methods 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
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0619—Neurons
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/36—Lipids
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/46—Amines, e.g. putrescine
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- 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
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- 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
- C12N2501/39—Steroid hormones
- C12N2501/392—Sexual steroids
-
- 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
-
- 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
- C12N2533/52—Fibronectin; Laminin
-
- 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
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of neuron induction, and particularly discloses a method and a culture medium for chemically inducing cortical neurons, wherein the culture medium is a chemical induction system which does not use animal serum and does not contain animal source components, and comprises a serum-free basic culture medium and a secretase inhibitor. The induction method has definite chemical components and high differentiation efficiency. Furthermore, the obtained human cortical neurons have the characteristics of low immunogenicity, short differentiation time and stable electrophysiological activity. Compared with the current international induction method of serum plus small molecule combination, the invention completely eliminates the potential danger caused by animal-derived components in the cell culture process, and is especially suitable for in vitro screening of nervous system disease drugs and treatment of neurodegenerative diseases, thus having great economic and social effects.
Description
Technical Field
The invention belongs to the technical field of neuron induction, and particularly relates to a method and a culture medium for chemically inducing cortical neurons.
Background
Ectoderm is the outermost layer formed during embryo development, and as organogenesis begins, ectodermal cells gradually differentiate into important systems such as brain, spinal cord, sensory organs, etc. The nervous system is an important system responsible for thinking, emotion, perception, movement and other functions. Ectodermal cells are related to the occurrence of various degenerative diseases, such as neurodegenerative diseases caused by aging and death of neurons, are common aging diseases at present, the treatment and nursing cost of the diseases is extremely high, and no specific medicine can be effectively treated in the market. Neurodegenerative diseases include Amyotrophic Lateral Sclerosis (ALS), parkinson's Disease (PD), alzheimer's Disease (AD), and the like. The difficulty in the study of neurodegenerative diseases and spinal cord injuries is that the neural cells of the central nervous system are nonrenewable, and the scarcity of in vitro disease models is the limiting factor in basic research, and such diseases are caused by irreversible damage to the central nervous system. Neural cells include neural stem cells, mature neurons, astrocytes, and oligodendrocytes, among other different neural cell types. Investigation of different cell types may reveal the pathogenesis and progression of the disease. Cell differentiation to study developmental processes is the basis for pathological studies. Knowledge of the genes that regulate stem cell differentiation will help to understand the cause of disease formation and develop corresponding disease treatment strategies by understanding the normal cellular differentiation and development processes. Taking cortical neural cells as an example, wherein the cerebral cortex covers the gray matter of the two hemispheres of the brain, which is the material basis for the activity of the higher nerve, is composed of neurons, nerve fibers and glia. Wherein the lesions and injuries of cortical neurons are related to cerebral stroke, epilepsy, and are important targeting targets for a variety of central nervous system diseases.
At present, traditional cell lines are mostly used for drug development in the related field, a rodent nerve cell line is used for drug screening, normal response of human beings to the drugs cannot be reflected, most of cortical cell lines in the current drug screening field are derived from primary rodent cells, and obvious species differences exist between mice and human beings, which is also one of reasons for deviation of drug screening; in addition, the source of the human cortical neuron cells is insufficient, the uniformity difference of the human neural stem cells of remains donation or embryo source is obvious, the limitation of ethical use is provided, and the industrial order requirement of high-flux screening can not be met; the factors greatly restrict the accuracy and efficiency of development of nervous system medicaments.
