CN110205283B - A method for inducing human amniotic epithelial cells to differentiate into retinal pigment epithelial cells and its application - Google Patents
A method for inducing human amniotic epithelial cells to differentiate into retinal pigment epithelial cells and its application Download PDFInfo
- Publication number
- CN110205283B CN110205283B CN201910310260.1A CN201910310260A CN110205283B CN 110205283 B CN110205283 B CN 110205283B CN 201910310260 A CN201910310260 A CN 201910310260A CN 110205283 B CN110205283 B CN 110205283B
- Authority
- CN
- China
- Prior art keywords
- epithelial cells
- cells
- human amniotic
- cell
- amniotic epithelial
- 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
- 241000282414 Homo sapiens Species 0.000 title claims abstract description 82
- 210000003425 amniotic epithelial cell Anatomy 0.000 title claims abstract description 81
- 238000000034 method Methods 0.000 title claims abstract description 74
- 210000000844 retinal pigment epithelial cell Anatomy 0.000 title claims abstract description 50
- 230000001939 inductive effect Effects 0.000 title claims abstract description 37
- 210000004027 cell Anatomy 0.000 claims abstract description 156
- 230000004069 differentiation Effects 0.000 claims abstract description 46
- 241000700159 Rattus Species 0.000 claims abstract description 34
- 239000000203 mixture Substances 0.000 claims abstract description 33
- 230000002207 retinal effect Effects 0.000 claims abstract description 30
- 208000015122 neurodegenerative disease Diseases 0.000 claims abstract description 25
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims abstract description 22
- RTKIYFITIVXBLE-QEQCGCAPSA-N trichostatin A Chemical group ONC(=O)/C=C/C(/C)=C/[C@@H](C)C(=O)C1=CC=C(N(C)C)C=C1 RTKIYFITIVXBLE-QEQCGCAPSA-N 0.000 claims abstract description 17
- RTKIYFITIVXBLE-UHFFFAOYSA-N Trichostatin A Natural products ONC(=O)C=CC(C)=CC(C)C(=O)C1=CC=C(N(C)C)C=C1 RTKIYFITIVXBLE-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229960003966 nicotinamide Drugs 0.000 claims abstract description 10
- 235000005152 nicotinamide Nutrition 0.000 claims abstract description 10
- 239000011570 nicotinamide Substances 0.000 claims abstract description 10
- 230000006698 induction Effects 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims description 38
- 239000008055 phosphate buffer solution Substances 0.000 claims description 35
- 210000001691 amnion Anatomy 0.000 claims description 26
- 238000002360 preparation method Methods 0.000 claims description 21
- 208000002780 macular degeneration Diseases 0.000 claims description 17
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 15
- 206010064930 age-related macular degeneration Diseases 0.000 claims description 15
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 14
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 13
- 239000007924 injection Substances 0.000 claims description 13
- 238000002347 injection Methods 0.000 claims description 13
- 241001465754 Metazoa Species 0.000 claims description 12
- 239000006143 cell culture medium Substances 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 9
- 239000000872 buffer Substances 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 8
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 7
- 210000001136 chorion Anatomy 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 210000002966 serum Anatomy 0.000 claims description 7
- 229940054269 sodium pyruvate Drugs 0.000 claims description 7
- 208000007014 Retinitis pigmentosa Diseases 0.000 claims description 6
- 102000004142 Trypsin Human genes 0.000 claims description 6
- 108090000631 Trypsin Proteins 0.000 claims description 6
- 239000001569 carbon dioxide Substances 0.000 claims description 6
- 238000004113 cell culture Methods 0.000 claims description 6
- 239000003797 essential amino acid Substances 0.000 claims description 6
- 235000020776 essential amino acid Nutrition 0.000 claims description 6
- 210000002826 placenta Anatomy 0.000 claims description 6
- 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 6
- 239000012588 trypsin Substances 0.000 claims description 6
- 238000007796 conventional method Methods 0.000 claims description 5
- 210000005059 placental tissue Anatomy 0.000 claims description 5
- 229960002833 aflibercept Drugs 0.000 claims description 4
- 108010081667 aflibercept Proteins 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 4
- 239000003937 drug carrier Substances 0.000 claims description 4
- 230000003203 everyday effect Effects 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- NWNOWCGAHDFNFJ-RUCXOUQFSA-N (2s)-2,5-bis(azanyl)-5-oxidanylidene-pentanoic acid Chemical compound OC(=O)[C@@H](N)CCC(N)=O.OC(=O)[C@@H](N)CCC(N)=O NWNOWCGAHDFNFJ-RUCXOUQFSA-N 0.000 claims description 3
- 208000010412 Glaucoma Diseases 0.000 claims description 3
- 241000282412 Homo Species 0.000 claims description 3
- 229940120638 avastin Drugs 0.000 claims description 3
- 210000004204 blood vessel Anatomy 0.000 claims description 3
- 238000005138 cryopreservation Methods 0.000 claims description 3
- 210000000981 epithelium Anatomy 0.000 claims description 3
- 229960003876 ranibizumab Drugs 0.000 claims description 3
- 229960005322 streptomycin Drugs 0.000 claims description 3
- 210000003954 umbilical cord Anatomy 0.000 claims description 3
- 241000283690 Bos taurus Species 0.000 claims description 2
- 241000282693 Cercopithecidae Species 0.000 claims description 2
- 108060005980 Collagenase Proteins 0.000 claims description 2
- 102000029816 Collagenase Human genes 0.000 claims description 2
- 206010061818 Disease progression Diseases 0.000 claims description 2
- 108010052014 Liberase Proteins 0.000 claims description 2
- 102000004882 Lipase Human genes 0.000 claims description 2
- 108090001060 Lipase Proteins 0.000 claims description 2
- 239000004367 Lipase Substances 0.000 claims description 2
- 241000124008 Mammalia Species 0.000 claims description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 2
- 241001494479 Pecora Species 0.000 claims description 2
- 108090000284 Pepsin A Proteins 0.000 claims description 2
- 102000057297 Pepsin A Human genes 0.000 claims description 2
- 108091005804 Peptidases Proteins 0.000 claims description 2
- 102000035195 Peptidases Human genes 0.000 claims description 2
- 239000004365 Protease Substances 0.000 claims description 2
- 238000013475 authorization Methods 0.000 claims description 2
- 210000000601 blood cell Anatomy 0.000 claims description 2
- 229950000025 brolucizumab Drugs 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 claims description 2
- 229960002424 collagenase Drugs 0.000 claims description 2
- 102000038379 digestive enzymes Human genes 0.000 claims description 2
- 108091007734 digestive enzymes Proteins 0.000 claims description 2
- 230000005750 disease progression Effects 0.000 claims description 2
- 230000006862 enzymatic digestion Effects 0.000 claims description 2
- 238000006911 enzymatic reaction Methods 0.000 claims description 2
- 235000019421 lipase Nutrition 0.000 claims description 2
- 238000010297 mechanical methods and process Methods 0.000 claims description 2
- 229940111202 pepsin Drugs 0.000 claims description 2
- 239000002504 physiological saline solution Substances 0.000 claims description 2
- STWNGMSGPBZFMX-UHFFFAOYSA-N pyridine-3-carboxamide Chemical compound NC(=O)C1=CC=CN=C1.NC(=O)C1=CC=CN=C1 STWNGMSGPBZFMX-UHFFFAOYSA-N 0.000 claims description 2
- 241000282472 Canis lupus familiaris Species 0.000 claims 1
- 241000283086 Equidae Species 0.000 claims 1
- 241000699670 Mus sp. Species 0.000 claims 1
- 210000001508 eye Anatomy 0.000 abstract description 19
- 238000001514 detection method Methods 0.000 abstract description 14
- 108090000623 proteins and genes Proteins 0.000 abstract description 9
- 101000729271 Homo sapiens Retinoid isomerohydrolase Proteins 0.000 abstract description 6
- 102100031176 Retinoid isomerohydrolase Human genes 0.000 abstract description 6
- 102000013760 Microphthalmia-Associated Transcription Factor Human genes 0.000 abstract description 5
- 108010050345 Microphthalmia-Associated Transcription Factor Proteins 0.000 abstract description 5
- 208000030533 eye disease Diseases 0.000 abstract description 5
- 108050002823 Bestrophin Proteins 0.000 abstract description 4
- 102000012304 Bestrophin Human genes 0.000 abstract description 4
- 101000620359 Homo sapiens Melanocyte protein PMEL Proteins 0.000 abstract description 4
- 102100022430 Melanocyte protein PMEL Human genes 0.000 abstract description 4
- 206010057430 Retinal injury Diseases 0.000 abstract description 3
- 230000004304 visual acuity Effects 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 31
- 238000005406 washing Methods 0.000 description 26
- 210000005252 bulbus oculi Anatomy 0.000 description 22
- 210000003583 retinal pigment epithelium Anatomy 0.000 description 21
- 210000001519 tissue Anatomy 0.000 description 17
- 238000000338 in vitro Methods 0.000 description 15
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 14
- 239000001993 wax Substances 0.000 description 14
- 230000014509 gene expression Effects 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 13
- 230000029087 digestion Effects 0.000 description 12
- 201000010099 disease Diseases 0.000 description 12
- 238000007710 freezing Methods 0.000 description 11
- 230000008014 freezing Effects 0.000 description 11
- 210000001525 retina Anatomy 0.000 description 11
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 10
- 239000012188 paraffin wax Substances 0.000 description 10
- 210000001671 embryonic stem cell Anatomy 0.000 description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 8
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 8
- 239000012091 fetal bovine serum Substances 0.000 description 8
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 8
- 108010062347 HLA-DQ Antigens Proteins 0.000 description 7
- 108010058597 HLA-DR Antigens Proteins 0.000 description 7
- 102000006354 HLA-DR Antigens Human genes 0.000 description 7
- 229930040373 Paraformaldehyde Natural products 0.000 description 7
- 230000018044 dehydration Effects 0.000 description 7
- 238000006297 dehydration reaction Methods 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- 229920002866 paraformaldehyde Polymers 0.000 description 7
- 238000002054 transplantation Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 230000004438 eyesight Effects 0.000 description 6
- 238000010166 immunofluorescence Methods 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 230000000638 stimulation Effects 0.000 description 6
- 239000011534 wash buffer Substances 0.000 description 6
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 5
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 5
- 108010019160 Pancreatin Proteins 0.000 description 5
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 5
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 230000006378 damage Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 210000003754 fetus Anatomy 0.000 description 5
- 210000001654 germ layer Anatomy 0.000 description 5
- 210000000110 microvilli Anatomy 0.000 description 5
- 229940055695 pancreatin Drugs 0.000 description 5
- 239000012089 stop solution Substances 0.000 description 5
- 230000000007 visual effect Effects 0.000 description 5
- 239000008096 xylene Substances 0.000 description 5
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 4
- 201000004569 Blindness Diseases 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- 206010057249 Phagocytosis Diseases 0.000 description 4
- 102100035846 Pigment epithelium-derived factor Human genes 0.000 description 4
- 235000011114 ammonium hydroxide Nutrition 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 239000012154 double-distilled water Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 4
- 230000008782 phagocytosis Effects 0.000 description 4
- 210000000608 photoreceptor cell Anatomy 0.000 description 4
- 108090000102 pigment epithelium-derived factor Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 239000008399 tap water Substances 0.000 description 4
- 235000020679 tap water Nutrition 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 208000006550 Mydriasis Diseases 0.000 description 3
