CN108048400B - 一种猴子单倍体神经干细胞的获得方法 - Google Patents
一种猴子单倍体神经干细胞的获得方法 Download PDFInfo
- Publication number
- CN108048400B CN108048400B CN201711435471.5A CN201711435471A CN108048400B CN 108048400 B CN108048400 B CN 108048400B CN 201711435471 A CN201711435471 A CN 201711435471A CN 108048400 B CN108048400 B CN 108048400B
- Authority
- CN
- China
- Prior art keywords
- haploid
- culture
- monkey
- stem cells
- culture medium
- 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
- 210000001178 neural stem cell Anatomy 0.000 title claims abstract description 47
- 241000282693 Cercopithecidae Species 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 18
- 210000004027 cell Anatomy 0.000 claims abstract description 33
- 230000004069 differentiation Effects 0.000 claims abstract description 30
- 239000001963 growth medium Substances 0.000 claims abstract description 26
- 210000001671 embryonic stem cell Anatomy 0.000 claims abstract description 20
- 102000004142 Trypsin Human genes 0.000 claims abstract description 9
- 108090000631 Trypsin Proteins 0.000 claims abstract description 9
- 230000006698 induction Effects 0.000 claims abstract description 9
- 239000012588 trypsin Substances 0.000 claims abstract description 9
- 210000005036 nerve Anatomy 0.000 claims abstract description 7
- 108010055896 polyornithine Proteins 0.000 claims abstract description 7
- 238000004114 suspension culture Methods 0.000 claims abstract description 7
- 230000006907 apoptotic process Effects 0.000 claims abstract description 6
- 229920002714 polyornithine Polymers 0.000 claims abstract description 6
- 210000000130 stem cell Anatomy 0.000 claims abstract description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000000684 flow cytometry Methods 0.000 claims abstract description 5
- IDDDVXIUIXWAGJ-DDSAHXNVSA-N 4-[(1r)-1-aminoethyl]-n-pyridin-4-ylcyclohexane-1-carboxamide;dihydrochloride Chemical compound Cl.Cl.C1CC([C@H](N)C)CCC1C(=O)NC1=CC=NC=C1 IDDDVXIUIXWAGJ-DDSAHXNVSA-N 0.000 claims abstract description 4
- 210000003783 haploid cell Anatomy 0.000 claims abstract description 4
- 210000001020 neural plate Anatomy 0.000 claims abstract description 3
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 8
- 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 8
- 238000004113 cell culture Methods 0.000 claims description 6
- AQGNHMOJWBZFQQ-UHFFFAOYSA-N CT 99021 Chemical compound CC1=CNC(C=2C(=NC(NCCNC=3N=CC(=CC=3)C#N)=NC=2)C=2C(=CC(Cl)=CC=2)Cl)=N1 AQGNHMOJWBZFQQ-UHFFFAOYSA-N 0.000 claims description 5
- 101000942967 Homo sapiens Leukemia inhibitory factor Proteins 0.000 claims description 5
- 241000282553 Macaca Species 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 4
- 229930182816 L-glutamine Natural products 0.000 claims description 4
- 229930182555 Penicillin Natural products 0.000 claims description 4