In 2006, the mountain stretch team invented a "cocktail" method consisting of four transcription factors OCT4, SOX2, KLF4 and c-Myc, which was able to successfully reprogram terminally differentiated skin fibroblasts into stem cells with a differentiation multipotency, which were termed induced multipotency stem cells (induced pluripotent cells) (Cell, 2006, 124 (4) pp.663-676; cell,2007, 131 (5) pp.861-872). These stem cells have differentiation potential similar to embryonic stem cells (embryonic stem cells) and are capable of forming the three most basic germ layers for human development: ectoderm, mesoderm and endoderm, and eventually form a variety of adult cells. The invention breaks through the ethical limitation of using human embryonic stem cells in medicine. Taking the nervous system as an example, in the field of regenerative medicine, currently, the induction of neural stem cells and neurons adopts a SMAD pathway dual inhibition method (Dual SMAD inhibition) (Chambers SM, et al, nat Biotechnol,2009, 27 (3): 275-80), and the neural stem cells obtained by the method can differentiate into other types of neuronal cells, and the principle is that the BMP and TGFBeta pathway is inhibited to simulate the signaling pathway in early embryonic development, thereby inducing the generation of the neural stem cells. Of these, LDN-193189 and SB431542 are two widely used chemical small molecule inhibitors that act on ALK2 and ALK3 in the BMP4 pathway, and ALK5 in the TGFBeta pathway, respectively, to achieve the effects of engrafting endoderm and mesoderm formation, thereby inducing ectodermal development and neurogenesis. By this method, induced neuronal cells can be obtained in the presence of a serotypes component (Knockout Serum Replacement) (Chambers SM, et al, nat Biotechnol.,2013, 30 (7): 715-720). The research of the induced nerve cells provides a new idea for the treatment of neurodegenerative diseases and drug screening.
Based on the technical problems existing in the prior art, a chemical induction cortical neuron culture medium and an induction method are necessary to be studied.
Disclosure of Invention
In view of the above, the present invention aims to provide a method and a culture medium for chemically inducing cortical neurons, which solve the potential danger caused by the existence of animal-derived components in the cell culture process in the prior art, thereby being suitable for in vitro screening of drugs for nervous system diseases and treatment of neurodegenerative diseases.
In order to solve the problems, the scheme of the invention is as follows:
the invention provides an application of an inhibitor Crinigacetat in inducing differentiation of neural stem cells into cortical neuron cells; the inhibitor Crenigacetat is used in an amount of 1 to 20. Mu.M, preferably 7.5. Mu.M.
The invention also provides a cortical neuron induction culture medium, which comprises a serum-free basal medium and an inhibitor Crengancestat, wherein the dosage of the inhibitor Crengancestat is 1-20 mu M.
Further, the inhibitor Crenigacetat is used in an amount of 7.5uM.
Further, the serum-free basal medium comprises vitamins, inorganic salts, small molecule inhibitors, serum substitutes, and other ingredients; the vitamins comprise 1ug/ml vitamin E,1.2uM vitamin B12 and 64mg/L vitamin C; the inorganic salt comprises 1.2g/L sodium bicarbonate, 0.5g/L sodium chloride and 13.6 mu g/L sodium selenite; the small molecule inhibitors include 12.5uM LY2157299,5uM JW55; the other components comprise 12.5ug/ml D (+) -galactose, 6.3ng/ml progesterone, 23ug/ml putrescine, 20mg/L insulin; the serum substitute is one or more of serum albumin, transferrin and fatty acid, and the dosage is 100ng/ml.
The invention also provides a method for inducing the differentiation of the pluripotent stem cells into the cortical neuron cells by using the cortical neuron induction medium, which comprises the following steps:
1. pluripotent stem cells induce differentiation of neural stem cells: performing monolayer adherence culture on the pluripotent stem cells in a serum-free basic culture medium, and inducing the culture medium to carry out adherence culture to obtain the neural stem cells which form the annular uniform arrangement of the neural flowers;
2. neural stem cells induce differentiation of cortical neurons: and (3) inducing and differentiating the neural stem cells obtained in the step (S1) through a cortical neuron induction culture medium to obtain cortical neurons.
Further, in the above method, the adhesive culture has a basement membrane preparation, which is one or more of a base adhesive, laminin and vitronectin.
Further, in the above method, the step 1 further comprises identifying neural stem cell markers Nestin, ZO-1, SOX2, PAX6 and Nestin to verify the induction result of the neural stem cells.
Further, in the above method, step 2 further comprises identifying cortical neuron expression genes GAD1, GRID1, GRIN1, VGLUT1 and VGLUT2 to verify the induced differentiation result of cortical neurons.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a chemical induction system of human cortical neurons, which does not use animal serum or contain animal-derived components, and the chemical components of the induction system are clear, so that the differentiation efficiency is high.
