CN107475200B - 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 - Google Patents
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 Download PDFInfo
- Publication number
- CN107475200B CN107475200B CN201710668928.0A CN201710668928A CN107475200B CN 107475200 B CN107475200 B CN 107475200B CN 201710668928 A CN201710668928 A CN 201710668928A CN 107475200 B CN107475200 B CN 107475200B
- Authority
- CN
- China
- Prior art keywords
- neural crest
- cells
- stem cells
- crest stem
- culturing
- 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
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 61
- 210000000933 neural crest Anatomy 0.000 title claims abstract description 60
- 210000003594 spinal ganglia Anatomy 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000012258 culturing Methods 0.000 title claims abstract description 28
- 230000004069 differentiation Effects 0.000 claims abstract description 14
- 230000035755 proliferation Effects 0.000 claims abstract description 11
- 238000002955 isolation Methods 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 49
- 210000001519 tissue Anatomy 0.000 claims description 26
- 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 18
- 239000001963 growth medium Substances 0.000 claims description 16
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 12
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims description 12
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Natural products OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 11
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 9
- 108010025020 Nerve Growth Factor Proteins 0.000 claims description 9
- 229930182555 Penicillin Natural products 0.000 claims description 9
- 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 9
- 229940049954 penicillin Drugs 0.000 claims description 9
- 229960005322 streptomycin Drugs 0.000 claims description 9
- 239000011573 trace mineral Substances 0.000 claims description 9
- 235000013619 trace mineral Nutrition 0.000 claims description 9
- 101000890609 Bos taurus A-kinase anchor protein 5 Proteins 0.000 claims description 8
- 101000735561 Mus musculus Protein-arginine deiminase type-6 Proteins 0.000 claims description 8
- 102000008730 Nestin Human genes 0.000 claims description 8
- 108010088225 Nestin Proteins 0.000 claims description 8
- 210000005055 nestin Anatomy 0.000 claims description 8
- 102100037362 Fibronectin Human genes 0.000 claims description 7
- 108010067306 Fibronectins Proteins 0.000 claims description 7
- 101000664703 Homo sapiens Transcription factor SOX-10 Proteins 0.000 claims description 7
