CN109266610B - Method for promoting mesenchymal stem cells to differentiate into neurons - Google Patents
Method for promoting mesenchymal stem cells to differentiate into neurons Download PDFInfo
- Publication number
- CN109266610B CN109266610B CN201811095661.1A CN201811095661A CN109266610B CN 109266610 B CN109266610 B CN 109266610B CN 201811095661 A CN201811095661 A CN 201811095661A CN 109266610 B CN109266610 B CN 109266610B
- Authority
- CN
- China
- Prior art keywords
- mesenchymal stem
- stem cells
- human mesenchymal
- differentiation
- cells
- 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
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 35
- 210000002569 neuron Anatomy 0.000 title claims abstract description 25
- 230000001737 promoting effect Effects 0.000 title claims abstract description 17
- 210000004027 cell Anatomy 0.000 claims abstract description 61
- 230000004069 differentiation Effects 0.000 claims abstract description 40
- 230000006698 induction Effects 0.000 claims abstract description 39
- 208000020431 spinal cord injury Diseases 0.000 claims abstract description 8
- 230000008439 repair process Effects 0.000 claims abstract description 7
- 239000002609 medium Substances 0.000 claims description 50
- 238000012258 culturing Methods 0.000 claims description 22
- 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 22
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 claims description 16
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 claims description 16
- 239000001963 growth medium Substances 0.000 claims description 16
- 239000012580 N-2 Supplement Substances 0.000 claims description 11
- 229930182555 Penicillin Natural products 0.000 claims description 11
- 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 11
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 claims description 11
- 229940049954 penicillin Drugs 0.000 claims description 11
- 229930002330 retinoic acid Natural products 0.000 claims description 11
- 210000002966 serum Anatomy 0.000 claims description 11
- 229960005322 streptomycin Drugs 0.000 claims description 11
- 210000005036 nerve Anatomy 0.000 claims description 9
- 230000001939 inductive effect Effects 0.000 claims description 7
- 101000739876 Homo sapiens Brain-derived neurotrophic factor Proteins 0.000 claims description 6
- 102000051542 human BDNF Human genes 0.000 claims description 6
- 229940077456 human brain-derived neurotrophic factor Drugs 0.000 claims description 6
- 229920000729 poly(L-lysine) polymer Polymers 0.000 claims description 6
- 239000003102 growth factor Substances 0.000 claims description 5
- 210000003954 umbilical cord Anatomy 0.000 claims description 5
- 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 claims description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 4
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 4
- 101800003838 Epidermal growth factor Proteins 0.000 claims description 4
- 102000004887 Transforming Growth Factor beta Human genes 0.000 claims description 4
- 108090001012 Transforming Growth Factor beta Proteins 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 229940116977 epidermal growth factor Drugs 0.000 claims description 4
- 239000000725 suspension Substances 0.000 claims description 4
- 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 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 235000015110 jellies Nutrition 0.000 claims description 3
- 239000008274 jelly Substances 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 239000003855 balanced salt solution Substances 0.000 claims description 2
- 210000004204 blood vessel Anatomy 0.000 claims description 2
- 230000010261 cell growth Effects 0.000 claims description 2
- 230000006862 enzymatic digestion Effects 0.000 claims description 2
- 238000001976 enzyme digestion Methods 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims 1
- 239000003550 marker Substances 0.000 abstract description 20
- 102000004243 Tubulin Human genes 0.000 abstract description 11
- 108090000704 Tubulin Proteins 0.000 abstract description 11
- 208000036815 beta tubulin Diseases 0.000 abstract description 11
- 230000001537 neural effect Effects 0.000 abstract description 11
- 108010025020 Nerve Growth Factor Proteins 0.000 abstract description 3
- 102000007072 Nerve Growth Factors Human genes 0.000 abstract 1
- 239000003900 neurotrophic factor Substances 0.000 abstract 1
- 239000012091 fetal bovine serum Substances 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 7
- 238000010166 immunofluorescence Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 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 4
- 238000004458 analytical method Methods 0.000 description 4
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 3
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 3
- 239000012981 Hank's balanced salt solution Substances 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 102100032912 CD44 antigen Human genes 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
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 2
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 2
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 2
- 102000015336 Nerve Growth Factor Human genes 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229940053128 nerve growth factor Drugs 0.000 description 2
- 230000000508 neurotrophic effect Effects 0.000 description 2
- 210000004248 oligodendroglia Anatomy 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 102100022900 Actin, cytoplasmic 1 Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 208000028389 Nerve injury Diseases 0.000 description 1
- 102000008730 Nestin Human genes 0.000 description 1
- 108010088225 Nestin Proteins 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 206010033892 Paraplegia Diseases 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 230000003140 astrocytic effect Effects 0.000 description 1