2. The human cortical neuron obtained by induction has the characteristics of low immunogenicity, short differentiation time and stable electrophysiological activity; compared with the current international induction method of serum plus small molecule combination, the invention not only greatly saves the production cost, but also shows the advantages of great purity and yield, and completely eliminates the potential danger brought by animal-derived components in the cell culture process, thereby being particularly suitable for in vitro screening of drugs for nervous system diseases and treatment of neurodegenerative diseases, and having great economic and social effects.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic representation of the molecular characterization of cells obtained by using NouvNeu002 for neural stem cell chemical induction according to the present invention.
FIG. 2 is a schematic diagram showing the results of identifying the expressed genes when the human neural stem cells induce the human cortical neuronal cells 7d according to the present invention.
FIG. 3 shows the differential expression results of Crinigacistat of the present invention on markers MAP2 and VGlut1 of cortical neurons at different concentrations.
FIG. 4 is a schematic representation of the change in cell morphology within 24 hours of induction in experimental and control groups of varying concentrations of Crenigacistat according to the present invention.
FIG. 5 is a graph showing the difference between the experimental group and the control group with different concentrations of Crnigacistat on apoptosis at the time of induction for 7 days.
Detailed Description
The following examples are illustrative of the invention but do not limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The invention provides a serum-free basal medium comprising vitamins, inorganic salts, small molecule inhibitors, serum substitutes and other components, wherein:
the vitamins comprise 1ug/ml vitamin E,1.2uM vitamin B12 and 64mg/L vitamin C; other vitamins may also be selected as desired.
The inorganic salt comprises 1.2g/L sodium bicarbonate, 0.5g/L sodium chloride and 13.6 mu g/L sodium selenite;
the small molecule inhibitors include 12.5uM LY2157299,5uM JW55;
the serum substitute is one or more of serum albumin, transferrin and fatty acid, and the dosage is 100ng/ml;
the other ingredients included 12.5ug/ml D (+) -galactose, 6.3ng/ml progesterone, 23ug/ml putrescine, 20mg/L insulin.
The invention also provides a culture medium for inducing and differentiating cortical neurons, which comprises the serum-free basic culture medium and a secretase inhibitor Crinigacitat, wherein the dosage of the secretase inhibitor Crinigacitat can be 1-20 mu M, and specifically can be 1 mu M,5 mu M,7.5 mu M,10 mu M,15 mu M and 20 mu M; preferably 7.5uM.
According to the invention, the multipotent stem cells are subjected to monolayer adherence culture in a serum-free culture medium, the serum-free culture medium does not contain serum, BMP or substances of TGF signal transduction paths and the like, and the nerve stem cells which form the nerve flower ring shape and are uniformly arranged are obtained by using the serum-free nerve induction culture medium for adherence culture for 20 days. Thereby further inducing differentiation using serum-free medium containing the specific secretase inhibitor crenigacistat to obtain cortical neurons.
Serum-free medium in the present invention means that it does not contain serum that is directly separated from blood. Serum is the clear liquid portion of plasma that contains no fibrinogen or blood cells and remains liquid after blood coagulation. Serum-free media may contain serum substitutes, examples of which include serum albumin, transferrin, fatty acids and the like purified materials, which are well known in the art as substitutes for serum.
Example 1 preparation of human neural cell-inducing basal medium NouvNeu-002 and cortical neuron-inducing medium NouvNeu-C
The basal medium for induction of human nerve cells was prepared according to the following formula, and is hereinafter referred to as NouvNeu-002:
du's modified eagle Medium (DMEM Medium), vitamin E (1 ug/ml), vitamin B12 (1.2 uM), vitamin C (L-ascorbic acid,64 mg/L), progesterone (Progesterone, 6.3 ng/ml), putrescine (Putressine, 23 ug/ml), sodium bicarbonate (NaHCO) 3 1.2 g/L), sodium Chloride (Sodium Chloride,0.5 g/L), sodium selenite (13.6. Mu.g/L), D (+) -galactose (D (+) -galactose,12.5 ug/ml), LY2157299 (12.5 uM), JW55 (5 uM), insulin (Insulin, 20 mg/L), recombinant human transferrin (100 ng/ml).