- 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 4
- 235000005324 Typha latifolia Nutrition 0.000 claims description 4
- 244000118869 coast club rush Species 0.000 claims description 4
- 235000020776 essential amino acid Nutrition 0.000 claims description 4
- 239000003797 essential amino acid Substances 0.000 claims description 4
- 210000005155 neural progenitor cell Anatomy 0.000 claims description 4
- 229940049954 penicillin Drugs 0.000 claims description 4
- 229960005322 streptomycin Drugs 0.000 claims description 4
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 4
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 claims 2
- 230000001537 neural effect Effects 0.000 abstract description 14
- 239000002609 medium Substances 0.000 abstract description 13
- 238000000338 in vitro Methods 0.000 abstract description 5
- 239000006143 cell culture medium Substances 0.000 abstract description 2
- 229960001484 edetic acid Drugs 0.000 abstract 2
- 238000010899 nucleation Methods 0.000 abstract 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 6
- 108010085895 Laminin Proteins 0.000 description 6
- 238000004115 adherent culture Methods 0.000 description 5
- 210000002569 neuron Anatomy 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 102000004058 Leukemia inhibitory factor Human genes 0.000 description 4
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 4
- 238000007792 addition Methods 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 102000046645 human LIF Human genes 0.000 description 4
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 102400001368 Epidermal growth factor Human genes 0.000 description 3
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 3
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229940116977 epidermal growth factor Drugs 0.000 description 3
- 229940126864 fibroblast growth factor Drugs 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 2
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 2
- 101800003838 Epidermal growth factor Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000034615 Glial cell line-derived neurotrophic factor Human genes 0.000 description 2
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 description 2
- 101001052035 Homo sapiens Fibroblast growth factor 2 Proteins 0.000 description 2
- 210000001130 astrocyte Anatomy 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 210000003981 ectoderm Anatomy 0.000 description 2
- 210000002242 embryoid body Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 210000004248 oligodendroglia Anatomy 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- CFMYXEVWODSLAX-QOZOJKKESA-N tetrodotoxin Chemical compound O([C@@]([C@H]1O)(O)O[C@H]2[C@@]3(O)CO)[C@H]3[C@@H](O)[C@]11[C@H]2[C@@H](O)N=C(N)N1 CFMYXEVWODSLAX-QOZOJKKESA-N 0.000 description 2