- 229940125388 beta agonist Drugs 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- YRQNKMKHABXEJZ-UVQQGXFZSA-N chembl176323 Chemical compound C1C[C@]2(C)[C@@]3(C)CC(N=C4C[C@]5(C)CCC6[C@]7(C)CC[C@@H]([C@]7(CC[C@]6(C)[C@@]5(C)CC4=N4)C)CCCCCCCC)=C4C[C@]3(C)CCC2[C@]2(C)CC[C@H](CCCCCCCC)[C@]21C YRQNKMKHABXEJZ-UVQQGXFZSA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000001378 electrochemiluminescence detection Methods 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000008764 nerve damage Effects 0.000 description 1
- 210000000944 nerve tissue Anatomy 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 210000005055 nestin Anatomy 0.000 description 1
- 210000001178 neural stem cell Anatomy 0.000 description 1
- 230000004766 neurogenesis Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000000554 physical therapy Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000001228 trophic effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0619—Neurons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- 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
- 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
- C12N2501/10—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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1369—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from blood-borne mesenchymal stem cells, e.g. MSC from umbilical blood
-
- 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)
- Neurology (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Neurosurgery (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Wood Science & Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Developmental Biology & Embryology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Virology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Ophthalmology & Optometry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for promoting differentiation of mesenchymal stem cells into neurons, which comprises the following steps: 1) preparing human mesenchymal stem cells and subculturing; 2) allowing the human mesenchymal stem cells after subculture to differentiate into human mesenchymal stem cell-neurospheres in a differentiation medium; 3) differentiating the human mesenchymal stem cell-neurospheres into neurons in an induction medium. The differentiation into neurons was promoted by the addition of neurotrophic factors to the induction medium, and the results showed that the cells expressing the mature neural marker (MAP2ab) and the immature neural marker (β -tubulin III) were significantly increased relative to the blank control. The method of the invention provides a certain direction for a new treatment method for promoting the repair of spinal cord injury.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a method for promoting mesenchymal stem cells to be differentiated into neurons.
Background
Spinal Cord Injury (SCI) is a serious central system disorder, the most serious consequence of which is severe dysfunction of the limbs below the injured segment, and the resulting paraplegia can cause patients to lose their ability to self-care, which not only causes serious physical and psychological injuries to the patients, but also causes heavy burden to the family.
In the treatment of spinal cord injured patients today, it is common to reduce the extent of paralysis by means of the use of some kind of medication in combination with physical therapy. However, the quality of life of the patient cannot be improved well by such treatment.
How to repair and restore the function of central nerves after spinal cord injury is still a problem. Although the discovery of nerve growth factor brings hope to clinical drug treatment of central nerve injury and achieves certain effect, the molecular weight of the neurotrophic substances is too large to penetrate the blood brain barrier and enter central nerve tissues to exert the activity of the neurotrophic substances.
In spinal cord injury, transplantation may be an operable method for central nervous system repair. However, in order to realize transplantation therapy of cells, it is first necessary to study and evaluate the ability of cells to differentiate into neurons and to develop a method for promoting cell differentiation.
Accordingly, those skilled in the art have been devoted to developing a method of promoting differentiation of stem cells into neurons.
Disclosure of Invention
To achieve the above objects, the present invention provides a method of promoting differentiation of mesenchymal stem cells into neurons.
In a preferred embodiment of the present invention, the method comprises the steps of:
1) preparing human mesenchymal stem cells and subculturing;
2) allowing the human mesenchymal stem cells after subculture to differentiate into human mesenchymal stem cell-neurospheres in a differentiation medium;
3) differentiating the human mesenchymal stem cell-neurospheres into neurons in an induction medium.
Preferably, the step 3) is to break the human mesenchymal stem cell-neurosphere in the step 2), inoculate the broken human mesenchymal stem cell-neurosphere into a culture container which is double-coated by poly-L-lysine and laminin and contains the induction medium, and perform culture at 37 ℃ and 5% CO2Culturing and differentiating for 7-10 days.
Further, the induction medium was NB medium supplemented with 0.5umol/L all-trans retinoic acid, 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin, and 10ng/mL recombinant human BDNF.
In another preferred embodiment, the step 3) is to break up the human mesenchymal stem cell-neurosphere in the step 2), inoculate the broken human mesenchymal stem cell-neurosphere into a culture container which is double-coated by poly-L-lysine and laminin, firstly use a first induction culture medium containing BDNF to perform induced differentiation for 5-6 days,then using a second induction medium containing TGF-beta 1 to perform induced differentiation for 4-5 days, and the cells are cultured at 37 ℃ and 5% CO2And (5) culturing.
Further, after adding a second induction medium containing TGF-. beta.1, the cells were first incubated at 40 ℃ with 5% CO2Culturing for 3-4 hr, transferring to 37 deg.C, and culturing with 5% CO2And continuously culturing and differentiating for 4-5 days.
Further, the first induction medium was NB medium supplemented with 0.5umol/L all-trans retinoic acid, 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin, and 10ng/mL recombinant human BDNF; the second induction medium was NB medium supplemented with 0.5umol/L all-trans retinoic acid, 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin, and 1-5ng/L TGF-. beta.1.
Further, the step 1) is specifically to disinfect the umbilical cord with alcohol, remove blood vessels in balanced salt solution, and cut the mesenchymal tissue in the Wharton's jelly to 0.4-0.6cm3Size, centrifuging to obtain mesenchymal tissue part, and washing by using serum-free DMEM/F12 culture medium; washing the precipitate obtained after centrifugation for enzymatic digestion; counting cells of the suspension liquid after enzyme digestion, and then carrying out inoculation culture to obtain the human mesenchymal stem cells; passages were performed when cells grew to 70% -80% confluency.
Preferably, step 2) is performed using the human mesenchymal stem cells of the fourth to sixth generations of step 1).
Further, step 2) comprises: separating the human mesenchymal stem cells cultured in the step 1) by an enzyme method, and culturing the human mesenchymal stem cells in a tissue culture low-adhesion plastic flask containing a differentiation culture medium; wherein the differentiation culture medium is NB culture medium containing 20ng/mL epidermal growth factor, 20ng/mL basic cell-forming growth factor and 1:50 diluted B27; cells were incubated at 37 ℃ with 5% CO2And (4) culturing, adding fresh epidermal growth factor, basic cell growth factor and B27 every 3-4 days, and replacing the differentiation culture medium once a week.
Another aspect of the present invention also provides an application of the method for promoting differentiation of mesenchymal stem cells into neurons in studying nerve repair after spinal cord injury.
By using the method disclosed by the invention, the proportion of mesenchymal stem cells differentiated into neurons can be effectively improved. Compared with a blank control group, the proportion of cells expressing a mature nerve marker (MAP2ab) and an immature nerve marker (beta-tubulin III) in the cells subjected to induced differentiation by using an induction medium containing BDNF is remarkably improved and can respectively reach 8 +/-1.9% and 38.6 +/-2.9%. In the cells induced and differentiated by using the first induction medium containing BDNF and the second induction medium containing TGF-beta 1, the proportion of the cells expressing the mature nerve marker (MAP2ab) and the immature nerve marker (beta-tubulin III) is also obviously improved compared with that of the blank control group, and the proportion of the two positive cells is further improved compared with that of the cells induced and differentiated by using only the induction medium containing BDNF. The method of the invention provides a certain direction for a new treatment method for promoting the repair of spinal cord injury.
The conception, the specific steps, and the technical effects produced by the present invention will be further described in conjunction with the accompanying drawings to fully understand the objects, the features, and the effects of the present invention.
Drawings
Fig. 1 shows two cell morphologies in the primary culture of human mesenchymal stem cells according to a preferred embodiment of the present invention, wherein fig. 1a is a long bar shape and fig. 1b is a circular shape.
FIG. 2 is the cell morphology of the third generation passage cell of the human mesenchymal stem cell cultured to 80% fusion rate according to a preferred embodiment of the present invention;
FIG. 3 is a diagram illustrating the initial aggregation of human mesenchymal stem cells 1-2 days after re-seeding of the human mesenchymal stem cells into a neurosphere differentiation medium according to a preferred embodiment of the present invention.
Fig. 4 is a diagram showing that the human mesenchymal stem cell according to a preferred embodiment of the present invention shows a plurality of spheroids of floating cells 3 to 4 days after transformation.
Fig. 5 is the result of the 3 rd generation CD44 staining of human mesenchymal stem cells according to a preferred embodiment of the present invention.
FIG. 6 is a nesting positive marker map of neurospheres derived from human mesenchymal stem cells according to a preferred embodiment of the present invention.
Fig. 7 is a graph showing the results of immunofluorescence histochemistry assay of human mesenchymal stem cells differentiated into β -tubulin III (immature neural marker) -positive cells according to a preferred embodiment of the present invention.
Fig. 8 is a graph showing the results of immunofluorescence histochemistry method of differentiation of human mesenchymal stem cells into GFAP (astrocyte marker) -positive cells according to a preferred embodiment of the present invention.
Fig. 9 is a graph showing the results of immunofluorescence histochemistry assay of human mesenchymal stem cells differentiated into CalC (oligodendrocyte marker) positive cells according to a preferred embodiment of the present invention.
Fig. 10 is a graph showing the results of immunofluorescence histochemistry assay of human mesenchymal stem cells differentiated into MAP2ab (mature neural marker) -positive cells according to a preferred embodiment of the present invention.
FIG. 11 is a graph showing the results of protein-blot analysis of the protein levels associated with the differentiation of human mesenchymal stem cells into different neural marker-positive cells according to a preferred embodiment of the present invention.
Detailed Description
The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.
The experimental methods used in the embodiments of the present invention are conventional in the art unless otherwise specified. The reagents used in the embodiments of the present invention can be obtained by purchase, unless otherwise specified.
Human mesenchymal stem cells have many advantages of both embryonic stem cells and adult stem cells, and have advantages over other stem cells in that: the plasticity is strong; is readily obtainable by a less invasive means and is capable of rapid proliferation; immune compatibility, the patient's own human mesenchymal stem cells can be used for autologous transplantation. Therefore, the present invention uses human mesenchymal stem cells as starting materials for differentiation studies.
In neurogenesis, micro-environmental factors have a very important influence on proliferation and differentiation. Among them, BDNF is widely distributed in the developing and mature nervous system and plays an important role in the development, survival and repair of nerve cells.
The effect of differentiation of human mesenchymal stem cells into neurons was observed by the present study through the combination of different trophic factors with different conditions.
Example 1 preparation of human mesenchymal Stem cells
The umbilical cord of the newborn was obtained, placed in hanks' balanced salt solution (HBSS, Gibco, USA), and stored at 4 ℃. The umbilical cord was sterilized with 75% alcohol for 30 seconds. In HBSS, umbilical vessels are cleared. The mesenchymal tissue present in Wharton's jelly was cut to about 0.5cm3And centrifuged at 1,200rpm for 5 minutes. After centrifugation, the supernatant was removed, and the precipitated portion of the mesenchymal tissue was washed with serum-free DMEM/F12 medium (Gibco, USA), followed by centrifugation at 1,200rpm for 5 minutes. The pellet fraction was enzymatically separated using 0.075% collagenase type II (Sigma) at 37 ℃ for 18 hours, and then further digested using 0.125% trypsin/EDTA (Gibco) at 37 ℃ for 30 minutes. The suspension was neutralized using DMEM/F12 containing 10% (v/v) fetal bovine serum, and the cells in the suspension were counted under a microscope. Adjusting to 5-7 × 103Per cm2Inoculating into tissue culture flask, and culturing at 37 deg.C with 5% CO2The culture was performed while maintaining a sub-confluent state. When cells were grown to 70% confluence, the cells were incubated at 1: 3, subculture. Subsequent studies were performed using cells from fourth to sixth passage.
As shown in fig. 1a and 1b, in the primary culture of human mesenchymal stem cells, two cell morphologies, one being a long strip and one being a round one, appeared. After three to five passages, the human mesenchymal stem cells exhibit a flat cell morphology of a monolayer, and the morphology of all cells tends to be similar. FIG. 2 shows the cell morphology in the third generation subculture to 80% confluency.
Example 2 formation of human mesenchymal Stem cell-neurosphere from human mesenchymal Stem cell
Separating the fourth generation of human mesenchymal stem cells with 0.125% trypsin/0.02% EDTA, wherein the separated human mesenchymal stem cells are 2 × 105Per cm2The density of (a) was inoculated into a tissue culture low-adhesion plastic flask containing a differentiation medium. The differentiation medium was NB medium (Invitrogen) containing 20ng/mL Epidermal Growth Factor (EGF), 20ng/mL basic fibroblast growth factor (bFGF) and 1:50 dilution of B27 (Gibco).
Cells were incubated at 37 ℃ with 5% CO2And (5) culturing. Fresh growth factors were added every 3-4 days and the medium was changed once a week. Sphere formation was observed 4-5 days after initial differentiation. Cells were cultured in differentiation medium until passage.
FIG. 3 shows the cell morphology of human mesenchymal stem cells re-seeded into neurosphere differentiation medium for 1-2 days, and the cells begin to aggregate to form human mesenchymal stem cells-neurospheres. Spheres of many floating cells appeared 3-4 days after transformation (i.e., around 7 days after reseeding), as shown in fig. 4.
The passaging is performed enzymatically every 10-12 days, i.e., with 0.025% trypsin plus 0.6% glucose. These neurosphere-like structures continue to expand for 4-6 weeks (approximately 3-4 generations) before the neurons terminally differentiate.
Almost all passage 3 human mesenchymal stem cells showed strong CD44 staining (fig. 5). Neurospheres derived from human mesenchymal stem cells were confirmed by nestin antibody (fig. 6).
Example 3 further differentiation of human mesenchymal Stem cell-neurosphere (BDNF Induction)
Human mesenchymal stem cell-neurospheres cultured in example 2 were collected, broken up, and re-seeded into poly-L-lysine and laminin double-coated six-well chamber slides (numcs) containing induction medium. Cells were incubated at 37 ℃ with 5% CO2Culturing and differentiating for 7-10 days.
Wherein the induction medium was NB medium supplemented with 0.5umol/L all-trans retinoic acid (Sigma), 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin (all purchased from Gibco) and 10ng/mL recombinant human BDNF (rhBDNF, R & D Systems).
In the control group, the experimental conditions were the same as described above except that the induction medium was replaced with a control induction medium without BDNF, i.e., NB medium supplemented with 0.5umol/L all-trans retinoic acid (Sigma), 1% FBS, 5% horse serum, 1% N2 supplement, and 1% penicillin/streptomycin (all purchased from Gibco).
The differentiated cells were collected for analysis:
human mesenchymal stem cells differentiated into beta-tubulin III (immature neural marker), GFAP (astrocytic marker), CalC (oligodendrocyte marker) and MAP2ab (mature neural marker) positive cells, and the results of immunofluorescence histochemistry for positive cells of different markers were shown in FIGS. 7-10. By Hoechst33342+And (3) dyeing, quantifying various differentiated positive cells, and calculating a quantitative result by the following method: corresponding number of positive cells/Hoechst 33342+The number of stained cells was 100%, and the results are shown in Table 1.
Table 1: quantitative percentage of positive cells
| Induction medium | Beta-tubulin III | GFAP | CalC | MAP2ab |
| Control | 19.2±3.0％ | 42.8±3.8％ | 27±2％ | 4.4±1.8％ |
| BDNF Induction | 38.6±2.9％ | 15.8±4.5％ | 20.6±4.6％ | 8±1.9％ |
The results show that beta-tubulin III positive cells and MAP2ab positive cells were significantly higher produced using the neural specific induction method than using the conventional induction method.
Protein imprinting further verified:
protein imprinting is a routine procedure in the art, and in this study was treated with a primary antibody against β -tubulin III (1:500), MAP2ab (1:500), followed by a secondary antibody linked to peroxidase. Development is performed by an enhanced chemiluminescence method. Beta-agonist protein (beta-Actin) was used as an internal control. Antibodies were all from Chemicon.
The results of the protein blot verification are shown in fig. 11, and the results match the immunohistochemical results.
The results show that after BDNF is added, the proportion of cells expressing a mature nerve marker (MAP2ab) and an immature nerve marker (beta-tubulin III) is obviously improved compared with that of a blank control group, namely, the proportion of umbilical cord derived neural stem cells which are transformed into neurons is obviously improved by adding a brain derived nerve growth factor group.
Example 4 further differentiation of human mesenchymal Stem cell-neurosphere (BDNF + TGF-. beta.1 Combined Induction)
Human mesenchymal stem cell-neurospheres cultured in example 2 were collected, ground, and re-seeded into poly-L-lysine and laminin double-coated six-well chamber slides (chamber slides, NUNC) containing BDNF induction medium. Cells were incubated at 37 ℃ with 5% CO2Culturing and differentiating for 5-6 days. After that time, the user can use the device,replacing BDNF induction culture medium with TGF-beta 1 induction culture medium, firstly, at 40 deg.C and 5% CO2Culturing for 3-4 hr, transferring to 37 deg.C, and culturing with 5% CO2And continuously culturing and differentiating for 4-5 days.
Wherein the BDNF induction medium is NB medium supplemented with 0.5umol/L all-trans retinoic acid (Sigma), 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin (all purchased from Gibco) and 10ng/mL recombinant human BDNF (rhBDNF, R & D Systems);
TGF-. beta.1 Induction medium was NB medium supplemented with 0.5umol/L all-trans retinoic acid (Sigma), 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin (all purchased from Gibco), and 1-5ng/L TGF-. beta.1 (Sigma).
In the control group, the experimental conditions were the same as described above except that both BDNF-inducing medium and TGF-. beta.1-inducing medium were replaced with BDNF-free control inducing medium, i.e., NB medium supplemented with 0.5umol/L all-trans retinoic acid (Sigma), 1% FBS, 5% horse serum, 1% N2 supplement, and 1% penicillin/streptomycin (all purchased from Gibco).
The differentiated cells were collected for analysis:
the positive cells of different markers were observed by immunofluorescence histochemistry and were examined by Hoechst33342+And (3) dyeing, quantifying various differentiated positive cells, and calculating a quantitative result by the following method: corresponding number of positive cells/Hoechst 33342+The number of stained cells was 100%, and the results are shown in Table 2.
Table 2: quantitative percentage of positive cells
The results show that beta-tubulin III positive cells and MAP2ab positive cells were significantly higher produced using the neural specific induction method than using the conventional induction method. Also, the proportion of cells expressing the mature neural marker (MAP2ab) and the immature neural marker (β -tubulin III) was increased to some extent, compared to the BDNF alone induction method in example 3.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (7)
1. A method of promoting differentiation of mesenchymal stem cells into neurons, comprising the steps of:
1) preparing human mesenchymal stem cells and subculturing;
2) allowing the human mesenchymal stem cells after subculture to differentiate into human mesenchymal stem cell-neurospheres in a differentiation medium;
3) differentiating the human mesenchymal stem cell-neurospheres into neurons in an induction medium; specifically, the human mesenchymal stem cells-neurospheres in the step 2) are smashed and inoculated into a culture container which is double-coated by poly-L-lysine and laminin and contains the induction culture medium, and the culture container is heated at 37 ℃ and 5% CO2Culturing and differentiating for 7-10 days;
wherein the induction medium is NB medium supplemented with 0.5umol/L all-trans retinoic acid, 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin, and 10ng/mL recombinant human BDNF.
2. A method of promoting differentiation of mesenchymal stem cells into neurons, comprising the steps of:
1) preparing human mesenchymal stem cells and subculturing;
2) allowing the human mesenchymal stem cells after subculture to differentiate into human mesenchymal stem cell-neurospheres in a differentiation medium;
3) differentiating the human mesenchymal stem cell-neurospheres into neurons in an induction medium; specifically, the human mesenchymal stem cells-neurospheres in the step 2) are smashed and connectedPlanting into culture container with double coatings of poly-L-lysine and laminin, inducing differentiation for 5-6 days with first inducing culture medium containing BDNF, and inducing differentiation for 4-5 days with second inducing culture medium containing TGF-beta 1, and culturing at 37 deg.C with 5% CO2Culturing;
wherein the first induction medium is an NB medium supplemented with 0.5umol/L all-trans retinoic acid, 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin, and 10ng/mL recombinant human BDNF; the second induction medium was NB medium supplemented with 0.5umol/L all-trans retinoic acid, 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin, and 1-5ng/L TGF-. beta.1.
3. The method for promoting differentiation of mesenchymal stem cells into neurons according to claim 2, wherein the cells are first cultured at 40 ℃ and 5% CO after the addition of the second induction medium containing TGF- β 12Culturing for 3-4 hr, transferring to 37 deg.C, and culturing with 5% CO2And continuously culturing and differentiating for 4-5 days.
4. The method for promoting differentiation of mesenchymal stem cells into neurons according to claim 1 or 2, wherein the step 1) is specifically to sterilize umbilical cord with alcohol, remove blood vessels in balanced salt solution, and cut the mesenchymal tissue in Wharton's jelly to 0.4-0.6cm3Size, centrifuging to obtain mesenchymal tissue part, and washing by using serum-free DMEM/F12 culture medium; washing the precipitate obtained after centrifugation for enzymatic digestion; counting cells of the suspension liquid after enzyme digestion, and then carrying out inoculation culture to obtain the human mesenchymal stem cells; passages were performed when cells grew to 70% -80% confluency.
5. The method for promoting differentiation of mesenchymal stem cells into neurons according to claim 1 or 2, wherein step 2) is performed using the human mesenchymal stem cells of the fourth to sixth generations of step 1).
6. The method of claim 5A method for promoting differentiation of mesenchymal stem cells into neurons, wherein the step 2) comprises: separating the human mesenchymal stem cells cultured in the step 1) by an enzyme method, and culturing the human mesenchymal stem cells in a tissue culture low-adhesion plastic flask containing a differentiation culture medium; wherein the differentiation culture medium is NB culture medium containing 20ng/mL epidermal growth factor, 20ng/mL basic cell-forming growth factor and 1:50 diluted B27; cells were incubated at 37 ℃ with 5% CO2And (4) culturing, adding fresh epidermal growth factor, basic cell growth factor and B27 every 3-4 days, and replacing the differentiation culture medium once a week.
7. Use of the method of promoting differentiation of mesenchymal stem cells into neurons according to claim 1 or 2 for studying nerve repair after spinal cord injury.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811095661.1A CN109266610B (en) | 2018-09-19 | 2018-09-19 | Method for promoting mesenchymal stem cells to differentiate into neurons |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811095661.1A CN109266610B (en) | 2018-09-19 | 2018-09-19 | Method for promoting mesenchymal stem cells to differentiate into neurons |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109266610A CN109266610A (en) | 2019-01-25 |
| CN109266610B true CN109266610B (en) | 2021-06-25 |
Family
ID=65197675
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811095661.1A Active CN109266610B (en) | 2018-09-19 | 2018-09-19 | Method for promoting mesenchymal stem cells to differentiate into neurons |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109266610B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269887B (en) * | 2020-02-28 | 2021-11-16 | 上海市东方医院（同济大学附属东方医院） | Method for differentiating human pluripotent stem cells to ectoderm |
| CN112899234B (en) * | 2021-04-02 | 2022-08-19 | 中国人民解放军总医院第一医学中心 | Parkinson disease concomitant depression cell model and preparation method and application thereof |
| CN113278624A (en) * | 2021-05-24 | 2021-08-20 | 中山大学·深圳 | Synthetic modified Olig2mRNA and application thereof |
| WO2022257001A1 (en) * | 2021-06-08 | 2022-12-15 | 宁波金未生物科技有限公司 | Method for promoting differentiation of mesenchymal stem cells into neurons and use thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103352026A (en) * | 2013-07-24 | 2013-10-16 | 黑龙江天晴干细胞有限公司 | Method for cultivating autologous umbilical cord mesenchymal stem cells by adopting human umbilical cord blood rich platelet lysate |
| CN104004713A (en) * | 2014-05-14 | 2014-08-27 | 安沂华 | Method for inducing and culturing umbilical cord mesenchymal stem cells into nerve cells |
| CN104419661A (en) * | 2013-08-28 | 2015-03-18 | 中国医学科学院基础医学研究所 | Preparation method of neural stem cell |
| CN104774808A (en) * | 2015-03-27 | 2015-07-15 | 安沂华 | Method for inducible differentiation of umbilical cord mesenchymal stem cells into gamma-aminobutyric acid-ergic neuron |
| CN104789531A (en) * | 2015-03-27 | 2015-07-22 | 安沂华 | Method for inducing umbilical cord mesenchymal stem cells to be differentiated into dopaminergic neurons |
| CN105814196A (en) * | 2013-10-14 | 2016-07-27 | 哈达斯特医疗研究服务和开发有限公司 | Method of obtaining terminally differentiated neuronal lineages and uses thereof |
| WO2017179767A1 (en) * | 2016-04-12 | 2017-10-19 | 서울대학교 산학협력단 | Method for inducing differentiation of adipose stem cells into neural stem cells, neurons and gamma-aminobutyric acid neurons, and method for inducing differentiation of human stem cells that secrete large amounts of growth factors from human bone marrow derived mesenchymal stem cells |
-
2018
- 2018-09-19 CN CN201811095661.1A patent/CN109266610B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103352026A (en) * | 2013-07-24 | 2013-10-16 | 黑龙江天晴干细胞有限公司 | Method for cultivating autologous umbilical cord mesenchymal stem cells by adopting human umbilical cord blood rich platelet lysate |
| CN104419661A (en) * | 2013-08-28 | 2015-03-18 | 中国医学科学院基础医学研究所 | Preparation method of neural stem cell |
| CN105814196A (en) * | 2013-10-14 | 2016-07-27 | 哈达斯特医疗研究服务和开发有限公司 | Method of obtaining terminally differentiated neuronal lineages and uses thereof |
| CN104004713A (en) * | 2014-05-14 | 2014-08-27 | 安沂华 | Method for inducing and culturing umbilical cord mesenchymal stem cells into nerve cells |
| CN104774808A (en) * | 2015-03-27 | 2015-07-15 | 安沂华 | Method for inducible differentiation of umbilical cord mesenchymal stem cells into gamma-aminobutyric acid-ergic neuron |
| CN104789531A (en) * | 2015-03-27 | 2015-07-22 | 安沂华 | Method for inducing umbilical cord mesenchymal stem cells to be differentiated into dopaminergic neurons |
| WO2017179767A1 (en) * | 2016-04-12 | 2017-10-19 | 서울대학교 산학협력단 | Method for inducing differentiation of adipose stem cells into neural stem cells, neurons and gamma-aminobutyric acid neurons, and method for inducing differentiation of human stem cells that secrete large amounts of growth factors from human bone marrow derived mesenchymal stem cells |
Non-Patent Citations (3)
| Title |
|---|
| Human umbilical cord mesenchymal stem cells derived from Wharton"s jelly differentiate into cholinergic-like neurons in vitro;JunLiang等;《Neuroscience Letters》;20130104(第532期);59-63 * |
| 人脐带间充质干细胞定向诱导分化成类神经元的实验研究;周志刚等;《中国病理生理杂志》;20150209;第31卷(第02期);229-233 * |
| 碱性成纤维细胞生长因子与表皮生长因子对神经干细胞增殖与分化的影响;毕研贞等;《中国组织工程研究与临床康复》;20070218(第07期);1345-1348 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109266610A (en) | 2019-01-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109266610B (en) | Method for promoting mesenchymal stem cells to differentiate into neurons | |
| Bianco et al. | Neurotrophin 3 promotes purification and proliferation of olfactory ensheathing cells from human nose | |
| EP2099901B1 (en) | Use of a composition contaning human umbilical cord blood-derived mesenchymal stem cell for inducing differentiation and proliferation of neural precursor cells or neural stem cells to neural cells | |
| Enzmann et al. | Enhanced induction of RPE lineage markers in pluripotent neural stem cells engrafted into the adult rat subretinal space | |
| Meng et al. | Co‐transplantation of bFGF‐expressing amniotic epithelial cells and neural stem cells promotes functional recovery in spinal cord‐injured rats | |
| Wang et al. | The differentiation of rat adipose-derived stem cells into OEC-like cells on collagen scaffolds by co-culturing with OECs | |
| Represa et al. | EGF‐responsive neural stem cells are a transient population in the developing mouse spinal cord | |
| Zeng et al. | Co-transplantation of schwann cells promotes the survival and differentiation of neural stem cells transplanted into the injured spinal cord | |
| WO2019228518A1 (en) | Differentiation medium and method for preparing oligodendrocyte precursor | |
| CN100554409C (en) | Method for differentiating mesenchymal stem cells into neural cells | |
| Cameron et al. | Post-thaw viability and functionality of cryopreserved rat fetal brain cells cocultured with Sertoli cells | |
| Chen et al. | Neural progenitor cells derived from the adult rat subventricular zone: characterization and transplantation | |
| Li et al. | Induction of functional recovery by co-transplantation of neural stem cells and Schwann cells in a rat spinal cord contusion injury model | |
| WO2022257001A1 (en) | Method for promoting differentiation of mesenchymal stem cells into neurons and use thereof | |
| Schumm et al. | Improved neural progenitor cell survival when cografted with chromaffin cells in the rat striatum | |
| CN108866005A (en) | A kind of immortal human derived neural stem cell line and preparation method thereof | |
| CN112154203B (en) | Methods for efficient isolation and culture of neural stem cells | |
| CN1966080B (en) | Neural stem cell injection for treating senile dementia and Parkinson's disease | |
| US20190374681A1 (en) | Method for isolating human brain tissue-derived neural stem cell at high efficiency | |
| CN111304167B (en) | Neuron precursor cells derived from human adipose-derived stem cells, and preparation method and application thereof | |
| CN107365743B (en) | Culture method of oligodendrocyte precursor cells | |
| CN101273983B (en) | Use of neurocyte differentiated from stem cell induced by isobavachin | |
| Lucena et al. | Plasticity of Mesenchymal Stem Cells from Mouse Bone Marrow in the Presence of Conditioned Medium of the Facial Nerve and Fibroblast Growth Factor‐2 | |
| CN118086207A (en) | A method for inducing hair follicle stem cells to differentiate into neural stem cells | |
| Khazaei et al. | Transplantation of human-induced pluripotent stem cell-derived neural precursor cells for treatment of spinal cord injury |
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 | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240722 Address after: 318014 Building D, building 11, Dream Park, No. 818, east section of Kaifa Avenue, Sanjia street, Taizhou bay new area, Taizhou City, Zhejiang Province Patentee after: Zhejiang Yihe Cell Biotechnology Co.,Ltd. Country or region after: China Address before: No.485 xiangshanhe Road, Dandong Street, Xiangshan County, Ningbo City, Zhejiang Province Patentee before: NINGBO JINWEI BIOTECHNOLOGY Co.,Ltd. Country or region before: China |