1-20. Mu.M Crenagantat (optimal concentration of 7.5. Mu.M) was added to NouvNeu-002 cell culture medium. Cortical neuron induction medium NouvNeu-C was formed.
Control experiments, reference was made to Jue Wang et al (Brain Research,2016,1634.) for cortical neuron induction using DAPT, control medium was N2B27 medium, hereinafter CK, which was formulated as follows: 50% Du's modified eagle F12 medium (DMEM-F12), 50% neural basal medium (Neurobasal), 0.1% N2 additive (N2 supplement), 2% B27 additive (B27 supplement), and 10uM DAPT.
EXAMPLE 2 Induction culture of human neural Stem cells
Human pluripotent stem cells include embryogenic pluripotent stem cells, such as H9 cell lines and human induced pluripotent stem cells. Wherein the human induced pluripotent stem cells are obtained by reprogramming from CD34+ cells according to the "reprogramming medium and culture method of reprogramming Cheng Youdao pluripotent stem cells" (ZL 201910050800.7).
Human pluripotent stem cells include embryogenic pluripotent stem cells, such as H9 cell lines and human induced pluripotent stem cells. Wherein the human induced pluripotent stem cells are obtained by reprogramming from CD34+ cells according to the "reprogramming medium and culture method of reprogramming Cheng Youdao pluripotent stem cells" (patent publication No. CN 109628383A).
The human pluripotent stem cells are coated with a T25 cell culture flask by using Matrigel (STEMCELL Technologies), are plated and then are placed in a 37 ℃ incubator for more than one hour. According to 1X 10 6 Cells were inoculated in T25 flasks for expansion and passaging.
When nerve induction is carried out, a 6-hole culture plate is coated by using 50ug/ml polylysine (SIGMA, P6407), and the culture plate is placed in a constant temperature oven at 37 ℃ for incubation for more than 3 hours after being plated; further coating with 5ug/ml Laminin (Laminin, I2020, SIGMAALDRICH), plating, and incubating in an incubator at 37 ℃ for more than 3 hours. When the pluripotent stem cells reached 70% coverage, digestion was stopped with EDTA at 37 ℃ for 5 minutes, using DMEM. After cell washing and centrifugation, the cells were centrifuged according to 2X 10 5 The ratio of each flask was re-inoculated into T25 plates. Induction was performed using NouvNeu002 at 37℃under 5% CO 2 (Panasonic, MCO-18 AC), medium was changed daily until neural stem cell rosettes formed.
Neural stem cells were digested with EDTA at 37 ℃ for 5 minutes, and cell digestion was stopped with DMEM. After cell washing and centrifugation, the cells were centrifuged according to 2X 10 5 The ratio of each flask was re-inoculated into 50ug/ml polylysine and 5ug/ml laminin coated T25 plates for further culture or differentiation. Culture conditions were 37℃and 5% CO 2 (Panasonic，MCO-18AC)。
Immunofluorescent staining and identification are carried out on the neural stem cells which are passaged after the NouvNeu002 system induction is completed: fixing cells for 40 minutes at room temperature by adopting 4% paraformaldehyde, and washing twice by using a DPBS buffer solution; then permeabilizing with 0.1% Triton X-100 for 5min, and washing twice with DPBS buffer; cells were then incubated overnight at 4℃with DPBS buffer containing 10% horse serum and 0.1% Triton X-100; then, the antibody diluted with DPBS buffer was added, incubated at 37℃for 2 hours, and after three washes with DPBS buffer, the photographs were taken using Leica DMi 8. Details of antibody use are shown in table 1. The results are shown in FIGS. 1a-1d, wherein FIG. 1a shows the structure of the neuro-garland reconstructed after in vitro passaging using neural stem cells obtained from NouvNeu 002; FIG. 1b shows neural stem cells obtained from NouvNeu002, the structure of the neurogarland of which expresses the neural stem cell marker Nestin; FIG. 1c shows neural stem cells obtained from NouvNeu002, the structure of the neuro-wreath of which expresses the neuro-wreath marker ZO-1; FIG. 1d shows the positioning assembly of FIGS. 1a-1 c; the above results show that nouvneuu 002 is capable of inducing the formation of neural stem cells in vitro.