- 229950010357 tetrodotoxin Drugs 0.000 description 2
- CFMYXEVWODSLAX-UHFFFAOYSA-N tetrodotoxin Natural products C12C(O)NC(=N)NC2(C2O)C(O)C3C(CO)(O)C1OC2(O)O3 CFMYXEVWODSLAX-UHFFFAOYSA-N 0.000 description 2
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 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 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 101500025419 Homo sapiens Epidermal growth factor Proteins 0.000 description 1
- 101100310657 Mus musculus Sox1 gene Proteins 0.000 description 1
- 108010088225 Nestin Proteins 0.000 description 1
- 102000007354 PAX6 Transcription Factor Human genes 0.000 description 1
- 101150081664 PAX6 gene Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 108700005079 Recessive Genes Proteins 0.000 description 1
- 102000052708 Recessive Genes Human genes 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 108091007416 X-inactive specific transcript Proteins 0.000 description 1
- 108091035715 XIST (gene) Proteins 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- CJGYSWNGNKCJSB-YVLZZHOMSA-N bucladesine Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](OC(=O)CCC)[C@@H]2N1C(N=CN=C2NC(=O)CCC)=C2N=C1 CJGYSWNGNKCJSB-YVLZZHOMSA-N 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000019975 dosage compensation by inactivation of X chromosome Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007831 electrophysiology Effects 0.000 description 1
- 238000002001 electrophysiology Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000005206 flow analysis Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 238000010448 genetic screening Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000031864 metaphase Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- BITYAPCSNKJESK-UHFFFAOYSA-N potassiosodium Chemical compound [Na].[K] BITYAPCSNKJESK-UHFFFAOYSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0623—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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- 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
- C12N2500/44—Thiols, e.g. mercaptoethanol
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/235—Leukemia inhibitory factor [LIF]
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
- C12N2533/32—Polylysine, polyornithine
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Neurology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Neurosurgery (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于生物工程技术领域,公开了一种猴子单倍体神经干细胞的获得方法,在原有普通猴子干细胞培养基的基础上进行优化培养,用0.05%的胰蛋白酶/EDTA处理单倍体胚胎干细胞克隆,成为单细胞状态,用EB培养基进行悬浮培养,形成拟胚体球;从分化第一天开始,培养基中要加ROCK抑制剂Y‑27632来抑制细胞凋亡;在第五天EB培养基换为神经诱导培养基;将初步形成神经外胚层的EB球种在基底胶铺底的培养皿中,加入神经诱导培养基进行分化;挑取神经花环,种在30μg/ml多鸟氨酸和3μg/ml层粘连蛋白铺底的培养皿中;培养14天后,进行流式细胞术,分选单倍体细胞。本发明第一次实现了在体外获得猴子单倍体神经干细胞,并具有向神经下游方向分化的能力。
Description
技术领域
本发明属于生物工程技术领域,尤其涉及一种猴子单倍体神经干细胞的获得方法。
背景技术
由于猴子单倍体胚胎干细胞系很难建系(目前文献报道的仅有一个课题组建立该细胞系)。猴子单倍体胚胎干细胞分化能力较弱,而且在单倍体神经分化过程中会存在严重的二倍化现象。所以目前尚未有报道证明能通过体外分化的方法得到猴子单倍体神经干细胞。
综上所述,现有技术存在的问题是:通过体外分化的方法得到猴子单倍体神经干细胞数据技术空白。
发明内容
针对现有技术存在的问题,本发明提供了一种猴子单倍体神经干细胞的获得方法。由于单倍体神经干细胞只有一套基因组,在遗传学筛选过程中可以避免隐性基因性状被遮盖的情况。同时由于神经干细胞有分化成下游神经元和神经胶质细胞的能力,所以该细胞系可以用于各种神经疾病靶点基因的筛选。是非常良好的筛选工具。起初用正常的灵长类干细胞培养液培养并分化发现猴子单倍体胚胎干细胞的神经分化能力很弱,分化到神经方向的细胞很少。于是就大胆猜测可能是由于单倍体只有一半基因组,控制多能性的蛋白剂量不够。于是就参考前人发表的工作,提升猴子单倍体胚胎干细胞的多能性。保障向神经干细胞分化的正常进行。进一步发现,随着多能性的提升,猴子单倍体胚胎干细胞的单倍性更稳定,分化出来的神经干细胞生存能力更强。
本发明是这样实现的,一种猴子单倍体神经干细胞的获得方法,所述猴子单倍体神经干细胞的获得方法包括以下步骤:
步骤一,在原有普通猴子干细胞培养液中加入10ng/ml的人白血病抑制因子(LIF),0.5μM的PD0325901,3μM的CHIR99021,10μM的SB203580和10μM的SP600125。
步骤二,用0.05%的胰蛋白酶/EDTA处理单倍体胚胎干细胞克隆,成为单细胞状态,用EB培养基进行悬浮培养,形成拟胚体球;
步骤三,从分化第一天开始,培养基中要加ROCK抑制剂Y-27632来抑制细胞凋亡;在第五天EB培养基换为神经诱导培养基;
步骤四,将初步形成神经外胚层的EB球种在基底胶铺底的培养皿中,加入神经诱导培养基进行分化;
步骤五,挑取神经花环,种在30μg/ml多鸟氨酸和3μg/ml层粘连蛋白铺底的培养皿中;
步骤六,培养14天后,进行流式细胞术,分选单倍体细胞。
进一步,所述步骤五培养皿中的培养基加入人碱性成纤维细胞生长因子和表皮生长因子的神经干细胞培养基。
进一步,所述步骤六在分选前,单倍体神经祖细胞经过大致4次传代来使细胞得到富集。
本发明的另一目的在于提供一种利用所述猴子单倍体神经干细胞的获得方法获得的猴子单倍体神经干细胞。
本发明第一次实现了在体外获得猴子单倍体神经干细胞,并证明其具有向神经下游方向分化的能力。因为基本上各种神经退行性疾病和神经紊乱性疾病都是下游神经元细胞的病变导致的,结合单倍体只有一套染色体组的特点,具有向下游分化能力的猴子单倍体神经干细胞可以用来进行这些疾病靶点基因的筛选。
附图说明
图1是本发明实施例提供的猴子单倍体神经干细胞的获得方法流程图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
下面结合附图对本发明的应用原理作详细的描述。
如图1所示,本发明实施例提供的猴子单倍体神经干细胞的获得方法包括以下步骤:
S101:在改进的单倍体猴子干细胞生长的培养液中培养猕猴单倍体胚胎干细胞。培养液包含20%KOSR和0.1mM非必需氨基酸的D/F12培养基,培养基中别的添加物为0.1mMβ-巯基乙醇,100U/ml的青霉素,0.1mg/ml的链霉素,2mM的L-Glutamine,5ng/ml的成纤维细胞生长因子(bFGF),10ng/ml的人白血病抑制因子(LIF),0.5μM的PD0325901,3μM的CHIR99021,10μM的SB203580和10μM的SP600125;
S102:培养中的猕猴单倍体胚胎干细胞用0.05%的胰蛋白酶消化成单细胞,用EB培养液重悬后在悬浮培养皿中悬浮培养;
S103:分化的第一天在EB培养液里加10μM的Y-27632以减少细胞凋亡;在分化的第5天EB培养液换成神经诱导培养液(NIM);在分化的第7天,悬浮培养的EB贴敷于matrigel包被的贴壁培养皿;大约一周之后,贴壁的集落中间会出现玫瑰花环样的细胞;
S104:手动挑出玫瑰花环并置于包被多聚鸟氨酸和层粘连蛋白的贴壁培养皿;此时培养液换成含有bFGF和EGF的神经干细胞培养液,扩增。
下面结合具体实施例对本发明的应用原理作进一步的描述。
实施例1:
1、一种多能性猴子胚胎干细胞培养液的制备
经过改进,单倍体猴子干细胞生长的培养液包含20%KOSR和0.1mM非必需氨基酸的D/F12培养基,培养基中别的添加物为0.1mMβ-巯基乙醇,100U/ml的青霉素,0.1mg/ml的链霉素,2mM的L-Glutamine,5ng/ml的成纤维细胞生长因子(bFGF),10ng/ml的人白血病抑制因子(LIF),0.5μM的PD0325901,3μM的CHIR99021,10μM的SB203580和10μM的SP600125。
2、猕猴单倍体神经干细胞的获得
培养中的猕猴单倍体胚胎干细胞用0.05%的胰蛋白酶消化成单细胞,用EB培养液重悬后在悬浮培养皿中悬浮培养。分化的第一天在EB培养液里加10μM的Y-27632以减少细胞凋亡。在分化的第5天EB培养液换成神经诱导培养液(NIM)。在分化的第7天,悬浮培养的EB贴敷于matrigel包被的贴壁培养皿。大约一周之后,贴壁的集落中间会出现玫瑰花环样的细胞。手动的挑出玫瑰花环并置于包被多聚鸟氨酸和层粘连蛋白的贴壁培养皿。此时培养液换成含有bFGF和EGF的神经干细胞培养液,进一步扩增。
2、流式分析和流式分选
待流式分选的细胞用0.05%的胰蛋白酶消化成单细胞,然后用Hoechst 33342(5μg/ml)染色,37℃水浴孵育15min。细胞筛过滤后用BD FACS AriaII进行分选“n”峰处的细胞。待流式分析的细胞用0.05%的胰蛋白酶消化成单细胞,用75%的分析级乙醇4℃过夜固定。第二天用5μg/ml的碘化丙啶染色,同时加入2mg/ml RNA酶,37℃水浴孵育15min。细胞筛过滤后用BD LSRII SORP进行相关分析。
3、单倍体神经干细胞的分化潜能
为了验证所获得的单倍体神经干细胞具有神经分化的潜能,单倍体神经干细胞按照2万/cm2的密度铺在用多聚鸟氨酸和层粘连蛋白包被的贴壁培养皿。对于神经元细胞的分化,培养液换成DMEM/F12,添加N2B27,胶质源性神经营养因子(GDNF),脑源性神经营养因子(BDNF),双丁酰环腺苷酸(cAMP)和抗坏血酸(VC)。在此培养液的基础上加入1%的胎牛血清,就能分化到星形胶质细胞和少突胶质细胞。