- 102100038808 Transcription factor SOX-10 Human genes 0.000 claims description 7
- 108010055896 polyornithine Proteins 0.000 claims description 7
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 claims description 6
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 claims description 6
- 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 claims description 6
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 6
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 claims description 6
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 claims description 6
- 102000034615 Glial cell line-derived neurotrophic factor Human genes 0.000 claims description 6
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 claims description 6
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 6
- 102000015336 Nerve Growth Factor Human genes 0.000 claims description 6
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 6
- 229930003268 Vitamin C Natural products 0.000 claims description 6
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 claims description 6
- 229940053128 nerve growth factor Drugs 0.000 claims description 6
- 210000002569 neuron Anatomy 0.000 claims description 6
- 229920002866 paraformaldehyde Polymers 0.000 claims description 6
- 239000011718 vitamin C Substances 0.000 claims description 6
- 235000019154 vitamin C Nutrition 0.000 claims description 6
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 5
- 241000699670 Mus sp. Species 0.000 claims description 5
- 235000020776 essential amino acid Nutrition 0.000 claims description 5
- 239000003797 essential amino acid Substances 0.000 claims description 5
- 210000001178 neural stem cell Anatomy 0.000 claims description 5
- 229920002714 polyornithine Polymers 0.000 claims description 5
- 102000004590 Peripherins Human genes 0.000 claims description 4
- 108010003081 Peripherins Proteins 0.000 claims description 4
- 101150008375 Pou4f1 gene Proteins 0.000 claims description 4
- 102000004338 Transferrin Human genes 0.000 claims description 4
- 108090000901 Transferrin Proteins 0.000 claims description 4
- 238000010166 immunofluorescence Methods 0.000 claims description 4
- 210000005047 peripherin Anatomy 0.000 claims description 4
- 239000000725 suspension Substances 0.000 claims description 4
- 239000012581 transferrin Substances 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 102000014429 Insulin-like growth factor Human genes 0.000 claims description 3
- 239000012580 N-2 Supplement Substances 0.000 claims description 3
- 102000007072 Nerve Growth Factors Human genes 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims description 3