Table 1: antibodies for immunofluorescent staining of cells
The transcriptional changes from different marker genes during neurogenesis were detected using Q-PCR, comparing pluripotent stem cells with neural stem cells induced using nouvnue 002. Total RNA extraction was performed using RNeasy Mini or Micro Kit (QIAGEN), respectively, and 1mg RNA was used to synthesize cDNA using SuperScript III First-Strand Synthesis System (Invitrogen). The labeling and reaction of the Quan-titative PCR was performed with SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa), and beta-action was used as an internal control. All data were analyzed using delta-Ct method. Three replicates were used for each set of experiments and variance statistics were performed. Primer sequences for identifying the coding genes for the different cell markers are shown in table 2. The results are shown in FIGS. 1e-1g, which demonstrate that NouvNeu 002-induced neural stem cells express neural stem cell markers.
TABLE 2 different marker Gene primers during the Induction of pluripotent Stem cells into neural Stem cells
| Gene | Primer(s) |
| SOX2-F | CATGCAGGTTGACACCGTTGG |
| SOX2-R | ATGGATTCTCGGCAGACTGATTCA |
| PAX6-F | TCTTTGCTTGGGAAATCCG |
| PAX6-R | CTGCCCGTTCAACATCCTTAG |
| Nestin-F | TCAAGATGTCCCTCAGCCTGGA |
| Nestin-R | TGGCACAGGTGTCTCAAGGGTAG |
| ACTIN-F | TCCCTGGAGAAGAGCTACGA |
| ACTIN-R | AGCACTGTGTTGGCGTACAG |
EXAMPLE 3 Induction of human cortical neuronal cells
The induced culture of human cortical neuron cells uses polylysine (Poly-L-ornithine solution, sigma) to coat the first layer of cell culture flask, plating and then incubating in a 37 ℃ incubator for more than 16 hours, and laminin (Biolaminin 521 LN) to coat the second layer, plating and then incubating in a 37 ℃ incubator for more than 2 hours. According to 4 x 10 4 Individual/cm 2 The human neural stem cells were inoculated into a culture flask or a well plate for passaging. NouvNeu-C cultures containing 1/5/10/20. Mu.M Crinigacetat were used based on 37℃and 5% CO, respectively 2 Induction culture was performed in a cell incubator for concentration screening of crenigacistat. The control group was cultured using the same batch of cells in 10. Mu.M DAPT medium.
Example 4 identification of human cortical neuronal cells
4.1 identification of Crencapurtat-induced cortical neuron expression genes
The transcriptional changes of different marker genes during differentiation from neural stem cells to neurons were detected using Q-PCR. Total RNA extraction was performed using the NouvNeu and N2B27 systems, using RNeasy Mini or Micro Kit (QIAGEN) respectively, and 1mg RNA was used to synthesize cDNA using SuperScript III First-Strand Synthesis System (Invitrogen). The labeling and reaction of the Quan-titative PCR was performed with SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa), and beta-action was used as an internal control. All data were analyzed using delta-Ct method. Three replicates were used for each set of experiments and variance statistics were performed. The primer sequences for identifying the coding genes for the different cell markers are shown in table 3.
The analysis results are shown in FIG. 2, in which the action expressed gene is used as an internal reference, and DAPT-induced human cortical neurons are used as controls. The results show that the gene transcription level of Crenigacetat-induced human cortical neurons GAD1, GRID1 and GRIN1 is not significantly different from that of DAPT-induced human cortical neurons, while the gene transcription level of 1-10 mu M Crenigacetat-induced human cortical neurons GRIA1, VGLUT1 and VGLUT2 is higher than that of control group DAPT-induced human cortical neurons, which indicates that the maturity of the human cortical neurons obtained by induction of the invention is higher than that of the control group.