- 241001085205 Prenanthella exigua Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000010009 beating Methods 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 210000003785 decidua Anatomy 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000002427 irreversible effect Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 230000004770 neurodegeneration Effects 0.000 description 3
- 238000012014 optical coherence tomography Methods 0.000 description 3
- 230000008823 permeabilization Effects 0.000 description 3
- 210000002993 trophoblast Anatomy 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000206602 Eukaryota Species 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 101001078886 Homo sapiens Retinaldehyde-binding protein 1 Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 238000003559 RNA-seq method Methods 0.000 description 2
- 102100028001 Retinaldehyde-binding protein 1 Human genes 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000519995 Stachys sylvatica Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 235000011089 carbon dioxide Nutrition 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 238000002659 cell therapy Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000007621 cluster analysis Methods 0.000 description 2
- 239000013065 commercial product Substances 0.000 description 2
- 230000001079 digestive effect Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 230000007831 electrophysiology Effects 0.000 description 2
- 238000002001 electrophysiology Methods 0.000 description 2
- 210000005081 epithelial layer Anatomy 0.000 description 2
- DZGCGKFAPXFTNM-UHFFFAOYSA-N ethanol;hydron;chloride Chemical compound Cl.CCO DZGCGKFAPXFTNM-UHFFFAOYSA-N 0.000 description 2
- 210000000887 face Anatomy 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 208000017532 inherited retinal dystrophy Diseases 0.000 description 2
- 210000004692 intercellular junction Anatomy 0.000 description 2
- MLFHJEHSLIIPHL-UHFFFAOYSA-N isoamyl acetate Chemical compound CC(C)CCOC(C)=O MLFHJEHSLIIPHL-UHFFFAOYSA-N 0.000 description 2
- QWTDNUCVQCZILF-UHFFFAOYSA-N isopentane Chemical compound CCC(C)C QWTDNUCVQCZILF-UHFFFAOYSA-N 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108090000468 progesterone receptors Proteins 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000007873 sieving Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 238000009168 stem cell therapy Methods 0.000 description 2
- 238000009580 stem-cell therapy Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 210000001578 tight junction Anatomy 0.000 description 2
- 230000004393 visual impairment Effects 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- NCYCYZXNIZJOKI-IOUUIBBYSA-N 11-cis-retinal Chemical compound O=C/C=C(\C)/C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-IOUUIBBYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101100257359 Caenorhabditis elegans sox-2 gene Proteins 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 238000000116 DAPI staining Methods 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102100021238 Dynamin-2 Human genes 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 102100028072 Fibroblast growth factor 4 Human genes 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
- 102100028970 HLA class I histocompatibility antigen, alpha chain E Human genes 0.000 description 1
- 102100028967 HLA class I histocompatibility antigen, alpha chain G Human genes 0.000 description 1
- 108010010378 HLA-DP Antigens Proteins 0.000 description 1
- 102000015789 HLA-DP Antigens Human genes 0.000 description 1
- 108010024164 HLA-G Antigens Proteins 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 206010019899 Hereditary retinal dystrophy Diseases 0.000 description 1
- 101000817607 Homo sapiens Dynamin-2 Proteins 0.000 description 1
- 101001060274 Homo sapiens Fibroblast growth factor 4 Proteins 0.000 description 1
- 101000986085 Homo sapiens HLA class I histocompatibility antigen, alpha chain E Proteins 0.000 description 1
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 1
- 101000976622 Homo sapiens Zinc finger protein 42 homolog Proteins 0.000 description 1
- 101150062179 II gene Proteins 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 108700036276 KH902 fusion Proteins 0.000 description 1
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 206010025421 Macule Diseases 0.000 description 1
- 208000035719 Maculopathy Diseases 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 101100257363 Mus musculus Sox2 gene Proteins 0.000 description 1
- 101000976618 Mus musculus Zinc finger protein 42 Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 101000574028 Rattus norvegicus Progesterone receptor Proteins 0.000 description 1
- 201000007737 Retinal degeneration Diseases 0.000 description 1
- 102100040756 Rhodopsin Human genes 0.000 description 1
- 108090000820 Rhodopsin Proteins 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 1
- 102000000591 Tight Junction Proteins Human genes 0.000 description 1
- 108010002321 Tight Junction Proteins Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 241000223259 Trichoderma Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 206010047571 Visual impairment Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 102100023550 Zinc finger protein 42 homolog Human genes 0.000 description 1
- SFCVEUVVOJFYSX-UHFFFAOYSA-J [Pb+2].C(CC(O)(C(=O)[O-])CC(=O)[O-])(=O)[O-].C(C)(=O)[O-].[U+6] Chemical compound [Pb+2].C(CC(O)(C(=O)[O-])CC(=O)[O-])(=O)[O-].C(C)(=O)[O-].[U+6] SFCVEUVVOJFYSX-UHFFFAOYSA-J 0.000 description 1
- ABUBSBSOTTXVPV-UHFFFAOYSA-H [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O Chemical compound [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O ABUBSBSOTTXVPV-UHFFFAOYSA-H 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 210000003484 anatomy Anatomy 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 230000001772 anti-angiogenic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229960000397 bevacizumab Drugs 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 210000004703 blastocyst inner cell mass Anatomy 0.000 description 1
- 210000000625 blastula Anatomy 0.000 description 1
- 210000004155 blood-retinal barrier Anatomy 0.000 description 1
- 230000004378 blood-retinal barrier Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 229950005748 conbercept Drugs 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000012303 cytoplasmic staining Methods 0.000 description 1
- 230000004300 dark adaptation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 210000001047 desmosome Anatomy 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- AFABGHUZZDYHJO-UHFFFAOYSA-N dimethyl butane Natural products CCCC(C)C AFABGHUZZDYHJO-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 230000002357 endometrial effect Effects 0.000 description 1
- 210000004696 endometrium Anatomy 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 210000002336 epiblast cell Anatomy 0.000 description 1
- 210000002219 extraembryonic membrane Anatomy 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000009395 genetic defect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 208000005252 hepatitis A Diseases 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 235000015243 ice cream Nutrition 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 229940117955 isoamyl acetate Drugs 0.000 description 1
- 229960003299 ketamine Drugs 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 208000018769 loss of vision Diseases 0.000 description 1
- 231100000864 loss of vision Toxicity 0.000 description 1
- 229940076783 lucentis Drugs 0.000 description 1
- 210000001365 lymphatic vessel Anatomy 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 101710135378 pH 6 antigen Proteins 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 108091008695 photoreceptors Proteins 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000004258 retinal degeneration Effects 0.000 description 1
- 230000004283 retinal dysfunction Effects 0.000 description 1
- 230000004243 retinal function Effects 0.000 description 1
- 239000000790 retinal pigment Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000002660 stem cell treatment Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000000352 supercritical drying Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 208000006379 syphilis Diseases 0.000 description 1
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 208000029257 vision disease Diseases 0.000 description 1
- 230000004382 visual function Effects 0.000 description 1
- 210000004127 vitreous body Anatomy 0.000 description 1
Images
Classifications
-
- 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
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/06—Antiglaucoma agents or miotics
-
- 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
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
-
- 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
- C12N5/0621—Eye cells, e.g. cornea, iris pigmented 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
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
-
- 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
- C12N2501/065—Modulators of histone acetylation
-
- 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
- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
- C12N2506/025—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells from extra-embryonic cells, e.g. trophoblast, placenta
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Ophthalmology & Optometry (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Gynecology & Obstetrics (AREA)
- Reproductive Health (AREA)
- Dermatology (AREA)
- Immunology (AREA)
- Virology (AREA)
- Epidemiology (AREA)
- Pregnancy & Childbirth (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明涉及一种诱导人羊膜上皮细胞向视网膜色素上皮细胞分化的方法及其在治疗视网膜退行性疾病中的应用，该方法为向人羊膜上皮细胞中添加含有10‑100mM烟酰胺和1‑10μM曲古抑菌素A的诱导组合物，诱导产生的细胞高表达MITF、PMEL17、RPE65、Bestrophin等视网膜色素上皮细胞的典型基因。将利用该方法诱导产生的细胞注射到RCS大鼠视网膜下腔，通过ERG以及眼底检测发现大鼠眼睛电生理信号明显增强，眼底结构明显改善，眼球切片HE染色显示其视网膜厚度明显增加，经过上述方案诱导分化后的细胞经注射后对大鼠的视力有一定程度的恢复。本发明中的诱导复合物能够用于人羊膜上皮细胞向视网膜色素上皮细胞的分化以及治疗视网膜损伤相关的眼部疾病。The invention relates to a method for inducing the differentiation of human amniotic epithelial cells into retinal pigment epithelial cells and its application in treating retinal degenerative diseases. The method comprises adding 10-100 mM nicotinamide and 1-10 μM The induction composition of trichostatin A, the induced cells highly express typical genes of retinal pigment epithelial cells such as MITF, PMEL17, RPE65, Bestrophin, etc. The cells induced by this method were injected into the subretinal space of RCS rats, and ERG and fundus detection showed that the electrophysiological signals of the rats' eyes were significantly enhanced, and the fundus structure was significantly improved. After the protocol-induced differentiation cells were injected, the visual acuity of the rats was restored to a certain extent. The induced complex in the present invention can be used for the differentiation of human amniotic epithelial cells into retinal pigment epithelial cells and the treatment of retinal damage-related eye diseases.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for inducing differentiation of human amniotic epithelial cells into retinal pigment epithelial cells and application of the method in treatment of retinal degenerative diseases.
Technical Field
Retinal neurodegenerative diseases have become the leading cause of irreversible blindness worldwide, affecting the visual health of millions of people, such as Retinitis Pigmentosa (RP), Age-related Macular Degeneration (AMD), glaucoma, and the like. The disease is mainly characterized in that Retinal Pigment Epithelium (RPE) cells and Photoreceptor cells (Photoreceptor cells) are progressively damaged and lost to cause Retinal dysfunction, and finally, progressive irreversible visual function of a patient is reduced or lost. However, with the development of technology in recent years, methods such as gene therapy, stem cell therapy, and artificial vision have provided therapeutic possibilities for the above-mentioned diseases.