- 108010084091 heregulin beta1 Proteins 0.000 claims description 3
- 238000013508 migration Methods 0.000 claims description 3
- 230000005012 migration Effects 0.000 claims description 3
- 239000003147 molecular marker Substances 0.000 claims description 3
- 238000004264 monolayer culture Methods 0.000 claims description 3
- 239000003900 neurotrophic factor Substances 0.000 claims description 3
- 230000002093 peripheral effect Effects 0.000 claims description 3
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 3
- 208000003098 Ganglion Cysts Diseases 0.000 claims description 2
- 208000005400 Synovial Cyst Diseases 0.000 claims description 2
- 239000008188 pellet Substances 0.000 claims description 2
- 238000001179 sorption measurement Methods 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims 5
- 230000029087 digestion Effects 0.000 claims 2
- WAPPRYMYMNVAFR-UHFFFAOYSA-N 2-sulfanylethanol Chemical compound OCCS.OCCS WAPPRYMYMNVAFR-UHFFFAOYSA-N 0.000 claims 1
- 150000007513 acids Chemical class 0.000 claims 1
- 238000012136 culture method Methods 0.000 claims 1
- 210000002257 embryonic structure Anatomy 0.000 claims 1
- 238000000338 in vitro Methods 0.000 abstract description 4
- 210000001982 neural crest cell Anatomy 0.000 description 9
- 239000000243 solution Substances 0.000 description 6
- 238000000576 coating method Methods 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 3
- 238000004115 adherent culture Methods 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000009194 climbing Effects 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 210000000276 neural tube Anatomy 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 239000002771 cell marker Substances 0.000 description 2
- 210000003792 cranial nerve Anatomy 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 208000004840 megacolon Diseases 0.000 description 2
- 210000005036 nerve Anatomy 0.000 description 2
- 210000003061 neural cell Anatomy 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 210000002718 aborted fetus Anatomy 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000008143 early embryonic development Effects 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 230000001815 facial effect Effects 0.000 description 1
- 210000000609 ganglia Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 210000002752 melanocyte Anatomy 0.000 description 1
- 230000001617 migratory effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 210000004409 osteocyte Anatomy 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 208000027232 peripheral nervous system disease Diseases 0.000 description 1