Table 3: real-time fluorescent quantitative PCR primer sequence
4.2 identification of Crenigacistat-induced human cortical neuron expressed proteins
Immunofluorescence experiments were used to identify markers of crenigacistat-induced human cortical neuronal cells. Human cortical neuronal cells were induced in 24 well plates containing glass plates according to the method of example 4, after which they were washed twice with DPBS buffer; fixing cells for 10 minutes at room temperature by adopting 4% paraformaldehyde, and washing twice by using a DPBS buffer solution; then permeabilizing the mixture for 2 minutes at the temperature of 4 ℃ by using precooled absolute methanol, and washing the mixture twice by using DPBS buffer solution; the cells were then incubated overnight at 4℃with DPBS buffer containing 10% bovine serum albumin; then adding an antibody diluted by a DPBS buffer solution containing 2% bovine serum albumin, incubating for 1h at room temperature, and washing twice by the DPBS buffer solution; then adding a secondary antibody diluted by a DPBS buffer solution containing 2% bovine serum albumin, incubating for 45min at room temperature in a dark place, and washing twice by the DPBS buffer solution; finally, DAPI containing 5 mug/mL was added and incubated for 2min at room temperature, and after three washes with DPBS buffer, the photographs were taken. Details of antibody use are shown in table 4. Differential expression of markers MAP2 and VGlut1 from cortical neurons at different concentrations of Crinigacistat was identified, and the results showed that expression of markers MAP2 and VGlut1 from cortical neurons correlated positively with the concentration of Crinigacistat. As shown in fig. 3, fig. 3a-3c show marker expression of cortical neurons at 1uM crenigacttat concentration; FIG. 3a shows VGlut1 expression at 1uM Crinigacistat concentration; FIG. 3b shows MAP2 expression at a concentration of 1uM Cronigamatat; FIG. 3c shows a composite picture of FIGS. 3a and 3 b; FIGS. 3d-3f show marker expression of cortical neurons at 5uM Cronigacitat concentration; FIG. 3d shows VGlut1 expression at 5uM Crinigacistat concentration; FIG. 3e shows MAP2 expression at 5uM Cronigacistat concentration; FIG. 3f shows a composite picture of FIGS. 3d and 3 f; FIGS. 3g-3i show marker expression of cortical neurons at 10uM Cronigacitat concentration; FIG. 3g shows VGlut1 expression at a concentration of 10uM Crinigacistat; FIG. 3h shows MAP2 expression at a concentration of 10uM Cronigacistat; fig. 3i shows the composite picture of fig. 3g and 3 h. The results show that cortical neuron marker expression appears to correlate with crenigacistat concentration.
Table 4: antibodies for immunofluorescent staining of cortical neuronal cell marker proteins
4.3 identification of Crencapurtat-induced cortical neuronal morphology
Morphological differences in cortical neurons during crenigacistat and DAPT induced differentiation were observed using a high content imaging system (ImageXpress Micro Confocal High-Content Imageing System, molecular devices). A corresponding number of human neural stem cells were seeded in coated 96-well plates according to the method of example 4, induced culture was performed in a high content imaging system using NouvNeu-C medium containing 1/10/20. Mu.M Crengagestat, four parallel wells per concentration, and the control group used the same batch of cells containing 10. Mu.M DAPT medium to culture the four parallel wells. Four fields of view were established per well, imaged every 1h for a total of 48h. The results are shown in FIG. 4, and demonstrate that the induction of human cortical neurons by the present invention is faster than that by the control group.
Example 5 Crenigacetat-induced long-term apoptosis detection of human cortical neuronal cells
The transcriptional changes of different marker genes during differentiation from neural stem cells to neurons were detected using Q-PCR. The human-derived neural stem cells (abbreviated as NSC) obtained in example 2 were taken, and cells after induction for 7 days using NouvNeu-C medium containing 0/1/7.5/15/20. Mu.M Crenigacetat were subjected to total RNA extraction using RNeasy Mini or Micro Kit (QIAGEN), respectively, and 1mg of RNA was synthesized into cDNA using SuperScript III First-Strand Synthesis System (Invitrogen). The labeling and reaction of the Quan-titative PCR was performed with SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa), and beta-action was used as an internal control. All data were analyzed using delta-Ctmethod. Three replicates were used for each set of experiments and variance statistics were performed. Primer sequences for identifying the coding genes for the different cell markers are shown in table 5.