Due to the special physiological and anatomical features of the eye, the eye becomes a precedent of the leading edge treatment technology. First, since the presence of the blood-retinal barrier makes the eye a relatively independent organ, foreign grafts do not readily elicit a large immune response in the body, with some level of immune privilege; secondly, the volume of the eyeball is relatively small, so the number of cells required for treatment is small; thirdly, the eyeballs are positioned on the superficial layer of the body, and most of eye operations are visual operations, which is very beneficial to the implementation of related therapies; in addition, the anatomical structure and the physiological function of the eye are easy to observe and analyze, the structure of each tissue of the eye can be directly observed by means of a slit lamp microscope, an ultrasonic instrument, a fundus camera, optical coherence tomography and the like, and the visual functional change can be recorded and objectively evaluated in real time by applying the technologies of visual electrophysiology, fundus fluorescence shadow and the like; finally, the two eyes can be used as the self-contrast of the experiment, so that the treatment effect can be conveniently verified.
AMD is a retinal degenerative disease caused by a variety of pathogenic factors including heredity, lifestyle, and environment, and is the leading blindness-causing eye in the western developed countries. With the aggravation of the aging of the population in China and the prolongation of the life of people, the incidence of AMD in China is increased year by year, the number of people with disease is more and more, the eyesight of people over 50 years old is seriously damaged, and the eye disease is a blindness-causing eye disease which seriously damages the eyesight of people over 50 years old.
AMD is a disease in which irreversible vision is reduced or lost, primarily caused by damage to the retinal pigment epithelium (RPE cells) and retinal degeneration. The site of attack is a particularly important structure in the fundus oculi-the macula. anti-VEGF preparations for treating macular degeneration on the market at present comprise ranibizumab (Lucentis), bevacizumab (Avastin), Aflibercept (Aflibercept) and combaiccept (Conbercept), and angiogenesis is inhibited by combining VEGF to delay the progress of diseases, but the medicines can not radically treat AMD diseases.
Stem cell therapy for AMD may be a better option. Indeed, embryonic stem cell treatment of AMD dates back to 2012 at the earliest, and researchers used differentiation of embryonic stem cells into RPE transplantation to treat two people, AMD and macular dystrophy, respectively, with some effect (Akon Higuchi et al trends in biotechnology.2017). By 2017, Induced pluripotent Stem Cells (iPS), similar to embryonic Stem Cells, were also used for the treatment of AMD, a work published in the well-known journal of medicine, new england journal (autogonus Induced Stem-Cell-Derived regenerative Cells for mechanical generation.2017). However, embryonic stem cells have problems of limited sources, ethics, and the like, and induced pluripotent stem cells (iPS) have problems of complicated culture techniques, and the like.
Disclosure of Invention
Aiming at the defects of the prior art and the method, the invention provides a safe, economic and efficient method for inducing the differentiation of the human amniotic epithelial cells to the retinal pigment epithelial cells in vitro, and simultaneously, the induced cells are used for treating the retinal neurodegenerative diseases, thereby providing a new treatment scheme for the diseases.
AMD, a typical case of retinal neurodegenerative disease, is a disease whose cause is visual impairment by apoptosis of retinal pigment epithelium on the retina, which affects normal retinal function, and thus the present invention uses this disease as a disease model for treatment. Meanwhile, an RCS (the radial College of surgery) rat is selected as an animal model for treatment, the RCS rat is an animal model with congenital hereditary Retinal degeneration and is widely applied to the research of hereditary Retinal dystrophy diseases, and the genetic defect of the RCS rat causes that a Retinal Pigment epithelial layer (RPE) cannot Phagocytose Outer Segments (POS) secreted by Photoreceptor cells, so that metabolite accumulation is caused, and the function of Retinal-related cells is disordered, thereby causing visual injury. The disease progresses in RCS rats in the form of retinal morphological changes observed at 12 th birth, photoreceptor cells begin to decrease at 18 th day until complete loss of vision in 3 months after birth, so the cell injection treatment is selected at 18 th to 21 th day after birth of RCS rats.
In addition, the invention selects human amniotic epithelial cells as objects for induced differentiation, and the human amniotic epithelial cells are derived from the amnion on the post-partum waste placenta of the newborn. The placenta consists of the amniotic membrane, the chorion (fetal part) and the decidua (maternal part), wherein the decidua is derived from the endometrium of the mother, and the amniotic membrane and the chorion are derived from the fetus, wherein the chorion is the side close to the decidua of the mother, and the amniotic membrane is positioned on the surface of the chorion of the fetus, is connected with the umbilical cord and the skin of the baby, wraps amniotic fluid and the fetus, is also called the fetal membrane, is an early product of embryonic development closely related to the developing fetus, and is an important tissue for the material communication between the mother and the fetus.
Genetically, amniotic epithelial cells are generated from the inner cell mass formed when the fertilized egg begins to develop. Morulae formed early in zygote development, not before implantation into the uterus (3-4 days after fertilization), and consisted of approximately 100 more cells. The outer tens of cells become trophoblasts and eventually form chorions, while the inner tens of cells are the inner cell population and develop into embryos and amnions in the future. Approximately 8 days after fertilization, the human blastocyst is partially implanted into the endometrial stroma. The outer layer of blastula (trophoblast) differentiates into two layers embedded within the stroma, and the inner cell mass also differentiates into two layers: the epiblast and the hypoblast. The epiblast is the source of all three germ layers, eventually forming a developing embryo. Meanwhile, amniotic cavity occurs within the epiblast, and epiblast cells adjacent to the trophoblast are called amniotic cells. The amniotic cavity expands with time, and forms a layer with a thickness of about 0.02-0.05mm and an area of about 700-2The amnion has no blood vessel, nerve, muscle and lymphatic vessel, has certain toughness and elasticity, and is divided into five layers from inside to outside. The amnion comprises five layers of amnion epithelial layer (epithelium), basement membrane (basement membrane), compact layer (compact layer), fibroblast layer (fibroblast layer) and sponge layer (sponge layer), wherein the innermost layer of the amnion faces to amniotic cavity and wraps amniotic fluidThe cells are amniotic epithelial cells.
The amniotic epithelial cells and the embryonic stem cells have the same development tissue source and are differentiated from blastocyst inner cell masses from the development of fertilized eggs to the 8 th day, so that the characteristics of the embryonic stem cells are retained, and the amniotic epithelial cells and the embryonic stem cells have multipotential dryness and stronger differentiation capacity and plasticity. hAECs typically express a variety of embryonic stem cell-associated markers, including stage specific embryonic antigen-3 (SSEA-3), stage specific embryonic antigen-4 (SSEA-4), tumor rejection antigen-60 (TRA 1-60), and tumor rejection antigen-81 (TRA 1-81). Meanwhile, specific transcription factors OCT-4, SOX-2, Nanog, FGF4 and REX-1 of the pluripotent stem cells are also expressed.
In practical application, the human amniotic epithelial cells are derived from the amniotic membrane on the postpartum waste placenta of the newborn, the source is wide, the materials are easy to obtain, the price is low, no application limitation exists, no harm is caused to the baby or the mother, and obviously, the ethical problem caused by the application of the embryonic stem cells does not exist. Meanwhile, the human amniotic epithelial cells have the capacity of regulating in-vivo and in-vitro immune reactions, and researches show that the amniotic epithelial cells have the expression of HLA-Ib (HLA-E, HLA-G); MHC II gene: HLA-DP, DQ, DR are low or not expressed; does not express beta2Microglobulin; co-stimulatory factors CD80, CD86 were not expressed. Since hAECs secrete a variety of immunomodulatory factors, anti-angiogenic proteins, or anti-inflammatory factor-related proteins when cultured in vitro, human amniotic epithelial cells can be considered as immune privileged cells without antigen presentation, and can reduce the source of immune cells after transplantation, avoiding the occurrence of immune rejection. Based on these considerations, human amniotic epithelial cells are the most suitable source and type of seed cells for clinical cell therapy and regenerative medicine among various sources and kinds of pluripotent cells.
Accordingly, in one aspect, the present invention provides a method of inducing differentiation of human amniotic epithelial cells into retinal pigment epithelial cells, the method comprising the steps of:
(1) culturing human amniotic epithelial cells under appropriate conditions for 12-48 hr;
(2) adding an inducing composition to continue culturing for 5-20 days to induce the differentiation of the human amniotic epithelial cells into retinal pigment epithelial cells, wherein the inducing composition is a cell culture medium containing 10-100mM nicotinamide and 1-10 mu M trichostatin A.
In the above method, the type of the cell culture medium used in the present invention is not limited, and any medium may be used as long as it can be used for culturing human amniotic epithelial cells, and commercially available media that are already in the market may be used as they are or may be prepared by themselves. Preferred media include DMEM media and NPBM media.
In another embodiment of the present invention, the human amniotic epithelial cells in step (1) are P0 or P1 human amniotic epithelial cells.
In another embodiment of the present invention, the P0 or P1 human amniotic epithelial cells in step (1) are according to 104-106The amount of cells per well was inoculated in a culture vessel and cultured for 12 to 30 hours, after which the inducing composition was added. More preferably 1X 105-5×105The cell amount per well is seeded in a well plate and cultured for 12-24 hours, after which the inducing composition is added.
In another embodiment of the present invention, in the step (2), the cell culture solution is cultured for 10 to 20 days by adding the inducing composition, and the differentiation of the human amniotic epithelial cells into retinal pigment epithelial cells is induced. Wherein, the cell culture medium in the inducing composition is DMEM medium. The nicotinamide (Nicotinamide) CAS number 98-92-0, the commercial product can be selected from Sigma (cat.no. N3376), trichostatin A (Trichostatin A) CAS number 58880-19-6, and the commercial product can be selected from APExBIO (cat.no. A8183).
In another embodiment of the present invention, the inducing composition in step (2) is a cell culture medium containing 30-70mM nicotinamide and 3-7. mu.M trichostatin A.
In a preferred embodiment of the present invention, the inducing composition in the step (2) is prepared by the following method: adding nicotinamide (Nicotinaid) with the final concentration of 10-100mM and trichostatin A (Trichostatin A) with the final concentration of 1-10 mu M into a DMEM/F121: 1(1X) culture medium containing 15% of KSR (knock-out Serum replacement), 2mM L-glutamine (L-glutamine), 1mM non-essential amino acid (non-essential amino acid), 1mM sodium pyruvate (sodium pyruvate), 100units/ml penillin and 100 mu g/ml streptomycin to obtain the composition for inducing the differentiation of the human amniotic epithelial cells to the retinal pigment epithelial cells.
In a preferred embodiment of the present invention, there is provided a method for inducing differentiation of human amniotic epithelial cells into retinal pigment epithelial cells, the method comprising the steps of:
(1) get P0Or P1Human amniotic epithelial cells at 1 × 105-5×105The cell amount of the cells/hole is inoculated in a hole plate and cultured for 24-48 hours;
(2) after the induction composition is added, the solution is changed every day, and the mixture is placed in an incubator containing 5.5 percent of carbon dioxide at 37 ℃ for culturing for 10 to 14 days to induce the human amniotic epithelial cells to be differentiated into retinal pigment epithelial cells, wherein the induction composition is a cell culture medium containing 10 to 100mM nicotinamide and 1 to 10 mu M trichostatin A.