- 210000002856 peripheral neuron Anatomy 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- 101150077014 sox10 gene Proteins 0.000 description 1
- 238000011476 stem cell transplantation Methods 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 210000001186 vagus nerve Anatomy 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
- 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
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
-
- 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
- C12N2500/38—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
- C12N2500/40—Nucleotides, nucleosides or bases
-
- 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
- C12N2501/105—Insulin-like growth factors [IGF]
-
- 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
- C12N2501/10—Growth factors
- C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
本发明提供了一种背根神经节源神经嵴干细胞的分离，培养与分化的新型方法，包括以下步骤：（一）背根神经节来源的神经嵴干细胞的原代分离；（二）背根神经节来源的神经嵴干细胞的纯化与培养扩增；（三）背根神经节来源的神经嵴干细胞的诱导分化。本发明获取的神经嵴干细胞具有较强的增殖能力，体外持续培养数代后仍具有增殖能力。
Description
技术领域
本发明属于干细胞生物技术领域，涉及背根神经节来源的神经嵴干细胞的分离、培养和分化方法。
背景技术
神经嵴干细胞是胚胎发育过程中的一种重要细胞类型。起源于早期胚胎发育过程中神经管背部，通过迁移至胚胎的各个部位分化成多种细胞类型。神经嵴细胞主要分为颅源性神经嵴细胞和躯干神经嵴干细胞，躯干颅源性神经嵴细胞分为迷走神经嵴和骶骨神经嵴，神经嵴细胞迁移至躯体各个部位后，可分化成黑色素细胞、颅面部细胞、软骨细胞、骨细胞、平滑肌细胞、中枢与外周神经元以及胶质细胞。
背根神经节(DRG)是由躯干神经嵴干细胞迁移分化而形成的。DRG细胞可体外分离和培养，为周围神经系统神经干细胞移植提供细胞来源。近年来关于背根神经节来源的神经嵴干细胞的分离和培养有较多研究，主要利用流式分选的方法分离细胞，该方法可获得较纯的细胞源，但细胞损失较大。
目前临床上有较多因神经嵴细胞发育不正常导致的疾病，如巨结肠，因早期发育过程中神经嵴细胞迁移至肠的数量不足，导致肠神经系统不完整，从而导致疾病的发生。如果体外能获得高质量和数量的神经干细胞用于移植到病变的肠中，有望解决巨结肠等外周神经疾病问题。
发明内容
本发明的目的是提供一种背根神经节来源的神经嵴干细胞的分离、培养与分化方法。本发明的目的通过以下技术方案实现：
一种背根神经节源神经嵴干细胞的分离、培养与分化方法，其特征在于它包以下步骤：
(一)背根神经节来源的神经嵴干细胞的原代分离；
(二)背根神经节来源的神经嵴干细胞的纯化与培养扩增；
(三)背根神经节来源的神经嵴干细胞的诱导分化。
进一步的，所述的分离方法，包括以下步骤：取出生后6天的幼鼠，或E‐13.5天左右的鼠类胚胎，或人的流产胎儿，在无菌条件下，取出背根神经节组织，剪成2毫米左右小块后，加入PBS洗涤，800‐1000RMP离心5min，弃上清，加入原代培养基重悬后，转移至包被多聚鸟氨酸的培养板中，置于含5％CO2培养箱，于37度下培养恒温下培养，培养两天，待组织周围爬出细胞后，于显微镜下挑起组织块，并转移到新的多聚鸟氨酸包被的板中，继续同前面所述条件下培养，待组织周围爬出细胞后，去掉组织。爬出的细胞进入步骤(二)继续培养。
以上以小鼠C57品系为例，人胚胎为9周以后。
上述原代培养基为：DMEM/F12培养基、1％N2、2％B27、20ng/ml bFGF(碱性成纤维细胞生长因子)、20ng/ml EGF(内皮细胞生长因子)、100IU/L青霉素/链霉素。
所述步骤(二)包括以下步骤：根据神经嵴干细胞较强的迁移能力，保留神经节组织块爬出的细胞中的最外围的部分，连续贴壁转移组织块3‐4次，用增殖培养基培养爬出的外围细胞，两天换一次培养基，四天左右传代，并利用免疫荧光的方法，鉴定分离的细胞是否具有神经干细胞特异性分子标记(sox10，nestin，P75)。
上述增殖培养基为：DMEM/F12培养基、2％牛血清白蛋白、100IU/L青霉素/链霉素、1％L‐丙氨酸‐L‐谷胺酰胺、1％MEM non‐essential amino acids(非必须氨基酸)、0.1％trace elements A、0.1％trace elements B、0.1％trace elements C、0.18％2‐mercaptoethanol(β‐巯基乙醇)、10mg/ml transferrin(转铁蛋白)、50mg/ml(+)‐sodiuml‐ascorbate(维生素C)、10ng/ml Heregulin‐β1、200ng/ml LONGR3IGF‐I(长链胰岛素样生长因子)、8ng/ml bFGF(碱性成纤维生长因子)。
神经嵴干细胞特征的鉴定，其具体方法为：将细胞消化后传至poly‐ornithine包被的玻片上，待贴壁后，多聚甲醛固定5分钟，PBS洗三遍，1％的BSA室温封闭30分钟，P75、Nestin、SOX10一抗室温孵育两小时，PBS洗三遍，二抗孵育1小时，PBS洗三遍后，于荧光显微镜下观察。
上述去除杂细胞和组织块的方法，其特征在于，于显微镜下用剥离管刮除。
所述的传代扩增，一方面可以以单层培养的方法进行传代扩增，一方面可以以球的方式进行传代扩增上述的单层培养进行扩增，其具体方法为，细胞经消化后，传至poly‐ornithine包被的板中进行培养。上述的球的形式进行传代扩增，其具体方法为，细胞经消化后，传至低吸附的板中进行培养。
所述的背根神经节来源的神经嵴干细胞的分化方法，其具体方法为，传至第二代的神经嵴干细胞消化后，60％的密度铺于poly‐ornithine/fibronectin包被的板中，加入分化培养基培养，三天左右换一次液；培养至两周后，通过免疫荧光方法鉴定是否表达神经元标志蛋白(Tuj1，Brn3a，Peripherin)。
上述分化培养基为DMEM/F12、1％N2supplement、100IU/L青霉素/链霉素、10ng/mlBDNF(脑源性神经营养因子)、10ng/ml NGF(神经生长因子)、10ng/ml GDNF(神经胶质细胞源性的神经营养因子)、10ng/ml NT3(神经营养因子3)、50μg/ml ascorbic acid(维生素C)和0.5mM cAMP(环磷酸腺苷)。
所述的神经元特异表达分子鉴定，其具体方法为：取出玻片，多聚甲醛固定5分钟，PBS洗三遍，1％的BSA室温封闭30分钟，P75、Nestin、SOX10一抗室温孵育两小时，PBS洗三遍，二抗孵育1小时，PBS洗三遍后，于荧光显微镜下观察。
上述poly‐ornithine/fibronectin(多聚鸟氨酸/纤连蛋白)包被的板，poly‐ornithine包被室温包被1h后，pbs洗两遍，25ug/ml fibronectin 37度包被2小时。
本发明方法获得的神经节来源的神经嵴干细胞，具有较高的纯度(如图3所示，本发明获得的背根神经节来源的神经嵴干细胞90％以上表达神经嵴干细胞标志蛋白)。本发明无需经过流式分选，即可获得目标细胞(如图2所示)。本发明的神经嵴干细胞培养方法，可使分离的神经嵴细胞具有较强的增殖能力，并能维持神经嵴细胞特性(如图4所示，本发明获得的神经嵴干细胞传代至第五代时仍然具有较强的增殖能力)。本发明的神经嵴干细胞分化方法，可使分离的神经嵴细胞较高比例地分化成神经细胞(如图5所示，本发明获得的神经嵴干细经诱导分化后，可获得较高比例的神经元细胞)。本发明的背根神经节来源的神经嵴干细胞的分离、培养和分化方法，不仅可以获得出生后的鼠源神经嵴干细胞，也可获得胚胎期的神经嵴干细胞，不仅可以获得鼠源的神经嵴干细胞，还可获得人的神经嵴干细胞。
附图说明
图1背根神经节来源的神经嵴细胞获取流程图。
图2背根神经节来源的神经嵴干细胞培养相差图：神经嵴细胞原代爬出后，两到三天长满(A‐B)，传代后和成球生长的形态(C‐D)。标尺100um
图3背根神经节来源的神经嵴干细胞特性鉴定图。A,贴壁培养细胞P75(灰色，如箭头所示)、SOX10(灰白色，如箭头所示)和Nestin(灰色，如箭头所示)免疫荧光染色。B,成球培养的细胞P75(灰白色色，如箭头所示)、SOX10(灰白色，如箭头所示)和Nestin(灰色，如箭头所示)免疫荧光染色。C,D贴壁培养和成球培养的细胞均具有90％以上的神经嵴干细胞比例。标尺100um
图4背根神经节来源的神经嵴干细胞的增殖能力分析图：培养一代和五代的细胞生长曲线。
图5背根神经节来源的神经嵴干细胞的分化图。培养一代的细胞诱导分化成神经细胞，Tuj1(灰白色，如箭头所示)、Peripherin(灰色，如箭头所示)和Brn3a(灰白色，如箭头所示)免疫荧光染色。标尺100um
具体实施方法
本发明结合实施例和附图作进一步说明
实施例1，背根神经节来源的神经嵴干细胞的分离与纯化
取出生后6天左右的C57小鼠，酒精浸泡15min，超净台下切开背部，取出神经管，于显微镜下，取出神经管边缘的背根神经节，剪成2毫米左右的块状，pbs漂洗后，800rmp离心5分钟后收集组织块，原代培养基重悬组织块，转移至poly‐ornithine包被的板中，如图1流程图所示。原代培养基为：DMEM/F12培养基、1％N2、2％B27、20ng/ml bFGF(碱性成纤维细胞生长因子)、20ng/ml EGF(内皮细胞生长因子)、100IU/L青霉素/链霉素。置于含5％CO2的37度恒温培养箱中培养。培养两天后，组织块周围爬出细胞，如图2‐A所示，在显微镜下剥离组织块，保留最外层的细胞继续培养，如图2‐B所示，并换成增殖培养基，取出的组织块再次贴于poly‐ornithine包被的板中，继续爬出细胞，两日后去除组织块，保留外围细胞继续于增殖培养基中培养。连续贴壁共计3‐4次。增殖培养基为：DMEM/F12培养基、2％牛血清白蛋白、100IU/L青霉素/链霉素、1％L‐丙氨酸‐L‐谷胺酰胺、1％MEM non‐essential amino acids(非必须氨基酸)、0.1％trace elements A、0.1％trace elements B、0.1％traceelements C、0.18％2‐mercaptoethanol(β‐巯基乙醇)、10mg/ml transferrin(转铁蛋白)、50mg/ml(+)‐sodium l‐ascorbate(维生素C)、10ng/ml Heregulin‐β1、200ng/mlLONGR3IGF‐I(长链胰岛素样生长因子)、8ng/ml bFGF(碱性成纤维生长因子)。
实施例2，背根神经节来源的神经嵴干细胞的鉴定。
取培养2代和成球培养的细胞，PBS漂洗两遍，多聚甲醛固定15min，含0.3％(体积比)triton的封闭液(1％BSA)室温封闭30min，1:100稀释的一抗工作液(P75，Nestin，SOX10)室温孵育2h，PBS洗三遍后，二抗稀释液(1：500)室温孵育1小时，PBS漂洗三遍后，于荧光显微镜下观察拍照，通过image J软件统计表达阳性的细胞比例。如图3所示，本发明方法获得的背根神经节来源的神经嵴干细胞具有较高的纯度，本发明获得的背根神经节来源的神经嵴干细胞90％以上表达神经嵴干细胞标志蛋白。