4、免疫荧光染色
生长在玻片上的细胞用4%的多聚甲醛4℃过夜固定。PBS洗三遍后用0.3%的Triton X-100室温孵育一小时。然后用3%的牛血清白蛋白(BSA)室温孵育一小时。按照推荐浓度稀释一抗,4℃过夜孵育。二抗室温避光孵育一小时。
5、染色体核型分析
培养中的细胞用0.2ug/ml nocodazle处理3小时(神经干细胞)/2小时(胚胎干细胞)进行中期同步化。胰蛋白酶消化成单细胞后用0.075mM的KCL在37℃水浴30分钟。然后用固定液(甲醇:乙酸=3:1)4℃固定20分钟。重复固定一次。然后细胞滴在洁净的玻片上。用吉姆萨染液染色7分钟后自然晾干。
6、生长曲线
96孔板的每空加入1000个细胞。在生长的第1,2,3,4,5天分别加入10μl CCK8溶液和90μl神经干细胞培养液。一小时后用酶标仪记录数据。
7、电生理
单倍体神经干细胞在贴壁培养皿分化三周。用膜片钳记录全细胞钠钾电流。加入500nM河豚毒素(TTX)后钠离子电流明显受阻。
下面结合实验对本发明的应用效果作详细的描述。
本发明通过优化猴子单倍体胚胎干细胞的培养体系,并利用流式细胞术得到了能够稳定维持单倍性的猴子单倍体神经干细胞。通过分化实验,证明得到的猴子单倍体神经干细胞具有完整的分化能力。具体过程及结果如下:
1、细胞培养。猴子单倍体胚胎干细胞获赠于昆明理工大学谭韬研究员处,培养条件为:加了20%KOSR和0.1mM非必需氨基酸的D/F12培养基,培养基中别的添加物为0.1mMβ-巯基乙醇,100U/ml的青霉素,0.1mg/ml的链霉素,2mM的L-Glutamine,5ng/ml的成纤维细胞生长因子(bFGF),10ng/ml的人白血病抑制因子(LIF),0.5μM的PD0325901,3μM的CHIR99021,10μM的SB203580和10μM的SP600125。在这个培养体系中,猴子单倍体胚胎干细胞能维持较高的多能性、单倍性维持和分化潜能,并且其细胞状态要好于传统培养条件。
2、通过单倍体胚胎干细胞的体外分化获得单倍体神经干细胞。采用的方法为拟胚体(EB)随机分化的方法来获得单倍体神经干细胞。为了得到拟胚体,用0.05%的胰蛋白酶/EDTA处理单倍体胚胎干细胞克隆,使其成为单细胞状态,然后用EB培养基进行悬浮培养,以形成拟胚体球。从分化第一天开始,培养基中要加ROCK抑制剂Y-27632来抑制细胞凋亡。在第五天的时候,EB培养基换为神经诱导培养基。换成神经诱导培养基两天后,可以看到神经外胚层结构的出现,然后将初步形成神经外胚层的EB球种在基底胶铺底的培养皿中,加入神经诱导培养基进行进一步分化。4-5天后,形成初始神经上皮结构。在分化后15天左右,大量细胞聚集在一起,形成成熟神经上皮结构和神经花环结构。然后挑取神经花环,种在30μg/ml多鸟氨酸和3μg/ml层粘连蛋白(laminin)铺底的培养皿中,培养基为加了人碱性成纤维细胞生长因子(bFGF)和表皮生长因子(EGF)的神经干细胞培养基。培养14天后,进行流式细胞术,分选单倍体细胞。在分选前,单倍体神经祖细胞经过大致4次传代来使细胞得到富集。得到的神经祖细胞能够维持较好的单倍性,不经流式分选,传20代后仍能保持较高单倍体比例。且用这种方法得到的单倍体神经干细胞与正常的二倍体神经干细胞在生长能力上也没有差别。
3、对得到的猴子单倍体神经干细胞进行鉴定。首先对猴子单倍体神经干细胞进行免疫荧光染色,发现其表达神经干细胞特异性的标记基因Nestin、Sox1和Pax6。实时定量PCR结果也说明了同样的结果。另外发现猴子单倍体神经干细胞没有X染色体失活必需基因Xist的表达。同时对猴子单倍体神经干细胞进行了分化培养,评估其分化潜能。结果发现在培养基中添加特定生长因子3周左右其可以形成星形胶质细胞、少突胶质细胞和神经元。并且分化得到的神经元也是具有电生理功能的。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (2)
1.一种猴子单倍体神经干细胞的获得方法,其特征在于,所述猴子单倍体神经干细胞的获得方法包括以下步骤:
步骤一,在改进的单倍体猴子干细胞生长的培养液中培养猕猴单倍体胚胎干细胞,培养液是添加有0.1mM β-巯基乙醇,100U/ml的青霉素,0.1mg/ml的链霉素,2mM的L-Glutamine,5ng/ml的成纤维细胞生长因子bFGF,10ng/ml的人白血病抑制因子LIF,0.5μM的PD0325901,3μM的CHIR99021,10μM的SB203580,10μM的SP600125,20% KOSR和0.1mM非必需氨基酸的D/F12培养基;
步骤二,用0.05%的胰蛋白酶/EDTA处理单倍体胚胎干细胞克隆,成为单细胞状态,用EB培养基进行悬浮培养,形成拟胚体球;
步骤三,从分化第一天开始,培养基中要加ROCK抑制剂Y-27632来抑制细胞凋亡;在第五天EB培养基换为神经诱导培养基;
步骤四,将初步形成神经外胚层的EB球种在基底胶铺底的培养皿中,加入神经诱导培养基进行分化;
步骤五,挑取神经花环,种在30μg/ml多聚鸟氨酸和3μg/ml层粘连蛋白铺底的培养皿中;此时培养液换成含有bFGF和EGF的神经干细胞培养液,扩增;
步骤六,培养14天后,进行流式细胞术,分选单倍体细胞。
2.如权利要求1所述的猴子单倍体神经干细胞的获得方法,其特征在于,所述步骤六在分选前,单倍体神经祖细胞经过大致4次传代来使细胞得到富集。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201711435471.5A CN108048400B (zh) | 2017-12-26 | 2017-12-26 | 一种猴子单倍体神经干细胞的获得方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201711435471.5A CN108048400B (zh) | 2017-12-26 | 2017-12-26 | 一种猴子单倍体神经干细胞的获得方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN108048400A CN108048400A (zh) | 2018-05-18 |
CN108048400B true CN108048400B (zh) | 2021-04-09 |
Family
ID=62128050