In another embodiment of the invention, a method is provided for isolating amniotic epithelial cells from amniotic tissue, the method comprising the steps of:
(1) mechanically separating the placenta tissue to obtain an amniotic membrane;
(2) and digesting the washed amniotic membrane by using digestive enzyme, and centrifuging the digested liquid to obtain the human amniotic epithelial cells.
In one embodiment of the invention, the amniotic epithelial cells are derived from a human. The amniotic membrane may be isolated from isolated human placenta, washed with physiological buffer to remove blood cells, and mechanically removed of residual chorion and blood vessels. Isolation refers to the removal of cells from a tissue sample and separation from additional tissue. Single cells are isolated from intact human amniotic epithelial tissue using any conventional technique or method, including mechanical force (cutting or shearing force), enzymatic digestion with one or a combination of proteases, such as collagenase, trypsin, lipase, liberase and pepsin, or a combination of mechanical and enzymatic methods.
In a preferred embodiment of the invention, the human amniotic membrane is obtained by mechanically separating placenta tissue from a healthy parturient after cesarean section, in response to the consent of the parturient.
In another preferred embodiment of the present invention, the human amniotic epithelial cells obtained in step (2) above may be cultured under the following conditions: at 1 × 106-1×108Inoculating cells into a culture dish according to the density of each cell/flat plate, placing the culture dish in a carbon dioxide incubator for culture, replacing culture solution after human amniotic epithelial cells are attached to the wall, digesting the cells after the flat plates are full of the cells, and performing cryopreservation for subsequent induced differentiation.
The concentration of isolated human amniotic epithelial cells or the aforementioned population of viable cells that direct the induced differentiation of amniotic epithelial cells into retinal pigment epithelial cells may be performed by other methods known to those skilled in the art. These post-processing washing/concentration steps may be performed separately or simultaneously. In addition to the above methods, the viable cell population can be further purified or enriched after cell washing or after culture to reduce both contaminating and dead cells. Separation of cells in suspension can be achieved by the following techniques: buoyant density sedimentation centrifugation, differential adhesion to and elution from solid phases, immunomagnetic beads, fluorescent laser cell sorting (FACS), or other techniques. Examples of these different techniques and apparatus for performing these techniques can be found in the prior art and in commercial products.
In another aspect of the invention, the invention discloses the use of the retinal pigment epithelial cells or cell preparations thereof induced by the human amniotic epithelial cells for treating and/or improving retinal degenerative diseases.
In one aspect, the invention discloses application of a retinal pigment epithelial cell induced and differentiated by a human amniotic epithelial cell or a cell preparation thereof in preparing a medicament for treating and/or improving retinal degenerative diseases. The retinal pigment epithelial cells or cell preparations thereof induced to differentiate by the human amniotic epithelial cells can be used alone or in combination with other medicines for treating and/or improving the retinal degenerative diseases. An effective dose refers to an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount for a particular subject may vary depending on a number of factors, such as the disease to be treated, the overall health of the patient, the method of administration and the dosage and severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.
In another embodiment of the present invention, the animal having retinal degenerative disease is a mammal. In a more preferred embodiment, the animal is a cow, horse, sheep, monkey, dog, rat, mouse, rabbit or human. In a most preferred embodiment, the animal having a retinal degenerative disease is a human.
In another preferred embodiment of the present invention, the retinal degenerative disease includes Retinitis Pigmentosa (RP), Age-related Macular Degeneration (AMD), glaucoma, and the like.
In another embodiment of the invention, the cell preparation comprises retinal pigment epithelial cells induced to differentiate by human amniotic epithelial cells and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier according to the present invention refers to a substance having a suitable benefit/risk ratio, e.g. a pharmaceutically acceptable solvent, suspension or excipient, suitable for use in humans and/or animals, without undue adverse side effects (such as toxicity, irritation and allergic response), which facilitates cell survival and enables delivery of the formulated cells to humans or animals. The carrier is selected according to the appropriately intended mode of administration. The carrier of the present invention includes, but is not limited to, various physiological buffers such as physiological saline, phosphate buffer, artificial cerebrospinal fluid or whole serum, umbilical cord serum, etc.
In another embodiment of the invention, after the induced differentiation of the human amniotic epithelial cells into retinal pigment epithelial cells, the cells after the induced differentiation are collected, and the retinal pigment epithelial cells can be administered to the patient by any suitable method. In a preferred embodiment of the invention, subretinal injections are administered to an animal with a retinal degenerative disease to treat and/or ameliorate a disease progression. The cells can be administeredEither repeatedly or continuously. Generally, multiple administrations are usually given at intervals of at least 7-10 days to achieve better efficacy. The appropriate amount of retinal pigment epithelial cells to induce differentiation will vary depending on the age, sex, weight, health of the patient, and other factors. Typically, the dosage range for each administration is about 103-109Cells, more preferably in a dosage range of about 105-107A cell.
In another preferred embodiment of the present invention, the cells after inducing differentiation in vitro are collected by the following steps: and (2) adding trypsin into the culture container, putting the trypsin into an incubator for digestion, adding Fetal Bovine Serum (FBS) into the culture container after the cells are observed to be round under a microscope, gently blowing and beating the cells by using a pipette after the cells are uniformly mixed, transferring the digestive juice into a centrifuge tube for centrifugation, removing the supernatant, re-suspending the cell sediment by using a culture medium, and counting the cells.
In another preferred embodiment of the present invention, the cells after inducing differentiation in vitro are collected by the following steps: adding 0.25% trypsin into a culture container, placing the culture container into an incubator at 37 ℃ for digestion for 5-10min, adding Fetal Bovine Serum (FBS) after the cells are observed to be round under a microscope, mixing uniformly, blowing and beating the mixture by using a pipette tip gently, transferring the digestive juice into a centrifuge tube for centrifugation, removing supernatant, resuspending cell precipitation by using a small amount of DMEM/F121: 1(1X) culture medium, and counting the cells.
In another embodiment of the present invention, the retinal pigment epithelial cells that induce differentiation are administered to the patient in combination with one or more agents including ranibizumab, aflibercept, combispagu, Avastin, and Brolucizumab, among others.
The invention relates to a method for inducing human amniotic epithelial cells to differentiate into retinal pigment epithelial cells and application thereof in treating retinal degenerative diseases, wherein the method comprises the steps of adding an inducing composition containing 10-100mM nicotinamide and 1-10 mu M trichostatin A into the human amniotic epithelial cells, and inducing the generated cells to highly express typical genes of the retinal pigment epithelial cells such as MITF, PMEL17, RPE65, Bestrophin and the like; the cells induced and generated by the method are injected into the retina lower cavity of the RCS rat, the ERG and fundus detection show that the electrophysiological signals of the eyes of the rat are obviously enhanced, the structure of the fundus is obviously improved, the eye section HE staining shows that the thickness of the retina of the rat is obviously increased, and the cells induced and differentiated by the scheme have certain recovery on the vision of the rat after being injected. The inducing composition can be used for the differentiation of the human amniotic epithelial cells into the retinal pigment epithelial cells and the treatment of the eye diseases related to the retinal damage, and particularly can be used for the treatment of human retinal degenerative diseases. The method is simple and easy to implement, wide in amniotic epithelial cell source, easy to obtain materials, free of application limitation and ethical problems, and has wide prospects in clinical application of ophthalmic diseases.
Drawings
FIG. 1: detection of hAECs-RPE major marker. (A) A picture of cell morphology under bright field; (B) observing microvilli on the cell surface under a scanning electron microscope; (C) observing cell tight junction (upper left arrow), desmosome (single asterisk), microvilli (double asterisk) and melanin granules (lower right arrow) under a transmission electron microscope; (D-I) immunofluorescence to detect the expression of the RPE early marker MITF/PMEL17, intercellular junction and skeleton ZO-1/F-ACTIN, RPE maturation marker RPE65/Bestrophin (bright white spots around the round nuclear shadow, actually shown in green), and DAPI (round nuclear shadow, actually shown in blue in the figure) represents the location of the cell nucleus. Scale bar represents 50 μm (A, D-I),5 μm (B).
FIG. 2: the cells after in vitro induced differentiation were compared to the RNA-seq of human retinal pigment epithelial cells. heatmaps of 148 RPE signature genes for hECs-RPE and hRPE show that the gene expression patches were approximately identical by cluster analysis (gene expression levels are actually indicated by blue-yellow-red).
FIG. 3: inducing the polarity of differentiated cells in vitro, phagocytosis test and the secretion of relevant cytokines. (A-A') immunofluorescence Z-stack detecting the expression of cell polarity markers Na, K-ATPNase (bright white spots around the round nuclear shadow, actually shown as green), DAPI (round nuclear shadow, actually shown as blue) staining nuclei; (B-B') Z-stack detecting the phagocytosis of POS-Rhodopsin by cells (bright white spots around shadows, actually showing green); (C-D) ELISA was performed to detect changes in VEGF/PEDF expression in supernatant culture medium-apical and subbasal culture medium-basal of transwell chamber. The scale represents 50 μm, P <0.05, P < 0.001.
FIG. 4: 50ng/mL interferon-gamma stimulates the expression of HLA-DR and HLA-DQ on the cell surface before and after differentiation. (A-B) flow-detecting HLA-DQ/HLA-DR expression condition of the primary hAECs; (C-D) flow-assay of HLA-DQ/HLA-DR expression of hAECs-RPE.
FIG. 5: the ERG level can be obviously improved after the hAECs-RPE treatment. (A) Representative electrophysiology maps of ERG b-waves at different intensities for the treated (right) and non-treated (left) eyes; (B) average b-wave statistical plots for each group, n 6 (. about.p <0.001,. about.p <0.05)
FIG. 6: HE staining of rat eyeball sections after cell injection. (A) A schematic diagram of the structure of the retina of a wild type RCS rat; (B) the ONL layer (PRs) is obviously thickened at the injection site of hAECs-RPE; (C) the ONL layer and the whole retina thickness at the non-injection part are thinned; (D) treatment eye representative paraffin section HE staining map (the left arrow points to the cell injection site); (E) statistical plots of retinal overall thickness and ONL thickness at injection and non-injection sites. Scale bar represents 100 μm (A-C),500 μm (D) n ═ 6 (. about.P <0.001,. about.P <0.05)
FIG. 7: frozen sections of the eyeball with GFP-labeled cells were injected for immunofluorescence. (A) Two-photon confocal microscope observation shows that the mature RPE main marker RPE65 (white spot in the figure, actually shown as red) is combined with GFP (white spot in the figure, actually shown as green) representing hAECs-RPE and then is superposed; (B) two-photon confocal microscope observation of mature RPE main marker CRALBP (white light in the figure, actually shown as red) and hAECs-RPE representing GFP (white light in the figure, actually shown as green) combined and coincided. The scale represents 50 μm.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1 preparation of human amniotic cell test solution
Step 1: preparing an amniotic epithelial cell culture solution: adding 95ml of KSR into 500ml of DMEM/F121: 1 (1X); 6.5ml of 100mM L-Glutamine; 6.5ml of 100mM Sodium Pyruvate; 6.5ml 100mM MEM NEAA; EGF at 2000 × and double antibody at 100 × (Penicilin-Streptomyces) were added before use;
step 2: 2000 × EGF preparation: adding 1ml sterile ddH2O into 100ug EGF packaging tube, standing for 5-10min for dissolving, adding 4ml diluent (PBS containing 5% trehalose), mixing, and packaging into 1.5ml EP tube with each tube containing 100 μ l;
and step 3: preparing a digestion stop solution: DMEM/F121: 1(1X) + 10% FBS;
and 4, step 4: preparing a frozen stock solution: 40% FBS + 50% culture medium + 10% DMSO.
Example 2 isolation of human amniotic epithelial cells
1. Source of human amniotic membrane
After the authorization of the lying-in woman, placenta tissues after the cesarean section of healthy lying-in women (serological reactions such as HIV, syphilis, hepatitis A, hepatitis B, hepatitis C and the like are all shown to be negative) are taken, the placenta is cut by a cross knife, and the whole amnion is obtained by mechanical separation.
2. Isolation of human amniotic epithelial cells
Step 1: the amniotic membrane was washed three times with sterile PBS solution with double antibody (P/S), blood and other impurities were washed off, and the amniotic membrane was transferred to a 50ml centrifuge tube.
Step 2: 10ml of 0.25% pancreatin (bathed at 37 ℃ C. earlier) was added for digestion for 30s, inverted 20 times and the amniotic membrane was transferred to another 50ml centrifuge tube.
And step 3: 15ml of 0.25% pancreatin (bathed at 37 ℃ in advance) was added to the centrifuge tube, and after digestion in a water bath at 37 ℃ for 10min, the amniotic membrane was transferred to another 50ml centrifuge tube.
And 4, step 4: adding 25ml of 0.25% pancreatin into a centrifuge tube, digesting in water bath at 37 ℃ for 40min, shaking 10 times every 10 minutes, forcibly reversing 10 times after finishing, adding an equal volume of digestion stop solution to stop digestion, rotating at the speed of 500g, centrifuging at room temperature for 10min, collecting cells, and re-suspending with 1ml of culture solution.
And 5: transferring amnion into another 50ml centrifuge tube, adding 25ml of 0.25% pancreatin, digesting at 37 ℃ for 40min, mixing uniformly for 10 times every 10min, turning over 10 times after finishing, adding an equal volume of digestion stop solution to stop digestion, rotating at 500g, centrifuging at room temperature for 10min, collecting cells, and resuspending with 1ml of culture solution.
Step 6: mixing the two re-suspended cells, adding 18ml culture solution (adding the double antibody and EGF in the culture solution in advance), uniformly mixing, sieving with a 200-mesh sieve, and sieving with a 400-mesh sieve.
Example 3 inoculation culture and cryopreservation of human amniotic epithelial cells
1. Cell counting culture: 1X 107Individual cells were plated onto 15cm dishes. And changing the culture solution after the cells adhere to the wall, and changing the culture solution once in three days later.
2. Freezing and storing cells: after the cells grew over the plate, the cells were digested and cryopreserved: 5ml of pancreatin is added into 15cm dish, observation is carried out under a mirror after 10min, when the cells become round and the cells are in a suspension state when the plate is shaken in a plane, the digestion is stopped by adding the same amount of digestion stop solution. The cells on the culture dish were blown down by a micropipette in the same direction, transferred into a 15ml centrifuge tube, centrifuged at 800g for 3min, collected and then counted. And adding the freezing solution into the freezing tube, marking the freezing date, the freezing batch and the number of the cells, putting the cells into the freezing tube, immediately putting the freezing tube into a freezing box, putting the freezing box into a refrigerator at minus 80 ℃, taking out the freezing box after 12 hours, and transferring the cells into a liquid nitrogen tank for storage.
Example 4 differentiation of human amniotic epithelial cells into retinal pigment epithelial cells:
step 1: preparation of a composition for inducing differentiation of human amniotic epithelial cells into retinal pigment epithelial cells: to a DMEM/F121: 1(1X) medium containing 15% KSR (knock-out Serum replacement), 2mM L-glutamine (L-glutamine), 1mM non-essential amino acid (non-essential amino acid), 1mM sodium pyruvate (sodium pyruvate), 100units/ml penillin, and 100. mu.g/ml streptomycin was added nicotinamide (Nicotinaid, CAS No. 98-92-0, Sigma, cat. No. N3376), and 1-10. mu.M Trichostatin A (Trichoderma A, CAS No. 58880-19-6, APBIO, cat. No. A8183) at a final concentration to obtain a composition for inducing differentiation of human amniotic epithelial cells into retinal pigment epithelial cells.
Step 2: the method for inducing the differentiation of the human amniotic epithelial cells to the retinal pigment epithelial cells comprises the following steps: get P0Or P1Human amniotic epithelial cells at a ratio of 1-2X 105The cell amount of the cells/well is inoculated in a six-well plate, the inducing composition is added after 24-48 hours, the solution is changed every day, and the culture is placed in an incubator containing 5.5% carbon dioxide at 37 ℃ for 10-20 days.
Example 5 Electron microscopy of differentiated intracellular melanin precursor substances, cell junction structures and surface microvilli
Transmission electron microscopy:
1. collection of cell samples
The cells grown in the culture dish after in vitro induced differentiation were scraped off, washed with 1X PBS solution and centrifuged to collect the cells.
2. Pretreatment sample for electron microscope
Step 1: fixing: 2.5% glutaraldehyde PBS buffer for 4 hours or more for more than 20 sample volumes;
step 2: rinsing with 0.1M PBS (0.5-1.0 ml) for 10-15min (depending on sample size) for 2 times;
and step 3: fixing 1% osmate fixed solution for 1 hr, and recovering after use;
and 4, step 4: ddH2O rinsed 10-15min (depending on sample size) 2 times;
and 5: fixing/dyeing with 2% uranium acetate for 30min, with the dosage of about 100 ul;
step 6: gradient dehydration (time depends on sample size, dosage is 0.5-1.0ml, sample amount): 50% ethanol for 10-15 min; 70% ethanol for 10-15 min; 90% ethanol for 10-15 min; 100% ethanol for 10-20 min; 100% acetone for 10-20min2 times;
and 7: and (3) infiltration: pure acetone + embedding agent (1:1), room temperature 2 hours; pure embedding agent, room temperature 2 hours;
and 8: embedding;
and step 9: polymerization: oven at 37 deg.C for 24 hr; oven at 45 deg.C for 24 hr; drying in a drying oven at 60 ℃ for 48 hours;
step 10: slicing by an ultrathin slicer, dyeing by uranium acetate-lead citrate, and observing by a transmission electron microscope.
Scanning electron microscope
(1) Fixing: the sample was placed in 2.5% glutaraldehyde at 4 ℃ overnight
(2) Discard glutaraldehyde, wash the sample with PBS three times for 20min each
(3) Fixation with 1% osmic acid at 4 ℃ for 1h
(4) Osmic acid was discarded and the samples were washed three times with PBS for 20min each
(5) And (3) dehydrating: sequentially adding 30%, 50%, 70%, 80%, 90% and 95% acetone for 10-15min, and adding 100% acetone for 10-15min for 2-3 times
(6) And (3) replacement: replacement with pure isoamyl acetate for more than 15min
(7) And (3) drying: critical point drying of CO2
(8) Loading the sample on a sample table, coating silver colloid, spraying gold in vacuum, and observing with a scanning electron microscope
As shown in FIG. 1, the shape of hAECs-RPE in bright field is regular polygon, and cells are tightly connected; the long microvilli of hAECs-RPE can be seen under a scanning electron microscope; the tight junctions among hAECs-RPE cells, microvilli and melanin granules can be more clearly seen under a transmission electron microscope.
Example 6 cellular immunofluorescence
(1) Cell fixation: taking out cells from the cell culture box, observing the cells, then discarding the culture solution, washing the cells for 2-3 times by 1 XPBS (phosphate buffer solution), adding 800 mu L of 4% paraformaldehyde into each hole, and fixing for 15min at room temperature;
(2) cell washing: discarding paraformaldehyde solution, washing with 1 × PBS for 3 times, each for 5 min;
(3) cell permeabilization: permeabilization of 1 XPBS with 0.25% Triton X-100 for 1-10min at room temperature (based on the cellular localization of the protein of interest);
(4) cell washing: discarding the permeabilization solution, washing with 1 × PBS for 3 times, each time for 5 min;
(5) and (3) sealing: adding 500-800 μ L blocking solution (850 μ L PBS +100 μ L10% BSA +50 μ L LHBS) to each well, blocking for 1h at room temperature (or overnight at 4 ℃);
(6) primary antibody incubation: the antibody was diluted 1 × PBS at a ratio of 1:50 to 1:200 and incubated at room temperature for 2h (or overnight at 4 ℃);
(7) cell washing: recovering or discarding primary anti-dilution solution, washing with 1 × PBS for 3 times, each for 5 min;
(8) and (3) secondary antibody incubation: diluting the fluorescent secondary antibody with 1 XPBS at the ratio of 1:200-1:500, and incubating for 1h at room temperature in a dark place;
(9) cell washing: discarding the fluorescent secondary antibody diluent, washing with 1 × PBS for 3 times, each for 5 min;
(10) DAPI staining: DAPI was diluted with 1 XPBS at a ratio of 1:1000 and cells were stained for 1min at room temperature;
(11) cell washing: discarding DAPI diluent, washing with 1 × PBS for 3 times, each for 5 min;
(12) sealing: taking out the cover glass from the 12-hole plate, sucking off PBS at the edge by using dust-free paper, and lightly buckling the cell growth side onto the mounting solution (the mounting solution is dripped on the glass slide in advance);
(13) and (4) observing and photographing: the pictures are observed and photographed under a fluorescence microscope or a two-photon laser confocal microscope.
As shown in FIG. 1, hAECs-RPE was detected by IF to express MITF (microphthalmia-associated transcription factor), ZO-1 (intercellular tight junction protein), PMEL17 (melanin precursor protein), F-ACTIN (cytoskeletal protein), and Bestrophin and RPE65, which are major markers of mature RPE.
Example 7 sequencing of cellular transcriptome (RNA-seq)
And (3) cracking the differentiated cell sample by using Trizol, blowing, beating, uniformly mixing, transferring to a 1.5mL centrifuge tube, preserving at-80 ℃, collecting RPE (RPE) and PRs (platelet-like peptide) sample cells for 3 batches, and testing by Hangzhou Tianke biotechnology limited company. The method mainly comprises the following steps:
(1) after extracting total RNA of a sample and digesting the DNA by DNaseI, enriching mRNA of a eukaryote by magnetic beads with oligo (dT) (if the eukaryote is a prokaryote, removing rRNA by using a kit and then entering the next step); (2) adding an interrupting reagent into a Thermomixer, interrupting mRNA into short fragments at a proper temperature, synthesizing first-strand cDNA by using the interrupted mRNA as a template, preparing a double-strand synthesis reaction system to synthesize second-strand cDNA, purifying and recovering by using a kit, repairing a sticky end, adding a base A at the 3' end of the cDNA and connecting a joint, selecting the size of the fragments, and finally performing PCR amplification;
(3) the constructed library was qualified by quality inspection using Agilent2100Bioanalyzer and ABI StepOneNus Real-Time PCR System and sequenced using Illumina HiSeqTM 4000 or other sequencer.
According to the literature report, 148 genes are selected as the main identified genes of the RPE, and the results are shown in figure 2, the cluster analysis of the hAECs-RPE and hAECs-RPE gene expression data shows that the gene expression color blocks are approximately the same, and the differentiation degree of the RPE cells differentiated by the hAECs-RPE and the hAECs-RPE is fully proved again.
Example 8 detection of retinal pigment epithelial cell function in vitro phagocytosis of photoreceptor outer segments
1. Reagent A preparation (1000mL)
20mmol/L Tris-HCl buffer: 20mmol Tris (2.4228g), 200g sucrose, 65mmol NaCl (3.8g), 1mmol MgCl2(MgCl2·6H2O0.2033 g), 10mmol glucose (C)6H12O6·H2O1.9817 g), 5mmol of aminoethanesulfonic acid (0.62575g), hydrochloric acid to adjust the pH to 7.4, and storage at 4 ℃.
2. Preparation of Outer Segments (OS)
Step 1: taking 6-8 rat eyeballs, and cutting out the cornea, crystal and vitreous body circularly. Fresh tissue (3mL) of the retina was removed in 20 mmol. multidot.L-1 Tris-HCl buffer and the retina was cut into 2 mm. times.2 mm tissue fragments. (Note: the cuvette was washed in buffer A to wash out the detached outer segment.)
Step 2: retinal tissue was transferred into a 5mL tube and gently shaken for 1min (QL-861Vortex Shaker) at 4 ℃ 1000rpm min-1Centrifuge for 5 min.
And step 3: transferring the centrifuged supernatant into a 5mL tube, gently washing the precipitate with 500uL of reagent A, mixing the supernatant with the centrifuged supernatant, and heating to 4 deg.C at 3000rpm min-1Centrifuge for 2 min.
And 4, step 4: repeat step 3 once.
3. In vitro phagocytosis assay
The POS pellets in all EP tubes were resuspended in 100uL DMEM/F-12 medium, and all EP tubes were washed once with 100uL DMEM/F-12 medium and pooled. The 200uL POS can be diluted to 800uL, evenly added into a culture plate in which retinal pigment epithelial cells generated by in vitro induction grow, and placed in an incubator containing 5.5% carbon dioxide at 37 ℃ for incubation for 6-8 hours.
4. Immunofluorescence of cell slide
As shown in example 6.
As a result, as shown in FIG. 3, Na, K-ATPNase (a marker of cell polarity) was mainly expressed in the upper layer of the cell (3.10A); POS from rat PRs is indicated by Rhodopsin and cells are able to phagocytose a green POS fragment (3.10B).
Example 9 VEGF/PEDF ELISA detection
Primary hAECs were cultured in 12-well cell culture plates in transwell chambers with 4 μm pore size, and after the cells were grown to 70% and cultured for 12 days with liquid change and differentiation every day, culture liquid was collected in and out of the chambers 48 hours later (day 14 of differentiation), and changes in the expression level of VEGF/PEDF in the culture liquid above and below the cells were measured by ELISA.
The specific experimental steps are as follows:
(1) sample adding: all reagents, standards and activated samples were prepared, 100. mu.L of standard, control or test sample was added to each well, the reaction plates were mixed well and incubated at 37 ℃ for 120 min.
(2) Washing the plate: the reaction plate was washed thoroughly 4-6 times with 1 × washing solution and blotted dry on quick filter paper.
(3) Add 100. mu.L of detection antibody to each well, mix the reaction plates well and incubate at 37 ℃ for 60 min.
(4) Washing the plate: the reaction plate was washed thoroughly 4-6 times with 1 × washing solution and blotted dry on quick filter paper.
(5) mu.L peroxidase was added to each well and the plate was incubated at 37 ℃ for 30 min.
(6) Washing the plate: the reaction plate was washed thoroughly 4-6 times with 1 × washing solution and blotted dry on quick filter paper.
(7) Add 100. mu.L chromogenic substrate to each well, react in the dark at 37 ℃ for 10-30 min.
(8) Add 100. mu.L of stop solution to each well and mix well.
(9) The absorbance (OD value) was measured at a wavelength of 450nm with a microplate reader within 30 min.
(10) And (3) calculating: drawing a standard curve (the concentration of the standard substance is an abscissa, and the OD value is an ordinate), calculating a linear regression equation of the standard curve by using the OD value and the concentration of the standard substance, calculating the concentration of the sample during detection according to the OD value of the sample, and multiplying the concentration by the dilution factor to obtain the actual concentration of the sample.
As a result, as shown in FIG. 3, the results of ELISA detection of the upper and lower cell cultures after 48 hours of culture showed that VEGF was mainly secreted in the lower culture (3.10C) and PEDF was mainly secreted in the upper culture (3.10D).
Example 10 flow cytometry identification of cell surface markers
1. Preparing a buffer solution:
washing buffer (PBS solution containing 2% FBS): 1ml FBS is made to 50ml with 1 XPBS solution
Fixative (4% PFA solution): heating 180ml double distilled water, 20 μ l 5M sodium hydroxide and 8g PFA (paraformaldehyde) to 65 ℃, stirring to completely dissolve, cooling to room temperature, adding 10ml 20 XPBS, fixing the volume to 200ml with double distilled water, and subpackaging at-20 ℃ for freezing.
2. Flow cytometry identification:
step 1: respectively taking primary human amniotic epithelial cells which are not subjected to in vitro induction differentiation and cells which are subjected to in vitro induction to be differentiated into retinal pigment epithelial cells, adding 0.25% trypsin for digestion, centrifuging for 5min at 4 ℃ and 1000rpm, discarding supernatant, adding 1ml of PBS (phosphate buffer solution) for resuspension of the cells, evenly dividing into four parts, placing into a 1.5ml centrifuge tube, centrifuging for 3min at 4 ℃ and 1000rpm, and discarding supernatant.
Step 2: add 800u l washing buffer heavy suspension cells, 4 degrees C1000 rpm centrifugal 3min, abandon the supernatant. Repeat step 2 twice.
And step 3: 50 mul of washing buffer solution is added into the cells, 5 mul of HLA-DQ isotype, HLA-DQ, HLA-DR isotype and HLA-DR antibody are added into each tube, and the mixture is evenly mixed and placed for 30min at 4 ℃ in a dark place.
And 4, step 4: after centrifugation at 1000rpm for 3min at 4 ℃ the supernatant was discarded and 800. mu.l of wash buffer was added to resuspend the cells. This was repeated twice.
And 5: add 500. mu.l of 4% PFA fixative to each tube and fix overnight at 4 ℃ in the dark.
Step 6: after taking out, the cells were centrifuged at 1000rpm for 3min at 4 ℃ and the supernatant was discarded, and 800. mu.l of a washing buffer was added to resuspend the cells. This was repeated twice.
And 7: add 500. mu.l of wash buffer to resuspend the cells and transfer to flow tube for detection by an up-conveyor.
As shown in FIG. 4, flow assay results of hAECs-RPE and hAECs of the same batch show that the HLA-DR/HLA-DQ results are negative, which indicates that the cells are still low in immunogenicity before and after differentiation, and the cells are beneficial to in vivo transplantation.
EXAMPLE 11 rat Ocular electrophysiological assay (ERG)
1. Dark adaptation
Prior to the experiment, the experimental RCS rats were placed in a completely dark environment and acclimatized in the dark for more than 12 hours, with attention paid to ventilation.
2. ERG detection
Step 1: ketamine is used for anesthesia, the dosage is 0.02-0.03/70g, and mydriasis drug is dripped into eyes to carry out mydriasis.
Step 2: and connecting the electrodes. 2 recording electrodes and reference electrodes respectively, and 1 ground electrode.
And step 3: dark response stimulation was performed in sequence with stimulation intensity: -40db, -25db, -10db, 0db, +5 db.
And 4, step 4: adaptation is carried out for 10 min.
And 5: carrying out bright response stimulation in sequence, wherein the stimulation intensity is as follows: -10db, -5db, 0db, +5db, +10 db.
We performed ERG assays around 4 weeks after cell transplantation and we used a self-control mode at the time of injection (cells injected in the right eye, blank left eye or DMEM/F12 injected). One day before the ERG detection, each group of rats is placed in a darkroom to dark adapt for more than 8 hours, then the abdominal cavity is anesthetized, the ERG detection is carried out after mydriasis, and the animals are kept warm during the detection. The results are shown in fig. 5, where the electrophysiological response of ERG b-waves at different intensities for the treated eye is significantly improved compared to the control eye.
Example 12 RCS rat eyeball Paraffin section and HE staining
1. Separating rat eyeballs:
the eyeballs of the rats killed by the cervical dislocation method are taken out and soaked in 1X PBS solution to remove other impurities such as fat around the eyeballs.
2. Solution preparation:
the preparation of the 4% PFA solution was carried out in the same manner as in example 4.
Hydrochloric acid ethanol solution: 7ml HCl (37%) +252ml 70% ethanol.
Diluted ammonia water: 200ml distilled water is added with 100 mul ammonia water to obtain 0.05% diluted ammonia water.
3. Eyeball paraffin embedding, section and HE staining
Step 1: dewatering and wax penetration:
placing the eyeball into an embedding box, and sequentially passing through a dehydration cylinder filled with the following solutions:
and (3) dehydrating: sequentially passing the embedding box filled with eyeball through 70% ethanol, 80% ethanol and 90% ethanol for 15min respectively, placing in two 95% ethanol dehydration cylinders for 30min, and placing in two 100% ethanol dehydration cylinders for 30 min;
and (3) transparency: sequentially placing in dehydration jar containing 50% xylene and 50% alcohol for 60min, and placing in two xylene dehydration jars for 60 min;
wax penetration: passing through two dehydration cylinders containing pure wax for 60 min.
Step 2: embedding:
taking out the embedding box by using tweezers, putting the embedding box and a paraffin mold into a wax cylinder, clamping the paraffin mold by using the tweezers, putting the paraffin mold at a wax dripping position, putting the paraffin mold at a 4 ℃ position after dripping a drop of wax, putting the tissue on the wax (the section faces downwards), putting a mould at the wax dripping position, covering the embedding box, adding the paraffin, putting the embedding box at the 4 ℃ position for solidification, and then supplementing the paraffin.
And step 3: slicing:
the wax block is trimmed according to the tissue part, and redundant wax blocks are cut off. The slicer is adjusted to make the blade and the wax block have proper distance and angle. The thickness of the slice was adjusted to 50 μm, the thickness of the slice was adjusted to 4 μm after the sample was cut, whether the desired tissue was cut or not was examined under the microscope, and then the slice was cut at a thickness of 4 μm. After carefully cutting the wax tape to a certain length, the wax tape was cut at a distance of 5 samples by a blade.
And 4, step 4: unfolding, sticking and baking:
the water temperature was maintained at 42 ℃ by opening the slide-out apparatus, the wax tape was held with a pair of tweezers and placed on the water surface, and the slide was spread in the water. And taking a clean glass slide, carefully fishing out the unfolded slice, and marking the date and the sample number on the ground surface at the other end. And adjusting the temperature of the sheet baking machine to 37 ℃, and putting the slices on the sheet baking machine for baking so that the slices are attached to the glass sheet. Baking for 4h, and collecting the slices, wherein the slices can be stored for a long time.
And 5: HE staining:
the cut eyeball slices sequentially pass through the following containers:
dewaxing: xylene 10min × 3;
hydration: 100% ethanol 5min × 2, 95% ethanol 2 min; washing with slow tap water flow for 5 min;
staining cell nuclei: hematoxylin (45 seconds to minutes, depending on the staining, the nucleus stains blue); washing with slow tap water flow for 5 min;
differentiation: hydrochloric acid ethanol solution for 2 seconds; washing with slow tap water flow for 5 min;
returning blue: putting into dilute ammonia water for 8 times; washing with slow tap water flow for 6 min; 95% ethanol for 4 min;
staining cytoplasm: eosin (45 sec, cytoplasmic staining red);
and (3) dehydrating: 95% ethanol 2min × 2, 100% ethanol 4min × 2;
and (3) transparency: xylene 5min × 3, after staining was completed the sections were placed in a fume hood for air drying of residual xylene and mounted with neutral resin.
The different groups of rats were harvested about 4 weeks after cell transplantation, sectioned after paraffin embedding of the eyeballs (thickness 3 μm), and the results after HE staining are shown in fig. 6, in which the structure of each retinal layer of the wild type RCS rat was intact, the Outer Nuclear Layer (ONL) of the non-wild type RCS rat was significantly thinned, and the overall thickness of the retina and the thickness of the outer nuclear layer at the cell injection site were significantly improved. Thus, the hAECs-RPE injection can obviously improve the structural damage of the retina.
Example 13 RCS rat eyeball cryosection and immunohistochemical experiments
1. Sampling and embedding of eyeball
The eyeballs of the rats killed by the cervical dislocation method are taken out, quickly placed in 1X PBS to wash off blood stains, and after impurities such as fat and the like around the eyeballs are removed, the rats are placed in an embedding box filled with an ice-cream embedding medium (OCT). The cassette was placed in dry ice pre-chilled isopentane until OCT and tissue were completely frozen.
2. Frozen section of eyeball sample
The embedded eyeball was fixed in a cryomicrotome, cut into 0.5 μm sections, and the sections were mounted on an adhesive slide.
3. Immunohistochemical testing of frozen sections of the eyeball
Step 1: the sections were allowed to stand at room temperature for 30min before staining to allow the sections to adhere well to the slide.
Step 2: the samples were placed in acetone at-20 ℃ and fixed for 10min (directly for subsequent operations if the tissue was fixed before embedding).
And step 3: the sections were removed and washed 3 times with PBS for 5min each.
And 4, step 4: the tissue is firstly enclosed by an oily pen, then primary antibody (the primary antibody is diluted by PBS of 5 percent HBS +1 percent BSA) is dripped on the tissue, and the tissue is placed in a wet box for overnight at 4 ℃;
and 5: taking out the slices, washing with PBS for 3 times, 5min each time;
step 6: remove the water on the section, add the fluorescent coupled secondary antibody (diluted with 5% HBS + 1% BSA in PBS) and incubate in a humid chamber for 1h in the dark at room temperature. (the subsequent operations from this step are all performed in the dark)
And 7: the sections were removed and washed 3 times with PBS for 5min each.
And 8: diluting DAPI with PBS, adding dropwise onto the slice, incubating at room temperature for 3min, and washing with PBS for 5min for 2 times;
and step 9: sealing liquid and sealing, and performing fluorescence microscope microscopic examination.
The cells were sampled about 4 weeks after transplantation, frozen sectioned and immunofluorescence photographed using a two-photon confocal microscope to detect cell survival and detect the major markers of RPE. As a result, as shown in fig. 7, the cells survived well 4 weeks after transplantation and expressed RPE major markers such as RPE65/CRALBP, indicating that RPE cells transplanted into the subretinal space survived for a long time and inserted into and integrated with the original retina to exert RPE function.
The invention provides a novel method for inducing differentiation of human amniotic epithelial cells to retinal pigment epithelial cells in vitro, wherein cells generated by induction highly express part of classical retinal pigment epithelial cell markers; in addition, the expression of HLA-DR and HLA-DQ antigens of the cells is still maintained at a low level after stimulation by the stimulating factors, which shows that the cells still have low immunogenicity after differentiation and are suitable for cell therapy as transplants. The ERG electrophysiological detection result of the RCS rat injected through the subretinal space shows that the electric signal intensity of the eyes of the RCS rat is obviously enhanced compared with that of a non-treatment group under the light stimulation, the fundus picture shows that the pigment layer of the RCS rat is recovered to a greater extent, the eyeball sampling section shows that the injected cells can be positioned at a specific position of the subretinal space and survive for a longer time, and the HE staining shows that the retina structure of the RCS rat injected is obviously recovered. Therefore, the inventors considered that the in vitro differentiation method can differentiate human amniotic epithelial cells into retinal pigment epithelial cells with high efficiency, and can recover the vision of RCS rats to some extent after injection. The inducing composition can be used for the differentiation of the human amniotic epithelial cells into the retinal pigment epithelial cells and the treatment of the eye diseases related to the retinal damage, and particularly can be used for the treatment of human retinal degenerative diseases.
In this specification the invention has been described with reference to specific embodiments, which are presented only to assist in understanding the method of the invention and its core ideas. The present invention has been described for illustrative purposes and is not to be construed as limited thereby, as modifications and variations can be readily made by those skilled in the art without departing from the principles of the present invention, and within the scope of the appended claims.
Claims (34)
1. A method of inducing differentiation of human amniotic epithelial cells to retinal pigment epithelial cells, the method comprising the steps of:
(1) culturing human amniotic epithelial cells under appropriate conditions for 12-48 hr;
(2) adding an inducing composition to continue culturing for 5-20 days to induce the differentiation of the human amniotic epithelial cells into retinal pigment epithelial cells, wherein the inducing composition is a cell culture medium containing 10-100mM nicotinamide and 1-10 mu M trichostatin A.
2. The method of claim 1, wherein: the cell culture medium used in the method is a culture medium for culturing the human amniotic epithelial cells, and the cell culture medium is self-prepared or directly uses a commercial culture medium existing in the market.
3. The method of claim 2, wherein: the cell culture medium in the method is a DMEM medium or an NPBM medium.
4. The method of claim 1, wherein: the human amniotic epithelial cells in the step (1) of the method are P0 or P1 human amniotic epithelial cells.
5. The method of claim 4, wherein: the P0 or P1 human amniotic epithelial cells in step (1) of the method are according to the formula 104-106The amount of cells per well was inoculated in a culture vessel and cultured for 12 to 30 hours.
6. The method of claim 5, wherein: in the method, the P0 or P1 human amniotic epithelial cells in the step (1) are arranged according to the proportion of 1x 105-5×105The cell amount per well was seeded in well plates and cultured for 12-24 hours.
7. The method of claim 1, wherein: in the step (2), the cell culture solution is added with an inducing composition and continuously cultured for 10-20 days to induce the differentiation of the human amniotic epithelial cells to retinal pigment epithelial cells.
8. The method of claim 1, wherein: the cell culture medium in the inducing composition in step (2) of the method is a DMEM medium.
9. The method of claim 1, wherein: the inducing composition in step (2) of the method is a cell culture medium containing 30-70mM nicotinamide and 3-7. mu.M trichostatin A.
10. The method of claim 1, wherein: the inducing composition in step (2) of the method is prepared by the following method: adding nicotinamide (Nicotinamide) with the final concentration of 10-100mM and trichostatin A (Trichostatin A) with the final concentration of 1-10 mu M into a DMEM/F121: 1(1X) culture medium containing 15% of KSR (knock out Serum replacement), 2mM L-glutamine (L-glutamine), 1mM non-essential amino acid (non-essential amino acid), 1mM sodium pyruvate (sodium pyruvate), 100units/ml penillin and 100 mu g/ml streptomycin to obtain the composition for inducing the differentiation of the human amniotic epithelial cells to the retinal pigment epithelial cells.
11. The method of claim 1, wherein: the method comprises the following steps:
(1) get P0Or P1Human amniotic epithelial cells at 1 × 105-5×105The cell amount of the cells/hole is inoculated in a hole plate and cultured for 24-48 hours;
(2) after the induction composition is added, the solution is changed every day, and the mixture is placed in an incubator containing 5.5 percent of carbon dioxide at 37 ℃ for culturing for 10 to 14 days to induce the human amniotic epithelial cells to be differentiated into retinal pigment epithelial cells, wherein the induction composition is a cell culture medium containing 10 to 100mM nicotinamide and 1 to 10 mu M trichostatin A.
12. The method according to any one of claims 1-11, wherein: the amniotic epithelial cells in the method are prepared by a method comprising the following steps:
(1) mechanically separating the placenta tissue to obtain an amniotic membrane;
(2) and digesting the washed amniotic membrane by using digestive enzyme, and centrifuging the digested liquid to obtain the human amniotic epithelial cells.
13. The method of claim 12, wherein: the amniotic membrane may be isolated from isolated human placenta, washed with physiological buffer to remove blood cells, and mechanically removed of residual chorion and blood vessels.
14. The method of claim 13, wherein: the human amnion is obtained by taking placenta tissue of healthy lying-in woman after cesarean section after the authorization of the lying-in woman, and obtaining the whole amnion by mechanical separation.
15. The method of claim 12, wherein: the method step (2) is performed by isolating single cells from the intact human amniotic epithelial tissue using any conventional technique or method including mechanical force, enzymatic digestion with one or a combination of proteases selected from collagenase, trypsin, lipase, liberase and pepsin or a combination of mechanical and enzymatic methods.