实施例3，背根神经节来源的神经嵴干细扩增能力分析
原代获取的神经干细胞以两种形式传代扩增培养，一种是单层贴壁培养，一种是成球悬浮培养，如图2(C‐D)所示两种培养方式均能在体外扩增数代。
取贴壁培养第一代和第五代的细胞，PBS漂洗两遍，4％(质量比)多聚甲醛固定15分钟，含0.3％triton的封闭液(1％BSA)室温封闭30分钟，1:200稀释的Ki67一抗工作液室温孵育2小时，PBS洗三遍后，二抗稀释液(1：500)室温孵育1小时，PBS漂洗三遍后，于荧光显微镜下观察拍照，通过image J软件统计Ki67阳性的细胞比例，如图4所示背根神经节来源的神经嵴干细胞分化潜能，本发明获得的神经嵴干细胞传代至第五代时仍然具有较强的增殖能力。
神经细胞的诱导：将培养至第二代的细胞接种到载有盖玻片的六孔板中，玻片经poly‐ornithine和Fibronectin包被(具体包被方法为，poly‐ornithine室温包被1小时，Fibronectin 37度包被2小时)，加入神经细胞分化培养基(DMEM/F12、1％N2supplement、100IU/L青霉素/链霉素、10ng/ml BDNF(脑源性神经营养因子)、10ng/ml NGF(神经生长因子)、10ng/ml GDNF(神经胶质细胞源性的神经营养因子)、10ng/ml NT3(神经营养因子3)、50μg/ml ascorbic acid(维生素C)和0.5mM cAMP(环磷酸腺苷))，每三天换一次液，培养两周后，4％多聚甲醛固定15min，含0.3％triton的封闭液(1％BSA)室温封闭30min，1:200稀释的一抗工作液(Tuj1，Brn3a，Peripherin)室温孵育2小时，PBS洗三遍后，二抗稀释液(1：500)室温孵育1小时，PBS漂洗三遍后，于荧光显微镜下观察拍照，通过Image J软件统计阳性的细胞比例，如图5所示，本发明获得的神经嵴干细经诱导分化后，可获得较高比例的神经元细胞。
以上所述仅为本发明的具体实施例，但本发明的结构特征并不局限于此，任何本领域的技术人员在本发明的领域内，所作的变化或修饰皆涵盖在本发明的保护范围之中。
Claims (6)
1.一种背根神经节源神经嵴干细胞的分离、培养与分化方法，其特征在于它包括以下步骤：
(一)背根神经节来源的神经嵴干细胞的原代分离；
(二)背根神经节来源的神经嵴干细胞的纯化与培养扩增；
(三)背根神经节来源的神经嵴干细胞的诱导分化；
步骤(一)包括以下步骤：
取出生后6天内的幼鼠，或E-13.5天之后的鼠胚胎，在无菌条件下，取出背根神经节组织，剪成2毫米左右小块后，加入PBS(磷酸盐缓冲液)洗涤，800-1000RMP离心5分钟，弃上清，加入原代培养基重悬后，转移至包被多聚鸟氨酸的培养板中，置于含5％CO2培养箱，于37度下培养恒温下培养，培养两天，待组织周围爬出细胞后，于显微镜下挑起组织块，并转移到新的多聚鸟氨酸包被的板中，继续同前面所述条件下培养，待组织周围爬出细胞后，去掉组织；所述背根神经节来源的神经嵴干细胞的原代培养基为：DMEM/F12培养基、1％N2、2％B27、20ng/mlbFGF(碱性成纤维细胞生长因子)、20ng/mlEGF(内皮细胞生长因子)、100IU/L青霉素/链霉素；
所述步骤(二)包括以下步骤：
根据神经嵴干细胞较强的迁移能力，保留神经节组织块爬出的细胞中的最外围的部分，连续贴壁转移组织块3-4次，用增殖培养基培养爬出的外围细胞，两天换一次培养基，四天左右传代，并利用免疫荧光的方法，鉴定分离的细胞是否具有神经干细胞特异性分子标记SOX10，Nestin，P75；所述增殖培养基为：DMEM/F12培养基、2％质量比的牛血清白蛋白、100IU/L青霉素/链霉素、1％质量比的L-丙氨酸-L-谷胺酰胺、1％MEM non-essentialaminoacids(非必须氨基酸)、0.1％体积比的trace elements A、0.1％体积比的trace elementsB、0.1％体积比的trace elements C、0.18％体积比的2-mercaptoethanol(β-巯基乙醇)、10mg/ml transferrin(转铁蛋白)、50mg/ml(+)-sodium l-ascorbate(维生素C)、10ng/mlHeregulin-β1、200ng/mlLONGR3 IGF-I(长链胰岛素样生长因子)、8ng/mlbFGF(碱性成纤维生长因子)；
所述鼠为小鼠。
2.如权利要求1所述的一种背根神经节源神经嵴干细胞的分离、培养与分化方法，其特征在于神经嵴干细胞特征的鉴定，其具体方法为：
将细胞消化后传至poly-ornithine(多聚鸟氨酸)包被的玻片上，待贴壁后，多聚甲醛固定5分钟，PBS洗三遍，1％的BSA室温封闭30min，P75、Nestin、SOX10一抗室温孵育两小时，PBS洗三遍，二抗孵育1小时，PBS洗三遍后，于荧光显微镜下观察。
3.如权利要求1所述的一种背根神经节源神经嵴干细胞的分离、培养与分化方法，其特征在于它的传代扩增一方面可以以单层培养的方法进行传代扩增，另一方面可以以球的方式进行传代扩增。
4.如权利要求3所述的一种背根神经节来源的神经嵴干细胞的分离、培养与分化方法，其特征在于所述的单层培养进行传代扩增，在细胞经消化后，传至poly-ornithine包被的板中进行培养；所述的球的形式进行传代扩增，在细胞经消化后，传至低吸附的板中进行培养。
5.如权利要求1所述的一种背根神经节源神经嵴干细胞的分离、培养与分化方法，其特征在于所述的背根神经节来源的神经嵴干细胞的分化方法，其具体方法为，传至第二代的神经嵴干细胞消化后，60％的密度铺于poly-ornithine/fibronectin(多聚鸟氨酸/纤连蛋白)包被的板中，加入分化培养基培养，三天左右换一次液；培养至两周后，通过免疫荧光方法鉴定是否表达神经元特异性分子标记Tuj1，Brn3a，Peripherin。
6.如权利要求5所述的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，其特征在于分化培养基为DMEM/F12、1％N2 supplement、100IU/L青霉素/链霉素、10ng/mlBDNF(脑源性神经营养因子)、10ng/mlNGF(神经生长因子)、10ng/mlGDNF(神经胶质细胞源性的神经营养因子)、10ng/mlNT3(神经营养因子3)、50μg/mlascorbic acid(维生素C)和0.5mM cAMP(环磷酸腺苷)。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710668928.0A CN107475200B (zh) | 2017-08-08 | 2017-08-08 | 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 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710668928.0A CN107475200B (zh) | 2017-08-08 | 2017-08-08 | 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 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107475200A CN107475200A (zh) | 2017-12-15 |
| CN107475200B true CN107475200B (zh) | 2020-12-22 |
Family
ID=60599181
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710668928.0A Active CN107475200B (zh) | 2017-08-08 | 2017-08-08 | 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 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107475200B (zh) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108753693A (zh) * | 2018-05-07 | 2018-11-06 | 广州沙艾生物科技有限公司 | 一种神经嵴干细胞制剂及其应用 |
| CN109266609A (zh) * | 2018-09-30 | 2019-01-25 | 广州赛莱拉干细胞科技股份有限公司 | 一种培养基及其在诱导羊膜间充质干细胞向神经样细胞分化中的应用 |
| CN109517795A (zh) * | 2018-12-13 | 2019-03-26 | 山西省农业科学院饲料兽药研究所 | 一种获取猪神经嵴干细胞的方法 |
| CN110241084B (zh) * | 2019-06-13 | 2024-08-16 | 香港中文大学深圳研究院 | 神经嵴细胞培养液、神经嵴间充质干细胞的制备方法及神经嵴间充质干细胞的应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2529003A1 (en) * | 2005-12-05 | 2007-06-05 | The Hospital For Sick Children | Neural crest stem cell and uses thereof |
| WO2013086236A2 (en) * | 2011-12-06 | 2013-06-13 | Advanced Cell Technology, Inc. | Method of directed differentiation producing corneal endothelial cells, compositions thereof, and uses thereof |
| CN106635990A (zh) * | 2016-12-16 | 2017-05-10 | 昆明医科大学 | 一种原代培养背根神经节卫星胶质细胞的方法 |
-
2017
- 2017-08-08 CN CN201710668928.0A patent/CN107475200B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2529003A1 (en) * | 2005-12-05 | 2007-06-05 | The Hospital For Sick Children | Neural crest stem cell and uses thereof |
| WO2013086236A2 (en) * | 2011-12-06 | 2013-06-13 | Advanced Cell Technology, Inc. | Method of directed differentiation producing corneal endothelial cells, compositions thereof, and uses thereof |
| CN106635990A (zh) * | 2016-12-16 | 2017-05-10 | 昆明医科大学 | 一种原代培养背根神经节卫星胶质细胞的方法 |
Non-Patent Citations (6)
| Title |
|---|
| HONG-YUN LI等.Isolation and Characterization of Neural Crest Progenitors from Adult Dorsal Root Ganglia.《Isolation and Characterization of Neural Crest Progenitors from Adult Dorsal Root Ganglia》.2007,第25卷(第8期),2053-2065. * |
| Isolation and Characterization of Neural Crest Progenitors from Adult Dorsal Root Ganglia;HONG-YUN LI等;《Isolation and Characterization of Neural Crest Progenitors from Adult Dorsal Root Ganglia》;20070524;第25卷(第8期);2053-2065 * |
| Ontogeny and Multipotency of Neural Crest-Derived Stem Cells in Mouse Bone Marrow, Dorsal Root Ganglia, and Whisker Pad;Narihito Nagoshi等;《Cell stem cell》;20080430;第2卷;392-403 * |
| Poly-L-ornithine enhances migration of neural stem_progenitor cells via promoting α-Actinin 4 binding to actin filaments;Hongfei Ge等;《Scientific Reports》;20161122;第6卷;1-11 * |
| 大鼠背根神经节源干细胞分离培养和鉴定;顾芸等;《交通医学》;20081231;第22卷(第5期);451-454 * |
| 背根节神经嵴干细胞的分离培养和分化的研究;顾芸;《中国博士学位论文全文数据库（·医药卫生科技辑）》;20120615(第6期);E059-7 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107475200A (zh) | 2017-12-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| 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 | |
| Tokumoto et al. | Comparison of efficiency of terminal differentiation of oligodendrocytes from induced pluripotent stem cells versus embryonic stem cells in vitro | |
| WO2010107159A1 (ko) | Znf281을 발현하는 제대혈 유래 줄기세포의 분리방법 및 대량배양 방법 | |
| US20200239842A1 (en) | Preparation and Expansion Methods for Human Pluripotent Stem Cell-Derived Human Retinal Pigment Epithelial Cells | |
| CN102449141A (zh) | 人脐带血来源的间充质干细胞的分离 | |
| CN101831401A (zh) | 体外诱导间充质干细胞分化为神经干细胞的方法 | |
| WO2018124605A1 (ko) | 기형종 형성이 억제된 전분화능 줄기세포 유래 신경 전구체구의 제조 방법 | |
| CN107142240A (zh) | 将消化道来源上皮细胞重编程为内胚层干/祖细胞的方法及应用 | |
| CN103013917A (zh) | 人羊膜间充质干细胞分化为神经元样细胞的诱导方法 | |
| CN104774808B (zh) | 将脐带间充质干细胞诱导分化成γ-氨基丁酸能神经元的方法 | |
| CN113151152A (zh) | 一种小鼠肺类器官的培养方法及其专用培养液 | |
| WO2018030719A1 (ko) | 유도만능줄기세포의 분화를 유도하여 각막 상피 세포를 배양하는 방법 및 시스템 | |
| KR101896803B1 (ko) | 인간 만능줄기세포로부터 중간엽 줄기세포로의 분화 유도 생산율을 증가시키는 방법 및 이에 의해 생성된 중간엽 줄기세포 | |
| CN114807034A (zh) | 一种人多能干细胞来源的Müller细胞的制备方法 | |
| WO2019198962A1 (ko) | 소변유래줄기세포로부터 제작된 유도만능줄기세포의 증식 및 분화 촉진 방법 | |
| CN107164325B (zh) | MSCs来源的少突胶质细胞的制备方法及试剂盒 | |
| CN109385399A (zh) | 一种羊水间充质干细胞分化为神经干细胞的方法 | |
| KR101204894B1 (ko) | 줄기세포의 외배엽성 세포로의 분화 방법 | |
| KR101613677B1 (ko) | 줄기 세포의 신경 전구 세포로의 분화용 조성물 및 이를 이용한 방법 | |
| CN114107206A (zh) | 基于hPSCs诱导分化得到的纹状体类器官及hPSCs诱导分化方法 | |
| CN103013919A (zh) | 胚源性神经干细胞原代细胞的分离及传代培养方法 | |
| TWI396737B (zh) | 豬胚幹細胞導向分化之方法及使用該細胞於藥物篩選之用途 | |
| EP1637590B1 (en) | Differentiated cells originating in precursor fat cells and method of acquiring the same | |
| KR100683199B1 (ko) | 신경전구세포를 콜린성 신경세포로 분화시키는 방법 및그에 사용되는 배지 | |
| EP1179046B1 (en) | Lineage-restricted precursor cells isolated from mouse neural tube and mouse embryonic stem cells |
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 |