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201711435471.5A Active CN108048400B (zh) | 2017-12-26 | 2017-12-26 | 一种猴子单倍体神经干细胞的获得方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN108048400B (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022110180A1 (en) * | 2020-11-30 | 2022-06-02 | Zhejiang Huode Bioengineering Company Limited | Generation of neural progenitor cells from embryonic stem cells or induced pluripotent stem cells |
CN114457113B (zh) * | 2022-03-07 | 2023-11-17 | 南开大学 | 一种抑制单倍体胚胎干细胞二倍化的方法 |
CN114958747B (zh) * | 2022-06-08 | 2024-08-20 | 中国科学院动物研究所 | 一种多能干细胞诱导产生兴奋性和抑制性神经元的方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU782846B2 (en) * | 1999-10-28 | 2005-09-01 | University Of Massachusetts | Gynogenetic or androgenetic production of pluripotent cells and cell lines, and use thereof to produce differentiated cells and tissues |
SG10201913367SA (en) * | 2015-07-29 | 2020-02-27 | New York Stem Cell Foundation Inc | Haploid human embryonic stem cell lines and somatic cell lines and methods of making the same |
-
2017
- 2017-12-26 CN CN201711435471.5A patent/CN108048400B/zh active Active
Also Published As
Publication number | Publication date |
---|---|
CN108048400A (zh) | 2018-05-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108048400B (zh) | 一种猴子单倍体神经干细胞的获得方法 | |
KR102573180B1 (ko) | 분화 배지 및 희소돌기아교세포 전구체를 제조하는 방법 | |
CN110872576A (zh) | 一种将小鼠成纤维细胞转分化为多巴胺能神经元的方法 | |
CN111471653A (zh) | 一种将非神经元细胞转化为神经元细胞的方法 | |
JP2020505945A (ja) | 奇形腫の形成が抑制された多分化能性幹細胞由来の神経前駆体球の製造方法 | |
CN108998410B (zh) | 蛋白激酶抑制剂在抑制单倍体细胞二倍化中的用途 | |
CN107475200B (zh) | 一种背根神经节源的神经嵴干细胞的分离、培养与分化方法 | |
CN115975914A (zh) | 利用化学小分子药物重编程诱导多能干细胞的方法 | |
KR101896803B1 (ko) | 인간 만능줄기세포로부터 중간엽 줄기세포로의 분화 유도 생산율을 증가시키는 방법 및 이에 의해 생성된 중간엽 줄기세포 | |
Minamino et al. | Isolation and propagation of neural crest stem cells from mouse embryonic stem cells via cranial neurospheres | |
CN108795842B (zh) | 一种3D悬浮诱导iPS细胞生成自体黑素细胞的方法及应用 | |
JP6218152B2 (ja) | 内耳細胞誘導方法 | |
CN114787341B (zh) | 从人多能干细胞制备间充质干细胞的方法及由此制备的间充质干细胞 | |
CN101294146A (zh) | 诱导神经干细胞分化的系统及诱导方法 | |
Chimge et al. | Generation of neural crest progenitors from human embryonic stem cells | |
KR101793722B1 (ko) | 성상세포의 생산방법 | |
CN107164325B (zh) | MSCs来源的少突胶质细胞的制备方法及试剂盒 | |
KR101613677B1 (ko) | 줄기 세포의 신경 전구 세포로의 분화용 조성물 및 이를 이용한 방법 | |
Philonenko et al. | Differentiation of human pluripotent stem cells into mesodermal and ectodermal derivatives is independent of the type of isogenic reprogrammed somatic cells | |
CN110938584B (zh) | 一种人胚胎干细胞向内皮细胞诱导分化的方法 | |
KR101766372B1 (ko) | Cxcr2 자극을 통한 인간 전분화능 줄기세포 유래 배아체의 외배엽 분화 방법 | |
KR101491444B1 (ko) | 포유동물의 생식선 줄기세포를 수득하는 방법 | |
KR101636966B1 (ko) | 배아줄기세포로부터 신경교전구세포, 성상세포 및 희소돌기 아교세포로의 분화방법 | |
KR101705245B1 (ko) | 마트리겔이 코팅된 신경줄기세포 배양용 플레이트 | |
KR101588110B1 (ko) | 만능줄기세포로부터 형성된 테라토마 유래 체내 신경줄기세포 생산 방법 |
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 |