16. The method of claim 12, wherein: the method comprises the following steps of (2) continuously culturing the obtained human amniotic epithelial cells under the culture conditions: at 1 × 106-1×108Inoculating cells into a culture dish according to the density of each cell/flat plate, placing the culture dish in a carbon dioxide incubator for culture, replacing culture solution after human amniotic epithelial cells are attached to the wall, digesting the cells after the flat plates are full of the cells, and performing cryopreservation for subsequent induced differentiation.
17. The method of claim 12, wherein: concentrating the separated human amniotic epithelial cells or the active cell population which enables the amniotic epithelial cells to be directionally induced and differentiated into retinal pigment epithelial cells.
18. Use of retinal pigment epithelial cells induced to differentiate by human amniotic epithelial cells or a cell preparation thereof in the preparation of a medicament for treating and/or ameliorating retinal degenerative diseases.
19. Use according to claim 18, characterized in that: an effective dose of retinal pigment epithelial cells or cell preparations thereof induced by human amniotic epithelial cells are used for treating and/or improving retinal degenerative diseases alone or in combination with other drugs.
20. Use according to claim 18, characterized in that: the treatment and/or improvement of the retinal degenerative disease refers to treatment and/or improvement of an animal with the retinal degenerative disease, which is a mammal.
21. Use according to claim 20, characterized in that: the animals with retinal degenerative diseases are cattle, horses, sheep, monkeys, dogs, rats, mice, rabbits or humans.
22. Use according to claim 21, characterized in that: the animal with the retinal degenerative disease refers to a human.
23. Use according to claim 18, characterized in that: the retinal degenerative diseases include Retinitis Pigmentosa (RP), Age-related Macular Degeneration (AMD), and glaucoma.
24. Use according to claim 18, characterized in that: after the human amniotic epithelial cells are induced to differentiate into retinal pigment epithelial cells, the induced and differentiated cells are collected and the retinal pigment epithelial cells are administered to the patient by any suitable method.
25. Use according to claim 24, characterized in that: subretinal injection is performed on an animal with a retinal degenerative disease to treat and/or ameliorate a disease progression.
26. Use according to claim 25, characterized in that: the animals with retinal degenerative diseases are injected with subretinal space, and the administration of cells can be repeated or continuous, and the multiple administration modes are respectively used at intervals of at least 7-10 days.
27. Use according to claim 24, characterized in that: the dosage range of each administration is 103-109A cell.
28. Use according to claim 27, characterized in that: the dosage range of each administration is 105-107A cell.
29. Use according to claim 18, characterized in that: the induced differentiation of retinal pigment epithelial cells is administered to the patient in combination with one or more agents including ranibizumab, aflibercept, combivicept, Avastin, and Brolucizumab.
30. Use according to any one of claims 18 to 29, characterized in that: the retinal pigment epithelial cells induced to differentiate from human amniotic epithelial cells or the cell preparation thereof are prepared according to the method of any one of claims 1 to 17.
31. A cell preparation comprising retinal pigment epithelial cells induced to differentiate by human amniotic epithelial cells and a pharmaceutically acceptable carrier.
32. The cell preparation of claim 31, wherein: pharmaceutically acceptable carriers include various physiological buffers.
33. The cell preparation of claim 32, wherein: the physiological buffer solution is selected from physiological saline, phosphate buffer solution, artificial cerebrospinal fluid or whole serum and umbilical cord serum.
34. The cell preparation of claim 31, wherein: the cell preparation is prepared according to the method of any one of claims 1-17.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810347953 | 2018-04-18 | ||
| CN2018103479533 | 2018-04-18 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110205283A CN110205283A (en) | 2019-09-06 |
| CN110205283B true CN110205283B (en) | 2021-04-23 |
Family
ID=67785450
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910310260.1A Active CN110205283B (en) | 2018-04-18 | 2019-04-17 | A method for inducing human amniotic epithelial cells to differentiate into retinal pigment epithelial cells and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110205283B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113999808A (en) * | 2021-10-29 | 2022-02-01 | 扬州大学 | A method for regulating immune function in dairy cow mammary epithelial cells with Escherichia coli |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101402943A (en) * | 2008-11-17 | 2009-04-08 | 中山大学中山眼科中心 | Method for in vitro abduction and cultivation of multi-potentiality stem cell |
| CN101688178A (en) * | 2007-04-18 | 2010-03-31 | 哈达锡特医学研究服务及发展有限公司 | Stem cell-derived retinal pigment epithelium |
| CN101824399A (en) * | 2009-03-05 | 2010-09-08 | 中日友好医院 | Method for inducing and differentiating into dopaminergic neuron from human amniotic sepithelial cell and application for separating obtained dopaminergic neuron |
| CN101914488A (en) * | 2010-08-04 | 2010-12-15 | 李荣旗 | Method for induced differentiation of human amniotic mesenchymal cells into insulin secreting cells |
| CN102307990A (en) * | 2008-12-03 | 2012-01-04 | 国际干细胞公司 | Methods of deriving differentiated cells from stem cells |
| CN103382457A (en) * | 2013-06-25 | 2013-11-06 | 清华大学深圳研究生院 | Application of trichostatin A in maintaining dryness of stem cells |
| CN105358677A (en) * | 2013-03-14 | 2016-02-24 | 布里格海姆妇女医院公司 | Compositions and methods for epithelial stem cell expansion and culture |
| CN105705021A (en) * | 2013-05-20 | 2016-06-22 | 位于西奈山的伊坎医学院 | Enriched and expanded human cord blood stem cells for treatment of hematological disorders |
| CN108373990A (en) * | 2018-03-19 | 2018-08-07 | 沈阳艾米奥生物工程技术研发中心有限公司 | People's amnioic epithelium stem cell is to melanocyte inductive differentiation medium and its method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9850463B2 (en) * | 2012-02-01 | 2017-12-26 | The Regents Of The University Of California | Methods of culturing retinal pigmented epithelium cells, including xeno-free production, RPE enrichment, and cryopreservation |
-
2019
- 2019-04-17 CN CN201910310260.1A patent/CN110205283B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101688178A (en) * | 2007-04-18 | 2010-03-31 | 哈达锡特医学研究服务及发展有限公司 | Stem cell-derived retinal pigment epithelium |
| CN101402943A (en) * | 2008-11-17 | 2009-04-08 | 中山大学中山眼科中心 | Method for in vitro abduction and cultivation of multi-potentiality stem cell |
| CN102307990A (en) * | 2008-12-03 | 2012-01-04 | 国际干细胞公司 | Methods of deriving differentiated cells from stem cells |
| CN101824399A (en) * | 2009-03-05 | 2010-09-08 | 中日友好医院 | Method for inducing and differentiating into dopaminergic neuron from human amniotic sepithelial cell and application for separating obtained dopaminergic neuron |
| CN101914488A (en) * | 2010-08-04 | 2010-12-15 | 李荣旗 | Method for induced differentiation of human amniotic mesenchymal cells into insulin secreting cells |
| CN105358677A (en) * | 2013-03-14 | 2016-02-24 | 布里格海姆妇女医院公司 | Compositions and methods for epithelial stem cell expansion and culture |
| CN105705021A (en) * | 2013-05-20 | 2016-06-22 | 位于西奈山的伊坎医学院 | Enriched and expanded human cord blood stem cells for treatment of hematological disorders |
| CN103382457A (en) * | 2013-06-25 | 2013-11-06 | 清华大学深圳研究生院 | Application of trichostatin A in maintaining dryness of stem cells |
| CN108373990A (en) * | 2018-03-19 | 2018-08-07 | 沈阳艾米奥生物工程技术研发中心有限公司 | People's amnioic epithelium stem cell is to melanocyte inductive differentiation medium and its method |
Non-Patent Citations (3)
| Title |
|---|
| Cell Programming for Future Regenerative Medicine;Frauke Hausburg等;《Regenerative Medicine - from Protocol to Patient》;20161231;第389-424页 * |
| Direct differentiation of hepatic stem-like WB cells into insulin-producing cells using small molecules;Jianping Liu等;《SCIENTIFIC REPORTS》;20130131;第3卷;第1-8页 * |
| 人羊膜上皮细胞的干细胞特征和临床应用潜力;朱丽华;《中国细胞生物学学报》;20181231;第40卷(第13期);第2166-2179页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110205283A (en) | 2019-09-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Keller et al. | Consensus recommendations for trabecular meshwork cell isolation, characterization and culture | |
| LU500561B1 (en) | In vitro construction method and use of liver organoids | |
| Kim et al. | Isolation and culture of neurons and astrocytes from the mouse brain cortex | |
| CN110478368A (en) | The purposes of umbilical cord mesenchymal stem cells conditioned medium | |
| CN110205283B (en) | A method for inducing human amniotic epithelial cells to differentiate into retinal pigment epithelial cells and its application | |
| CN114470001A (en) | Therapeutic use of human amniotic epithelial cells in autoimmune diseases | |
| CN110106147B (en) | Method for inducing differentiation of human amniotic epithelial cells into retinal photoreceptor cells and application thereof | |
| Price | The secretion of Mullerian inhibiting substance by cultured isolated Sertoli cells of the neonatal calf | |
| US20090047738A1 (en) | Feeder cell derived from tissue stem cell | |
| Ho et al. | Tissue culture of retinal pigment epithelium following isolation with a gelatin matrix technique | |
| Walker et al. | Establishment of a clinically relevant ex vivo mock cataract surgery model for investigating epithelial wound repair in a native microenvironment | |
| US20090226955A1 (en) | Immortalized retinal pigmented epithelial cells | |
| CN107206028A (en) | Eye disorders are treated using progenitor cells | |
| CN110951672B (en) | A mouse endometrial epithelial cell and its 3D differentiation culture model construction method | |
| Vats et al. | Retinal Explant Culture from Mouse, Human, and Nonhuman Primates and Its Applications in Vision Research | |
| CN114134107A (en) | Artificial ovary participated by mesenchymal stem cells and preparation method and application thereof | |
| CN108291199A (en) | Retinosis is treated using progenitor cells | |
| CN113577109A (en) | Anti-aging strategy based on embryonic stem cells | |
| Langenfeld et al. | An improved method for the isolation and culture of retinal pigment epithelial cells from adult rats | |
| JP2023511003A (en) | A differentiation method to secure large numbers of oligodendrocytes by degrading 3D organoids prepared from human pluripotent stem cells | |
| JP5014535B2 (en) | Cynomolgus monkey-derived embryonic stem cells | |
| CN108603168B (en) | Method for obtaining female reproductive stem cells from follicle extract | |
| Alsalloum et al. | Culture of human retinal explants as an ex vivo model for retinal gene therapy | |
| CN113774015B (en) | Method for in vitro induction of oogenic cell differentiation into oocyte | |
| CN113679741A (en) | Application of human amniotic epithelial stem cells in preparation of medicine for treating cisplatin-induced acute kidney 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 |