The analysis results are shown in fig. 5, and the results show that the maturity of the human cortical neurons obtained by induction of the invention is higher than that of the control group, wherein the results show that the markers of apoptosis are significantly increased when the concentration is higher than 7.5uM.
Table 5: apoptosis detection primers for use in long-term culture
The present invention is not limited to the details and embodiments described herein, and further advantages and modifications may readily be achieved by those skilled in the art, so that the present invention is not limited to the specific details, representative solutions and illustrative examples shown and described herein, without departing from the spirit and scope of the general concepts defined by the claims and the equivalents.
Claims (8)
1. Use of the secretase inhibitor crenigacitat for inducing differentiation of neural stem cells into cortical neuronal cells, characterized in that the inhibitor crenigacitat is used in an amount of 1-20 μm, wherein the neural stem cells are derived from an H9 cell line or a human induced pluripotent stem cell.
2. Use according to claim 1, characterized in that the inhibitor crenigacistat is used in an amount of 7.5 μm.
3. A cortical neuron induction culture medium, which is characterized by comprising a serum-free basic culture medium and an inhibitor Cregantat, wherein the dosage of the inhibitor Cregantat is 1-20 mu M, and the cortical neuron induction culture medium is characterized by comprising vitamins, inorganic salts, small molecular inhibitors, serum substitutes and other components;
the vitamin is vitamin E1 ug/ml, vitamin B12 1.2uM, vitamin C64 mg/L;
the inorganic salt is sodium bicarbonate of 1.2g/L, sodium chloride of 0.5g/L and sodium selenite of 13.6 mu g/L;
the small molecule inhibitor is 12.5uM LY2157299,5uM JW55;
the other components are 12.5ug/ml D (+) -galactose, 6.3ng/ml progesterone, 23ug/ml putrescine, 20mg/L insulin;
the serum substitute is one or more of serum albumin, transferrin and fatty acid, and the dosage is 100ng/ml.
4. A culture medium according to claim 3, wherein the inhibitor crenigacistat is used in an amount of 7.5 μm.
5. A method of inducing differentiation of pluripotent stem cells into cortical neuronal cells in accordance with claim 3, comprising the steps of:
s1, inducing and differentiating the neural stem cells by using the pluripotent stem cells: performing monolayer adherence culture on the pluripotent stem cells in a serum-free basic culture medium, and inducing the culture medium to carry out adherence culture to obtain the neural stem cells which form the annular uniform arrangement of the neural flowers;
s2, inducing and differentiating cortical neurons by using neural stem cells: inducing and differentiating the neural stem cells obtained in the step S1 through a cortical neuron induction culture medium to obtain cortical neurons,
wherein the pluripotent stem cells are H9 cell lines or human induced pluripotent stem cells.
6. The method of claim 5, wherein the adherent culture has a basement membrane preparation that is one or more of a primer, a laminin, and vitronectin.
7. The method of claim 5, wherein step S1 further comprises identifying neural stem cell markers Nestin, ZO-1, SOX2, PAX6, and Nestin to verify the induction of neural stem cells.
8. The method of claim 5, wherein step S2 further comprises identifying cortical neuron expression genes GAD1, GRID1, GRIN1, VGLUT1 and VGLUT2 to verify the induced differentiation result of cortical neurons.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011562592.8A CN112553160B (en) | 2020-12-25 | 2020-12-25 | Method and culture medium for chemically inducing cortical neurons |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011562592.8A CN112553160B (en) | 2020-12-25 | 2020-12-25 | Method and culture medium for chemically inducing cortical neurons |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112553160A CN112553160A (en) | 2021-03-26 |
| CN112553160B true CN112553160B (en) | 2023-10-27 |
Family
ID=75032733
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011562592.8A Active CN112553160B (en) | 2020-12-25 | 2020-12-25 | Method and culture medium for chemically inducing cortical neurons |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112553160B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105658787A (en) * | 2013-10-14 | 2016-06-08 | 加的夫大学学院咨询有限公司 | Neuronal stem cell differentiation |
| CN110317775A (en) * | 2018-03-30 | 2019-10-11 | 中国科学院上海生命科学研究院 | The culture medium prepared for hepatocyte cultures and liver organoid |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9730975B2 (en) * | 2014-11-25 | 2017-08-15 | The Penn State Research Foundation | Chemical reprogramming of human glial cells into neurons for brain and spinal cord repair |
| JP7196376B2 (en) * | 2016-01-27 | 2022-12-27 | メモリアル スローン ケタリング キャンサー センター | Differentiation of cortical neurons from human induced pluripotent stem cells |
-
2020
- 2020-12-25 CN CN202011562592.8A patent/CN112553160B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105658787A (en) * | 2013-10-14 | 2016-06-08 | 加的夫大学学院咨询有限公司 | Neuronal stem cell differentiation |
| CN110317775A (en) * | 2018-03-30 | 2019-10-11 | 中国科学院上海生命科学研究院 | The culture medium prepared for hepatocyte cultures and liver organoid |
Non-Patent Citations (1)
| Title |
|---|
| γ-分泌酶在神经干细胞分化过程中的调节作用;王江涛;《中国组织工程研究与临床修复》;20091231;第13卷(第14期);第2727-2732页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112553160A (en) | 2021-03-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Doi et al. | Isolation of human induced pluripotent stem cell-derived dopaminergic progenitors by cell sorting for successful transplantation | |
| KR102487142B1 (en) | How to differentiate pluripotent cells | |
| CN111793608B (en) | HS5 conditioned medium for directionally inducing differentiation of hipscs into neural cell system | |
| Su et al. | Direct conversion of fibroblasts into neural progenitor-like cells by forced growth into 3D spheres on low attachment surfaces | |
| KR101870240B1 (en) | Differentiation method of adipose-derived mesenchymal stem cells into neural stem cells, neural cells, and GABAergic neural cells | |
| JP2017511153A (en) | Production and use of midbrain dopaminergic neurons | |
| JP6824267B2 (en) | Methods for inducing differentiation of human-induced pluripotent stem cells into Leydig cells and their uses | |
| JP6474806B2 (en) | Conversion of somatic cells into neural crest cells by small molecules | |
| Jurga et al. | Neurogenic potential of human umbilical cord blood: Neural‐like stem cells depend on previous long‐term culture conditions | |
| WO2021232830A1 (en) | Application of tgf-β inhibitor in inducing neural stem cells and organoid formation | |
| Bojnordi et al. | Differentiation of spermatogonia stem cells into functional mature neurons characterized with differential gene expression | |
| EP1705245A1 (en) | Neural stem cells | |
| Sauerzweig et al. | A population of serumdeprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells | |
| CN115968401A (en) | Method for differentiating stem cells into dopaminergic progenitor cells | |
| US10472607B2 (en) | Culture medium and method for inducing differentiation of pluripotent stem cells into neuroepithelial cells | |
| Li et al. | Engraftable neural crest stem cells derived from cynomolgus monkey embryonic stem cells | |
| EP2581443B1 (en) | Method for differentiating human neural progenitor cells into dopaminergic neurons, and medium for differentiation thereof | |
| CN101294146B (en) | System for inducing nerve stem cell differentiation and inducing method thereof | |
| KR101896803B1 (en) | Method for increasing the rate of inducing differentiation of human pluripotent stem cells to mesenchymal stem cells, and mesenchymal stem cells produced by thereof | |
| CN112553160B (en) | Method and culture medium for chemically inducing cortical neurons | |
| Salehi et al. | Differentiation of human ES cell-derived neural progenitors to neuronal cells with regional specific identity by co-culturing of notochord and somite | |
| CN107164325B (en) | The preparation method and kit of the oligodendroglia in the source MSCs | |
| WO2022134031A1 (en) | Chemical induction method for photoreceptor neuron cells | |
| KR101613677B1 (en) | Composition for differentiating stem cells to neural precursor cells, and method using the same | |
| Lyu et al. | MiR-210-5p promotes the differentiation of human induced pluripotent stem cells into dopaminergic neural precursors by targeting SMAD4 and SUFU and treats parkinsonian rats |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |