CN116790476A - 用于体外制造胃底组织的方法和与其相关的组合物 - Google Patents
用于体外制造胃底组织的方法和与其相关的组合物 Download PDFInfo
- Publication number
- CN116790476A CN116790476A CN202310699246.1A CN202310699246A CN116790476A CN 116790476 A CN116790476 A CN 116790476A CN 202310699246 A CN202310699246 A CN 202310699246A CN 116790476 A CN116790476 A CN 116790476A
- Authority
- CN
- China
- Prior art keywords
- cells
- period
- fundus
- time
- hfgo
- 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.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 59
- 238000000338 in vitro Methods 0.000 title claims abstract description 14
- 239000000203 mixture Substances 0.000 title abstract description 7
- 238000004519 manufacturing process Methods 0.000 title abstract description 4
- 210000001900 endoderm Anatomy 0.000 claims abstract description 31
- 210000004027 cell Anatomy 0.000 claims description 147
- 102000013814 Wnt Human genes 0.000 claims description 62
- 108050003627 Wnt Proteins 0.000 claims description 62
- 239000012190 activator Substances 0.000 claims description 49
- 230000019491 signal transduction Effects 0.000 claims description 43
- 210000001711 oxyntic cell Anatomy 0.000 claims description 40
- 230000037361 pathway Effects 0.000 claims description 32
- 230000011664 signaling Effects 0.000 claims description 27
- 210000002438 upper gastrointestinal tract Anatomy 0.000 claims description 27
- 239000003112 inhibitor Substances 0.000 claims description 22
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 18
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 claims description 17
- 101000762379 Homo sapiens Bone morphogenetic protein 4 Proteins 0.000 claims description 17
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 claims description 14
- 229930002330 retinoic acid Natural products 0.000 claims description 14
- 229960001727 tretinoin Drugs 0.000 claims description 14
- 239000003102 growth factor Substances 0.000 claims description 12
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 claims description 11
- 102100024270 Transcription factor SOX-2 Human genes 0.000 claims description 11
- 108010047320 Pepsinogen A Proteins 0.000 claims description 10
- 239000002253 acid Substances 0.000 claims description 10
- 229940124647 MEK inhibitor Drugs 0.000 claims description 9
- 230000004156 Wnt signaling pathway Effects 0.000 claims description 9
- 239000002829 mitogen activated protein kinase inhibitor Substances 0.000 claims description 9
- 230000003248 secreting effect Effects 0.000 claims description 8
- 230000001939 inductive effect Effects 0.000 claims description 7
- 102100021904 Potassium-transporting ATPase alpha chain 1 Human genes 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 102100028412 Fibroblast growth factor 10 Human genes 0.000 claims description 5
- 101000917237 Homo sapiens Fibroblast growth factor 10 Proteins 0.000 claims description 5
- 239000011159 matrix material Substances 0.000 claims description 5
- 108010083204 Proton Pumps Proteins 0.000 claims description 4
- 150000007513 acids Chemical class 0.000 claims description 3
- 230000007045 gastrulation Effects 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims 1
- 230000009772 tissue formation Effects 0.000 claims 1
- 210000002220 organoid Anatomy 0.000 abstract description 43
- 210000001519 tissue Anatomy 0.000 abstract description 38
- 230000004069 differentiation Effects 0.000 abstract description 34
- 230000015572 biosynthetic process Effects 0.000 abstract description 10
- 210000002599 gastric fundus Anatomy 0.000 abstract description 8
- 210000000056 organ Anatomy 0.000 abstract description 6
- 230000001131 transforming effect Effects 0.000 abstract description 2
- 230000002496 gastric effect Effects 0.000 description 63
- 210000002784 stomach Anatomy 0.000 description 54
- 210000000981 epithelium Anatomy 0.000 description 45
- 108060000903 Beta-catenin Proteins 0.000 description 42
- 102000015735 Beta-catenin Human genes 0.000 description 42
- 230000014509 gene expression Effects 0.000 description 39
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 38
- 108090000623 proteins and genes Proteins 0.000 description 34
- 230000002441 reversible effect Effects 0.000 description 32
- 210000001778 pluripotent stem cell Anatomy 0.000 description 29
- 238000011161 development Methods 0.000 description 27
- 230000018109 developmental process Effects 0.000 description 27
- 241000699666 Mus <mouse, genus> Species 0.000 description 26
- AQGNHMOJWBZFQQ-UHFFFAOYSA-N CT 99021 Chemical compound CC1=CNC(C=2C(=NC(NCCNC=3N=CC(=CC=3)C#N)=NC=2)C=2C(=CC(Cl)=CC=2)Cl)=N1 AQGNHMOJWBZFQQ-UHFFFAOYSA-N 0.000 description 25
- 210000000130 stem cell Anatomy 0.000 description 25
- 210000001671 embryonic stem cell Anatomy 0.000 description 23
- 229960001340 histamine Drugs 0.000 description 19
- 210000002257 embryonic structure Anatomy 0.000 description 18
- 238000002474 experimental method Methods 0.000 description 18
- 210000004907 gland Anatomy 0.000 description 18
- 101710183548 Pyridoxal 5'-phosphate synthase subunit PdxS Proteins 0.000 description 15
- 238000011282 treatment Methods 0.000 description 15
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 14
- 230000000968 intestinal effect Effects 0.000 description 14
- 230000024245 cell differentiation Effects 0.000 description 13
- 238000001727 in vivo Methods 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 13
- 230000000694 effects Effects 0.000 description 12
- 239000002243 precursor Substances 0.000 description 12
- 108090001072 Gastricsin Proteins 0.000 description 11
- 102000034255 Pepsinogen C Human genes 0.000 description 11
- 210000003890 endocrine cell Anatomy 0.000 description 11
- 239000002609 medium Substances 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 description 10
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 10
- 230000001965 increasing effect Effects 0.000 description 10
- 210000003097 mucus Anatomy 0.000 description 10
- 101800001586 Ghrelin Proteins 0.000 description 9
- 102000012004 Ghrelin Human genes 0.000 description 9
- 238000011529 RT qPCR Methods 0.000 description 9
- 230000004913 activation Effects 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 230000006698 induction Effects 0.000 description 9
- 102000045246 noggin Human genes 0.000 description 9
- 108700007229 noggin Proteins 0.000 description 9
- 238000010606 normalization Methods 0.000 description 9
- 238000012353 t test Methods 0.000 description 9
- 101150111723 PDX1 gene Proteins 0.000 description 8
- 101100247004 Rattus norvegicus Qsox1 gene Proteins 0.000 description 8
- 230000000762 glandular Effects 0.000 description 8
- 230000004044 response Effects 0.000 description 8
- 108010083123 CDX2 Transcription Factor Proteins 0.000 description 7
- 102000006277 CDX2 Transcription Factor Human genes 0.000 description 7
- 241000283707 Capra Species 0.000 description 7
- 101000972282 Homo sapiens Mucin-5AC Proteins 0.000 description 7
- 102000004232 Mitogen-Activated Protein Kinase Kinases Human genes 0.000 description 7
- 102100022496 Mucin-5AC Human genes 0.000 description 7
- 241000283973 Oryctolagus cuniculus Species 0.000 description 7
- 102100041030 Pancreas/duodenum homeobox protein 1 Human genes 0.000 description 7
- 210000003484 anatomy Anatomy 0.000 description 7
- 210000001161 mammalian embryo Anatomy 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 238000010186 staining Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- SUBDBMMJDZJVOS-UHFFFAOYSA-N 5-methoxy-2-{[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]sulfinyl}-1H-benzimidazole Chemical compound N=1C2=CC(OC)=CC=C2NC=1S(=O)CC1=NC=C(C)C(OC)=C1C SUBDBMMJDZJVOS-UHFFFAOYSA-N 0.000 description 6
- 102100021022 Gastrin Human genes 0.000 description 6
- 101000753535 Homo sapiens Potassium-transporting ATPase subunit beta Proteins 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical class OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- 102100021944 Potassium-transporting ATPase subunit beta Human genes 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 230000012010 growth Effects 0.000 description 6
- 230000001404 mediated effect Effects 0.000 description 6
- 229960000381 omeprazole Drugs 0.000 description 6
- 101150037241 CTNNB1 gene Proteins 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 101000972278 Homo sapiens Mucin-6 Proteins 0.000 description 5
- 101000753506 Homo sapiens Potassium-transporting ATPase alpha chain 1 Proteins 0.000 description 5
- 101000819074 Homo sapiens Transcription factor GATA-4 Proteins 0.000 description 5
- 102100022493 Mucin-6 Human genes 0.000 description 5
- 101150037203 Sox2 gene Proteins 0.000 description 5
- 102100021380 Transcription factor GATA-4 Human genes 0.000 description 5
- 229960001596 famotidine Drugs 0.000 description 5
- XUFQPHANEAPEMJ-UHFFFAOYSA-N famotidine Chemical compound NC(N)=NC1=NC(CSCCC(N)=NS(N)(=O)=O)=CS1 XUFQPHANEAPEMJ-UHFFFAOYSA-N 0.000 description 5
- 210000002950 fibroblast Anatomy 0.000 description 5
- 101150003286 gata4 gene Proteins 0.000 description 5
- 210000004602 germ cell Anatomy 0.000 description 5
- 238000003125 immunofluorescent labeling Methods 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 230000003612 virological effect Effects 0.000 description 5
- 108010059616 Activins Proteins 0.000 description 4
- 102000005606 Activins Human genes 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 210000000712 G cell Anatomy 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 101001002317 Homo sapiens Gastrin Proteins 0.000 description 4
- 101000603702 Homo sapiens Neurogenin-3 Proteins 0.000 description 4
- 101100013973 Mus musculus Gata4 gene Proteins 0.000 description 4
- 238000000692 Student's t-test Methods 0.000 description 4
- 238000003917 TEM image Methods 0.000 description 4
- 230000002159 abnormal effect Effects 0.000 description 4
- 230000009858 acid secretion Effects 0.000 description 4
- 239000000488 activin Substances 0.000 description 4
- 239000000556 agonist Substances 0.000 description 4
- 239000005557 antagonist Substances 0.000 description 4
- 210000002469 basement membrane Anatomy 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 101150079068 irx3 gene Proteins 0.000 description 4
- 108010082117 matrigel Proteins 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 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 description 4
- 210000003014 totipotent stem cell Anatomy 0.000 description 4
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 102000010911 Enzyme Precursors Human genes 0.000 description 3
- 108010062466 Enzyme Precursors Proteins 0.000 description 3
- 102100028072 Fibroblast growth factor 4 Human genes 0.000 description 3
- 208000012895 Gastric disease Diseases 0.000 description 3
- 101000971300 Homo sapiens Class A basic helix-loop-helix protein 15 Proteins 0.000 description 3
- 101001060274 Homo sapiens Fibroblast growth factor 4 Proteins 0.000 description 3
- 101000977762 Homo sapiens Iroquois-class homeodomain protein IRX-5 Proteins 0.000 description 3
- 102100023529 Iroquois-class homeodomain protein IRX-5 Human genes 0.000 description 3
- 102100024193 Mitogen-activated protein kinase 1 Human genes 0.000 description 3
- 102000052547 Wnt-1 Human genes 0.000 description 3
- 230000001594 aberrant effect Effects 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 210000004504 adult stem cell Anatomy 0.000 description 3
- 210000000270 basal cell Anatomy 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 210000002459 blastocyst Anatomy 0.000 description 3
- 101150067309 bmp4 gene Proteins 0.000 description 3
- 101150083915 cdh1 gene Proteins 0.000 description 3
- 230000011712 cell development Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 210000002308 embryonic cell Anatomy 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000002304 esc Anatomy 0.000 description 3
- 210000001156 gastric mucosa Anatomy 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 230000005305 organ development Effects 0.000 description 3
- 230000008672 reprogramming Effects 0.000 description 3
- 210000001082 somatic cell Anatomy 0.000 description 3
- 230000010473 stable expression Effects 0.000 description 3
- 238000007619 statistical method Methods 0.000 description 3
- 239000013603 viral vector Substances 0.000 description 3
- UBWXUGDQUBIEIZ-UHFFFAOYSA-N (13-methyl-3-oxo-2,6,7,8,9,10,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-17-yl) 3-phenylpropanoate Chemical compound CC12CCC(C3CCC(=O)C=C3CC3)C3C1CCC2OC(=O)CCC1=CC=CC=C1 UBWXUGDQUBIEIZ-UHFFFAOYSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 101100257359 Caenorhabditis elegans sox-2 gene Proteins 0.000 description 2
- 102100021615 Class A basic helix-loop-helix protein 15 Human genes 0.000 description 2
- JMIFGARJSWXZSH-UHFFFAOYSA-N DMH1 Chemical compound C1=CC(OC(C)C)=CC=C1C1=CN2N=CC(C=3C4=CC=CC=C4N=CC=3)=C2N=C1 JMIFGARJSWXZSH-UHFFFAOYSA-N 0.000 description 2
- 101100317380 Danio rerio wnt4a gene Proteins 0.000 description 2
- 101100264065 Danio rerio wnt5b gene Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 108091008794 FGF receptors Proteins 0.000 description 2
- 102000044168 Fibroblast Growth Factor Receptor Human genes 0.000 description 2
- 102100035290 Fibroblast growth factor 13 Human genes 0.000 description 2
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 2
- 108010052343 Gastrins Proteins 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 101001053438 Homo sapiens Iroquois-class homeodomain protein IRX-2 Proteins 0.000 description 2
- 101001053430 Homo sapiens Iroquois-class homeodomain protein IRX-3 Proteins 0.000 description 2
- 101001133081 Homo sapiens Mucin-2 Proteins 0.000 description 2
- 101000606748 Homo sapiens Pepsin A-5 Proteins 0.000 description 2
- 101000984042 Homo sapiens Protein lin-28 homolog A Proteins 0.000 description 2
- 102100024434 Iroquois-class homeodomain protein IRX-2 Human genes 0.000 description 2
- 102100024374 Iroquois-class homeodomain protein IRX-3 Human genes 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 229930182816 L-glutamine Natural products 0.000 description 2
- 102000043136 MAP kinase family Human genes 0.000 description 2
- 108091054455 MAP kinase family Proteins 0.000 description 2
- 230000005723 MEK inhibition Effects 0.000 description 2
- 101710119980 Macrophage migration inhibitory factor Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108091092878 Microsatellite Proteins 0.000 description 2
- 102100034263 Mucin-2 Human genes 0.000 description 2
- 101100446513 Mus musculus Fgf4 gene Proteins 0.000 description 2
- 101100257363 Mus musculus Sox2 gene Proteins 0.000 description 2
- 241000204031 Mycoplasma Species 0.000 description 2
- 102100038553 Neurogenin-3 Human genes 0.000 description 2
- 101100519293 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) pdx-1 gene Proteins 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 102100039652 Pepsin A-5 Human genes 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102100032709 Potassium-transporting ATPase alpha chain 2 Human genes 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100025460 Protein lin-28 homolog A Human genes 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 101150077398 WNT-1 gene Proteins 0.000 description 2
- 101150027447 WNT-5B gene Proteins 0.000 description 2
- 101150025022 WNT11 gene Proteins 0.000 description 2
- 102000052548 Wnt-4 Human genes 0.000 description 2
- 101150000483 Wnt6 gene Proteins 0.000 description 2
- 101100485097 Xenopus laevis wnt11b gene Proteins 0.000 description 2
- 101100540688 Xenopus tropicalis wnt11b-1 gene Proteins 0.000 description 2
- 101100540689 Xenopus tropicalis wnt11b-2 gene Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 108010023082 activin A Proteins 0.000 description 2
- 108010023079 activin B Proteins 0.000 description 2
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- SESFRYSPDFLNCH-UHFFFAOYSA-N benzyl benzoate Chemical compound C=1C=CC=CC=1C(=O)OCC1=CC=CC=C1 SESFRYSPDFLNCH-UHFFFAOYSA-N 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- AOXOCDRNSPFDPE-UKEONUMOSA-N chembl413654 Chemical compound C([C@H](C(=O)NCC(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@H](CCSC)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](C)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@@H](N)CCC(O)=O)C1=CC=C(O)C=C1 AOXOCDRNSPFDPE-UKEONUMOSA-N 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- XHBVYDAKJHETMP-UHFFFAOYSA-N dorsomorphin Chemical compound C=1C=C(C2=CN3N=CC(=C3N=C2)C=2C=CN=CC=2)C=CC=1OCCN1CCCCC1 XHBVYDAKJHETMP-UHFFFAOYSA-N 0.000 description 2
- 238000007876 drug discovery Methods 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 230000013020 embryo development Effects 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 210000001654 germ layer Anatomy 0.000 description 2
- 238000003364 immunohistochemistry Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 210000004347 intestinal mucosa Anatomy 0.000 description 2
- 101150022285 irx2 gene Proteins 0.000 description 2
- -1 klf4 Proteins 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000031142 liver development Effects 0.000 description 2
- 230000008567 mammal embryogenesis Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002493 microarray Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 210000003550 mucous cell Anatomy 0.000 description 2
- 210000005036 nerve Anatomy 0.000 description 2
- 230000020477 pH reduction Effects 0.000 description 2
- 230000015031 pancreas development Effects 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000007310 pathophysiology Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000007727 signaling mechanism Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 208000001608 teratocarcinoma Diseases 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000034512 ubiquitination Effects 0.000 description 2
- 238000010798 ubiquitination Methods 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- 101150070086 wnt8b gene Proteins 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 101150024971 ATP4 gene Proteins 0.000 description 1
- 101150096411 AXIN2 gene Proteins 0.000 description 1
- 239000012103 Alexa Fluor 488 Substances 0.000 description 1
- 239000012110 Alexa Fluor 594 Substances 0.000 description 1
- 239000012114 Alexa Fluor 647 Substances 0.000 description 1
- 101000993093 Arabidopsis thaliana Heat stress transcription factor B-2a Proteins 0.000 description 1
- 101100189945 Arabidopsis thaliana PER63 gene Proteins 0.000 description 1
- 101150017509 Atp4b gene Proteins 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 102100035683 Axin-2 Human genes 0.000 description 1
- 101150023803 Bhlha15 gene Proteins 0.000 description 1
- 101100454433 Biomphalaria glabrata BG01 gene Proteins 0.000 description 1
- 101100126292 Caenorhabditis elegans irx-1 gene Proteins 0.000 description 1
- 101100510259 Caenorhabditis elegans klf-2 gene Proteins 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 102100028914 Catenin beta-1 Human genes 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 108091007854 Cdh1/Fizzy-related Proteins 0.000 description 1
- 102000038594 Cdh1/Fizzy-related Human genes 0.000 description 1
- 241001227713 Chiron Species 0.000 description 1
- 102000010792 Chromogranin A Human genes 0.000 description 1
- 108010038447 Chromogranin A Proteins 0.000 description 1
- 102100040835 Claudin-18 Human genes 0.000 description 1
- DWJXYEABWRJFSP-XOBRGWDASA-N DAPT Chemical compound N([C@@H](C)C(=O)N[C@H](C(=O)OC(C)(C)C)C=1C=CC=CC=1)C(=O)CC1=CC(F)=CC(F)=C1 DWJXYEABWRJFSP-XOBRGWDASA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 101100428953 Danio rerio wnt8a gene Proteins 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 201000009051 Embryonal Carcinoma Diseases 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 241001481760 Erethizon dorsatum Species 0.000 description 1
- 101150090015 FOXF1 gene Proteins 0.000 description 1
- 102100028073 Fibroblast growth factor 5 Human genes 0.000 description 1
- 102100037680 Fibroblast growth factor 8 Human genes 0.000 description 1
- 101710181403 Frizzled Proteins 0.000 description 1
- 102000005698 Frizzled receptors Human genes 0.000 description 1
- 108010045438 Frizzled receptors Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102100032606 Heat shock factor protein 1 Human genes 0.000 description 1
- 101710190344 Heat shock factor protein 1 Proteins 0.000 description 1
- 101150094793 Hes3 gene Proteins 0.000 description 1
- 101150029234 Hes5 gene Proteins 0.000 description 1
- 229940122957 Histamine H2 receptor antagonist Drugs 0.000 description 1
- 101000916173 Homo sapiens Catenin beta-1 Proteins 0.000 description 1
- 101000749329 Homo sapiens Claudin-18 Proteins 0.000 description 1
- 101001060267 Homo sapiens Fibroblast growth factor 5 Proteins 0.000 description 1
- 101001027382 Homo sapiens Fibroblast growth factor 8 Proteins 0.000 description 1
- 101000975496 Homo sapiens Keratin, type II cytoskeletal 8 Proteins 0.000 description 1
- 101000843556 Homo sapiens Transcription factor HES-1 Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 102100023972 Keratin, type II cytoskeletal 8 Human genes 0.000 description 1
- XXYGTCZJJLTAGH-UHFFFAOYSA-N LGK974 Chemical compound C1=NC(C)=CC(C=2C(=CC(CC(=O)NC=3N=CC(=CC=3)C=3N=CC=NC=3)=CN=2)C)=C1 XXYGTCZJJLTAGH-UHFFFAOYSA-N 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101100324687 Mus musculus Atp1b1 gene Proteins 0.000 description 1
- 101100310645 Mus musculus Sox15 gene Proteins 0.000 description 1
- 108091057508 Myc family Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 108700026495 N-Myc Proto-Oncogene Proteins 0.000 description 1
- 102100030124 N-myc proto-oncogene protein Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 101100436871 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) atp-3 gene Proteins 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 101150005926 Pc gene Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- GOOHAUXETOMSMM-UHFFFAOYSA-N Propylene oxide Chemical compound CC1CO1 GOOHAUXETOMSMM-UHFFFAOYSA-N 0.000 description 1
- 102000006270 Proton Pumps Human genes 0.000 description 1
- 206010037407 Pulmonary hypoplasia Diseases 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 101100016889 Rattus norvegicus Hes2 gene Proteins 0.000 description 1
- 101150099498 SOX1 gene Proteins 0.000 description 1
- 101150071739 Tp63 gene Proteins 0.000 description 1
- 102100030798 Transcription factor HES-1 Human genes 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- 102000013127 Vimentin Human genes 0.000 description 1
- 108010065472 Vimentin Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- ITBGJNVZJBVPLJ-UHFFFAOYSA-N WAY-316606 Chemical compound FC(F)(F)C1=CC=C(S(=O)(=O)C=2C=CC=CC=2)C=C1S(=O)(=O)NC1CCNCC1 ITBGJNVZJBVPLJ-UHFFFAOYSA-N 0.000 description 1
- 101150109862 WNT-5A gene Proteins 0.000 description 1
- 108700020987 Wnt-1 Proteins 0.000 description 1
- 102000043366 Wnt-5a Human genes 0.000 description 1
- 108700020483 Wnt-5a Proteins 0.000 description 1
- 101100257370 Xenopus laevis sox3-a gene Proteins 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- 229960002903 benzyl benzoate Drugs 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- HOQPTLCRWVZIQZ-UHFFFAOYSA-H bis[[2-(5-hydroxy-4,7-dioxo-1,3,2$l^{2}-dioxaplumbepan-5-yl)acetyl]oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HOQPTLCRWVZIQZ-UHFFFAOYSA-H 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 230000011748 cell maturation Effects 0.000 description 1
- 230000023715 cellular developmental process Effects 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000010226 confocal imaging Methods 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- 108010007093 dispase Proteins 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 238000011977 dual antiplatelet therapy Methods 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 210000003981 ectoderm Anatomy 0.000 description 1
- 230000017793 embryonic organ development Effects 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 230000020619 endoderm development Effects 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000009786 epithelial differentiation Effects 0.000 description 1
- 230000008202 epithelial morphogenesis Effects 0.000 description 1
- 210000000646 extraembryonic cell Anatomy 0.000 description 1
- 230000006832 extrinsic signaling Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 101150113268 ghrl gene Proteins 0.000 description 1
- 210000002149 gonad Anatomy 0.000 description 1
- 230000002710 gonadal effect Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 239000003485 histamine H2 receptor antagonist Substances 0.000 description 1
- 230000001146 hypoxic effect Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 101150073113 irx5 gene Proteins 0.000 description 1
- 101150105127 klf1 gene Proteins 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000001531 micro-dissection Methods 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 230000003990 molecular pathway Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000000472 morula Anatomy 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000012285 osmium tetroxide Substances 0.000 description 1
- 229910000489 osmium tetroxide Inorganic materials 0.000 description 1
- 238000001139 pH measurement Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 238000000059 patterning Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000011458 pharmacological treatment Methods 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 101150103310 pitx1 gene Proteins 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920000724 poly(L-arginine) polymer Polymers 0.000 description 1
- 108010011110 polyarginine Proteins 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 210000005238 principal cell Anatomy 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 210000001187 pylorus Anatomy 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000007261 regionalization Effects 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- DYPYMMHZGRPOCK-UHFFFAOYSA-N seminaphtharhodafluor Chemical compound O1C(=O)C2=CC=CC=C2C21C(C=CC=1C3=CC=C(O)C=1)=C3OC1=CC(N)=CC=C21 DYPYMMHZGRPOCK-UHFFFAOYSA-N 0.000 description 1
- 230000007781 signaling event Effects 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- IHQKEDIOMGYHEB-UHFFFAOYSA-M sodium dimethylarsinate Chemical compound [Na+].C[As](C)([O-])=O IHQKEDIOMGYHEB-UHFFFAOYSA-M 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 101150089855 sox3 gene Proteins 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000008280 toxic mechanism Effects 0.000 description 1
- 108091008023 transcriptional regulators Proteins 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 238000012762 unpaired Student’s t-test Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000005048 vimentin Anatomy 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 101150006165 wnt8 gene Proteins 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0679—Cells of the gastro-intestinal tract
-
- 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
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
-
- 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
- C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
-
- 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
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
-
- 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
- C12N2501/40—Regulators of development
- C12N2501/415—Wnt; Frizzeled
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/03—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from non-embryonic pluripotent stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
Abstract
本公开涉及用于体外制造胃底组织的方法和与其相关的组合物,用于通过定向分化将哺乳动物定形内胚层(DE)细胞转化为特定组织或器官的方法。特别地,本公开涉及由分化的定形内胚层形成的胃底组织和/或类器官的形成。
Description
本申请是申请号为201780024903.5的同名中国专利申请的分案申请。
相关申请的交叉引用
本申请要求2016年5月5日提交的美国临时专利申请62/332,194的优先权和权益,所述申请的内容通过引用整体并入。
政府支持条款
本发明依据All 16491和DK092456在政府支持下进行。政府具有本发明中的某些权利。
背景技术
虽然胃疾病在全球流行,但是存在很少用于研究人类胃的基底上皮组织的足够模型。人类胃底类型胃类器官(hFGO)的发展将是研究人类胃生理学、病理生理学和药物发现的分子基础的新型且强力的模型***。
发明内容
本公开涉及用于通过定向分化将哺乳动物定形内胚层(DE)细胞转化为特定组织或器官的方法。特别地,本公开涉及由分化的定形内胚层形成的胃底组织和/或类器官的形成。
附图说明
本领域技术人员将理解以下描述的附图仅出于说明性目的。附图并不旨在以任何方式限制本教导内容的范围。
图1.Wnt/β-连环蛋白信号传导对于小鼠中胚胎胃底的规范化是所需的。a,Pdx1和Sox2在胃窦(a)中表达,而Pdx1在胃底(f)中不存在,这通过在E18.5时的表达Atp4b的壁细胞来鉴定。b,来自Axin2:LacZ报道基因胚胎的E10.5前肠的X-gal染色显示Wnt活性局限于胃的前部区域但从后部胃被排除。c,胃上皮组织中β-连环蛋白的缺失致使Pdxl前部扩展到胃的基底区域中。d,在E18.5 ShhCre/+β-连环蛋白fl/fl(cKO)胚胎中,Pdx1在整个胃中表达,在含有壁细胞的上皮组织的一些剩余的片中除外。插图1a-c和2a-c分别显示对照和cKO胃中的框内区域。e,在cKO胃中,Ctnnb1表现出嵌合体缺失,并且壁细胞仅在Ctnnb1足够的上皮组织中分化。比例尺,250μm(a),200μm(c),以及500μm(d和e)。
图2.β-连环蛋白活化促进胃底从人类前肠祖细胞球形体发育。a,针对胃底和胃窦hGO两者的分化方案的示意图。b、c,在第9天,CHIR处理的类器官表现出PDX1减少,IRX2、IRX3和IRX5增加,并且胃标记物SOX2或GATA4没有变化。*,p<0.05;双尾学生t测试;n=3个生物平行测定,数据代表4个独立实验。d,hFGO与hAGO类似地成长,但是还表现出腺体出芽形态发生(白色箭头)。e,两种hGO均含有表达CDH1、KRT8和CTNNB1以及胃标记物GATA4和CLDN18的上皮组织。hAGO表现出几乎无所不在的PDX1表达,而hFGO并非如此。比例尺,50μm(c),500μm(d)以及100μm(e)。误差条表示s.e.m.。
图3.hGO中粘液和内分泌细胞谱系的分化。a,胃的胃底和胃窦腺体中存在的共有的和不同的谱系的示意图。b,胃窦和胃底hGO两者均含有MUC5AC阳性表面粘液细胞和MUC6阳性颈粘液细胞。c、d,hFGO含有表达泛内分泌标记物SYP的内分泌细胞。在hFGO中识别到各种不同的激素细胞类型,包括表达GHRL、SST和组胺的内分泌细胞。胃窦特异性G细胞标记物GAST在hAGO中表达,但是不在hFGO中表达;相反地,hFGO中富含GHRL。**,p<0.01;双尾学生t测试;分别在hAGO和hFGO中,n=8和24个生物平行测定,数据代表6个独立实验。e,hAGO,而不是hFGO,能够响应于促内分泌转录因子NEUROG3(+dox)的表达而产生胃窦特异性的表达GAST的内分泌细胞。*,p<0.01;双尾学生t测试;n=4个生物平行测定,数据代表3个独立实验。误差条表示s.e.m.。
图4.hFGO中主细胞的形成。a,hFGO具有MIST1和胃蛋白酶原C(PGC)阳性细胞两者。b,高放大率的图(a)中的框内区域,显示具有顶端PGC染色的细胞集群的腺体。c,与hAGO相比,hFGO具有显著增加的主细胞标记物PGA5(1,000倍)、PGC(100倍)和MIST1(>10倍)的表达。**,p<0.05;双尾学生t测试。n=3个生物平行测定,数据代表4个独立实验。d,含有密集的酶原颗粒的hFGO细胞的透射电子显微照片,指示主细胞。e,在MEK抑制剂(PD03)的存在或不存在下,与hAGO相比,在hFGO中的胃蛋白酶原蛋白含量。**,p<0.0001,与hAGO相比,双尾学生t测试,分别在hAGO、对照hFGO和hFGO(没有PD03)中,n=8、12和11个生物平行测定。比例尺,200μm(a),25μm(b),以及10μm(d)。误差条表示s.e.m.。
图5.对驱动hFGO中功能性壁细胞的分化的途径的识别。a,与在基线处的胃窦相比,壁细胞基因ATP4、ATP4B和GIF的表达在hFGO中表现出10至100倍增加,但是通过将hFGO暴露于两天的PD03/BMP4脉冲而明显地增加。**,p<0.05,与hAGO相比;#,p<0.05,与对照hFGO相比,双尾学生t测试,n=4个生物平行测定,数据代表15个独立实验。b,在用PD03/BMP4处理之后,对表达ATP4B的壁细胞的刺激分化。c,hFGO衍生的壁细胞与体内成熟小鼠胃底上皮组织中存在的那些相似。d,具有近似于壁细胞的小管结构的hFGO细胞的透射电子显微照片。e,人类胃底腺体的上皮组织和hFGO上皮组织被组织成表面上皮组织中的表达MUC5AC的细胞和腺体单元中的表达ATP4B的壁细胞。f,通过SNARF-5F的腔内注射,对响应于组胺的类器官中腔内pH的分析。hFGO中腔内pH快速下降,而hAGO未表现出应答。通过用法莫替丁或奥美拉唑预处理类器官来阻断酸化。分别在hFGO(组胺)、hFGO(组胺和法莫替丁)、hFGO(组胺和奥美拉唑)、以及hAGO(组胺)中,n=9、9、7以及4个生物平行测定;数据代表三个独立实验。g,在60分钟之后,组胺在分离的小鼠胃腺中和在hFGO中诱导呈小管类型模式的吖啶橙(AO)染料积累。比例尺,100μm(b),10μm(c),10μm(d),100μm(e;人类胃底),20μm(e;hFGO),以及10μm(g)。误差条表示s.e.m.。
图6.在体内发育的胃中限定分子区域。A,对胚胎小鼠胃(E14.5)中Sox2、Pdx1和Gata4的分析显示胃底(f)是Sox2+Gata4+Pdx1-,而胃窦(a)是Sox2+Gata4+Pdx1+。前胃(fs)表达Sox2,但是既不表达Gata4也不表达Pdx1。b,明视场立体显微照片显示通过qPCR分析的E14.5小鼠胃的解剖区域。fs,前胃;f,胃底;a,胃窦;d,十二指肠。c,针对已知的区域表达的标记物(Sox2,P63,Gata4,Pdx1以及Cdx2)通过qPCR分析b中的解剖区域以验证微解剖的准确性。对解剖的E14.5胃区域的qPCR分析显示,与胃窦相比,胃底中富含推定的胃底标记物Irx1、Irx2、Irx3、Irx5以及Pitx1。n=4个生物平行测定/解剖区域。比例尺,500μm。误差条表示s.d.。
图7.对β-连环蛋白cKO胚胎的分析。a,在E12.4和E14.5之前,在整个背侧胃上皮组织以及cKO胚胎的大部分近侧胃上皮组织中观察到异常Pdx1表达。b,对E14.5 cKO前肠的解剖区域(图6,b)的qPCR分析显示Pdx1在胃底和前胃区域中的显著上调。相反地,Irx2、Irx3和Irx5在这些近侧区域中明显地减少。*,p<0.05;双尾学生t测试,对于每种基因型,n=3个生物平行测定/解剖区域。c,E18.5解剖内脏的立体显微照片证明cKO胚胎表现出肺发育不全,如先前所报告的。胃肠道,特别是胃,的大小明显减小。d,E18.5时的免疫荧光染色揭示Ctnnb1的嵌合体缺失模式。框内区域在图1中示出,e,在E18.5 cKO胃中,缺少Ctnnb1染色的重组腺体不含有壁细胞,而在Ctnnb1阳性腺体中观察到稳健的壁细胞分化。比例尺,200μm(a),500μm(d)以及50μm(e)。误差条表示s.d.。
图8.hGO中胃底命运的稳定诱导和方案的效率。a,申请人研究了需要多长时间的CHIR处理来建立胃底特性。简短的CHIR处理(d6-9)和随后直至第34天的对照生长培养基中类器官的生长产生表达胃窦标记物PDX1的胃底类器官,从而表明短的CHIR处理不产生稳定的胃底命运。申请人然后测试是否需要更长地暴露于CHIR来保持胃底命运,并且发现仅至少达到第29天的连续处理可维持胃窦标记物PDX1的低表达。*,p<0.05,与对照胃窦hGO相比;双尾学生t测试。n=3个生物平行测定,数据代表2个独立实验。b、c,在方案过程中,PDX1在CHIR处理的类器官中保持较低,而IRX5表达持续升高。*,p<0.05;双尾学生t测试;n=3个生物平行测定/时间点。d,d6后部前肠球形体向早期胃类器官(d20)的转化在hAGO和hFGO方案两者中均大于80%有效。e,在d20,hFGO上皮组织是~90%GATA4+/SOX2+/PDX1-,而hAGO上皮组织是~90%GATA4+/SOX2+/PDX1+。**,p<0.001,双尾学生t测试,n=4个生物平行测定/实验,示出两个独立的实验。比例尺,100μm(c)和200μm(d)。
图9.从前肠祖细胞诱导肠命运的Wnt/β-连环蛋白活化的BMP依赖性。a,肠特异性转录因子CDX2在第9天或第20天在CHIR处理的hGO中不被显著地诱导。b,当与人类肠类器官(hIO)相比时,胃底或胃窦hGO均不表达与肠细胞类型相关联的基因,包括MUC2、CCK和SCT。*,p<0.05,与hIO相比;双尾学生t测试。n=3个生物平行测定。c,通过WNT和BMP的活性协调地控制前后命运。在BMP抑制剂头蛋白的存在下,所有的类器官维持前肠(SOX2+),无论Wnt/β-连环蛋白途径活性如何;然而,在BMP4的存在下,所有的类器官被后部化(CDX2+)。处于BMP抑制状态的Wnt(CHIR)的活化引起胃底模式(SOX2+、PDX1-、CDX2-),而WNT(CHIR)的活化和BMP4的添加引起肠命运(CDX2+)。*,p<0.05,与类似的头蛋白处理的条件相比;双尾学生t测试。n=3个生物平行测定。d,人类组织的免疫荧光染色揭示CLDN18是肠中不存在的胃特异性上皮标记物。比例尺,200μm。误差条表示s.e.m.。
图10.hFGO含有由相关联的间充质层支持的组织腺体。a,透射电子显微照片证明hFGO腺体表现出具有窄顶膜的组织构造。b,hFGO和hAGO两者均含有支持层FOXF1+/VIM+未分化的成纤维细胞。比例尺,5μm(a)和100μm(b)。
图11.人类胃类器官中的区域特异性细胞分化。a,胃窦和胃底hGO表现出粘液细胞标记物MUC5AC和MUC6的相当的表达。b,如在透射电子显微照片中所示,hFGO含有表现出与颈粘液细胞(分化的主细胞的前体)一致的颗粒模式的丰富细胞。c,衍生自NEUROG3缺陷hESC系的hGO中的NEUROG3的外源性表达诱导SYP阳性内分泌细胞的稳健分化。虽然hAGO和hFGO两者均形成表达GHRL和SST的内分泌细胞,但是GAST+G细胞的规范化仅在hAGO中观察到。d,hGO和人类胃活检组织中细胞谱系标记物的表达比较。qPCR分析证明hGO表现出若干谱系标记物(MUC5AC、ATP4B)的相当的表达水平,而其他基因在比完全分化的成熟人类胃中所存在的低得多的水平下表达(ATP4A、PGA5和PGC)。比例尺,5μm(b)和100μm(c)。误差条表示s.d.(a)和s.e.m.(b)。
图12.对鼠主细胞发育的分析。a,与早在后期胚胎阶段就表达功能性标记物(Atp4b)的壁细胞不同,主细胞基因产物直至很晚的发育阶段为止才可检测。在胚胎(E18.5)和幼年(P12)胃中,仍未表达Gif和Pgc,从而表明主细胞在发育中比胃上皮组织中的其他谱系成熟晚得多。b,虽然Pgc不存在,但是P12小鼠胃确实含有表达核Mist1(主细胞特异性标记物)的丰富腺细胞。因此,主细胞确实较早地被规范化,但是花费若干周来发展终端分化标记物的稳健表达。比例尺,100μm(a)和200μm(b)。
图13.对促进胃底hGO中壁细胞分化的途径的筛选。a,为了测试能够诱导壁细胞分化的生长因子/小分子,将hFGO暴露于指示的激动剂或拮抗剂两天(30至32),然后在第34天进行分析。在不同途径的筛选实验中,发现仅用PD03进行的MEK抑制稳健地诱导ATP4A/B的表达。b,从培养基减少或去除EGF不足以再产生MEK抑制的作用。c,PD03/BMP4的诱导壁细胞发育的能力对于胃底hGO是独有的,因为胃窦hGO不响应于PD03/BMP4而表达胃底标记物。d,暴露于PD03/BMP4快速增加胃底hGO中ATP4A和ATP4B的表达。e,用PD03/BMP4诱导壁细胞生成不显著地影响主细胞(PGA5和PGC)和内分泌细胞(CHGA)的分化。f,稳健的壁细胞分化需要在hFGO分化方案的每个阶段处的操纵,因为去除任何单个步骤导致ATP4A/B表达的丧失。误差条表示s.d.(a至c)和s.e.m.(d至f)。
图14.胃类器官中的现场体外pH监测。a,染料SNAFR5F在5至8的pH范围内表现出响应性,这使得其非常适于检测响应于壁细胞介导的酸分泌的生理变化。b,添加组胺之前(封闭的圆)和之后60分钟(开圆)记录的培养基和腔内pH测量值。胃窦hGO没有反应,而胃底hGO腔内pH响应于组胺而降低。通过用法莫替丁或奥美拉唑预处理类器官来抑制酸化。另外,奥美拉唑足以在暴露于组胺之前升高胃底类器官中的pH,从而表明胃底类器官中的基线酸分泌。培养基pH在任何类器官中均不改变。***,p<0.001,与组胺之前相比;$$$,p<0.001,与在没有组胺的情况下的腔内pH相比;###,p<0.001,与在使用组胺的情况下的腔内pH相比;双尾学生t测试。c,hFGO含有壁细胞密集的腺体,其中吖啶橙(AO)在沿腺体的内腔排列的几乎所有细胞中积累。d,在hFGO中的壁细胞中观察到呈小管类型模式的AO积累。比例尺,10μm。误差条表示s.d.。
图15.人类胃类器官的连续传代。a,用于确定hGO中的胃干细胞的存在的实验的示意性表示。b,当片段在仅含有EGF的培养基中生长时,它们不生长或扩展而形成新的类器官。然而,向培养基添加CHIR和FGF10,这足以支持个体片段生长为新形成的类器官。c,在两代之后,hFGO仍然表达与胃表型一致的基因,包括PGC、MUC6、MUC5AC以及GHRL。经历连续传代同时维持胃特性的此能力支持以下结论:hFGO含有其具有的特性类似于成人胃干细胞的细胞。d,虽然传代的hFGO表达与若干分化的胃细胞类型相关联的标记物,但是它们不表达与壁细胞相关联的基因,诸如ATP4B。另外,壁细胞的分化不可如它们在传代之前可以的那样通过MEK抑制来诱导。误差条表示s.d.
具体实施方式
除非另外指出,否则术语应根据相关领域的普通技术人员的常规用法来理解。
如本文所用,术语“胃底组织”意指含有胃底细胞类型的原体中存在的胃底类型的胃上皮组织,包括但不限于产生酸的壁细胞和产生蛋白酶的主细胞。
如本文所用,术语“定形内胚层(DE)细胞”意指原肠胚形成的过程产生的三个初级胚层中的一个。
如本文所用,术语“wnt信号传导途径”意指wnt/β-连环蛋白途径,并且是通过Wnt配体和通过β-连环蛋白起作用的卷曲细胞表面受体介导的信号转导途径。
如本文所用,关于途径(诸如“wnt途径”)的术语“活化剂”意指活化Wnt/β-连环蛋白途径、使得Wnt/β-连环蛋白靶标增加的物质。
如本文所用,术语“FGF信号传导途径活化剂”意指活化FGF途径、使得FGF靶标增加的物质。
如本文所用,术语“BMP信号传导途径抑制剂”意指干扰BMP途径并且致使BMP靶标降低的物质。
如本文所用,术语“生长因子”意指能够刺激细胞过程(包括但不限于生长、增殖、形态发生或分化)的物质。
如本文所用,术语“胃底谱系”意指原体胃中的胃底上皮组织中存在的细胞类型。
如本文所用,术语“SOX2+GATA+PDX1-上皮组织”意指表达所列的蛋白质的上皮组织。
如本文所用,术语标记物的“稳定表达”意指在生长环境改变后不改变的表达。
如本文所用,术语“全能干细胞(totipotent stem cells)”(还被称为全能性干细胞(omnipotent stem cells))是可分化成胚胎和胚胎外细胞类型的干细胞。此类细胞可构建完整的、可存活的生物体。这些细胞由***和***细胞的融合产生。由受精卵的前几次***产生的细胞也是全能的。
如本文所用,术语“多能干细胞(PSC)”(还通常被称为PS细胞)涵盖可分化成几乎所有细胞的任何细胞,即,衍生自三个胚层(生殖上皮组织),包括内皮层(内部胃内层、胃肠道、肺)、中胚层(肌肉、骨、血液、泌尿生殖器)和外胚层(表皮组织和神经细胞)中的任一个的细胞。PSC可以是全能细胞的子代,衍生自胚胎(包括胚胎生殖细胞)或通过强迫某些基因的表达来通过非多能细胞(诸如成人体细胞)的诱导获得。
如本文所用,术语“诱导的多能干细胞(iPSC)”(还通常缩写为iPS细胞)是指通过诱导某些基因的“受强迫的”表达来人工衍生自通常是非多能细胞(诸如成人体细胞)的一类多能干细胞。
如本文所用,术语“前体细胞”涵盖可用于本文所述的方法的任何细胞,通过所述方法,一种或多种前体细胞获得使自身更新或分化成一种或多种特化的细胞类型的能力。在一些实施方案中,前体细胞是多能的或具有变得多能的能力。在一些实施方案中,前体细胞经受外部因子(例如,生长因子)的处理来获得多能性。在一些实施方案中,前体细胞可以是全能干细胞;多能干细胞(诱导的或非诱导的);多潜能干细胞;以及单能干细胞。在一些实施方案中,前体细胞可来自胚胎、婴儿、儿童或成人。在一些实施方案中,前体细胞可以是经受处理、使得通过基因操纵或蛋白质/肽处理而被赋予多能性的体细胞。
在发育生物学中,细胞分化是较少特化的细胞变成较多特化的细胞类型的过程。如本文所用,术语“定向分化”描述较少特化的细胞变成特定的特化靶细胞类型的过程。特化靶细胞类型的特定性可通过可用于限定或改变原始细胞的命运的任何可应用的方法来确定。示例性方法包括但不限于基因操纵、化学处理、蛋白质处理以及核酸处理。
如本文所用,术语“细胞成分”是个体基因、蛋白质、表达mRNA的基因和/或任何其他可变的细胞组分或蛋白质活性,诸如例如通常由本领域技术人员在生物实验(例如,通过微阵列或免疫组织化学)中测量的蛋白质修饰(例如,磷酸化)的程度。与生命***和常见的人类疾病所基于的生物化学过程的复杂网络相关的重要发现和基因发现以及结构确定现在可有助于将细胞成分丰度数据应用为研究过程的一部分。细胞成分丰度数据可帮助识别生物标记物、区分疾病亚型以及识别毒性机制。
衍生自胚胎细胞的多能干细胞
在一些实施方案中,一个重要步骤是获得多能的或可被诱导而变得多能的干细胞。在一些实施方案中,多能干细胞衍生自胚胎干细胞,所述胚胎干细胞进而衍生自早期哺乳动物胚胎的全能细胞并且能够进行体外无限非分化增殖。胚胎干细胞是衍生自胚泡(一种早期胚胎)的内细胞团的多能干细胞。用于从胚细胞衍生胚胎干细胞的方法在本领域中是熟知的。人类胚胎干细胞H9(H9-hESC)用于本申请所述的示例性实施方案中,但是本领域的技术人员将理解,本文所述的方法和***可应用于任何干细胞。
可用于根据本发明的实施方案中的另外的干细胞包括但不限于由以下提供或以下中所述的那些:由在加利福尼亚大学旧金山分校(UCSF)处的人类胚胎干细胞研究中心的美国国立干细胞库(NSCB)拥有的数据库;在Wi Cell研究院处的WISC细胞库;威斯康星大学干细胞和再生医学中心(UW-SCRMC);Novocell公司(加利福尼亚州圣地亚哥);CellartisAB公司(瑞典哥德堡);胚胎干细胞国际私人投资有限公司(新加坡);以色列理工学院(以色列海法市);以及由普林斯顿大学和宾夕法尼亚大学拥有的干细胞数据库。可用于根据本发明的实施方案中的示例性胚胎干细胞包括但不限于SA01(SA001);SA02(SA002);ES01(HES-1);ES02(HES-2);ES03(HES-3);ES04(HES-4);ES05(HES-5);ES06(HES-6);BG01(BGN-01);BG02(BGN-02);BG03(BGN-03);TE03(13);TE04(14);TE06(16);UC01(HSF1);UC06(HSF6);WA01(H1);WA07(H7);WA09(H9);WA13(H13);WA14(H14)。
关于胚胎干细胞的更多细节可见于,例如,Thomson等人,1998,“《衍生自人类胚泡的胚胎干细胞系(Embryonic Stem Cell Lines Derived from Human Blastocysts)》,”《科学杂志(Science)》282(5391):1145-1147;Andrews等人,2005,“《胚胎干(ES)细胞和胚胎癌(EC)细胞:同一事物的对立面(Embryonic stem(ES)cells and embryonal carcinoma(EC)cells:opposite sides of the same coin)》,”《生化学会会刊(Biochem SocTrans)》33:1526-1530;Martin 1980,“《畸胎癌和哺乳动物胚胎发生(Teratocarcinomasand mammalian embryogenesis)》,”《科学杂志(Science)》209(4458):768-776;Evans和Kaufman,1981,“《来自小鼠胚胎的多能细胞的培养的建立(Establishment in culture ofpluripotent cells from mouse embryos)》,”《自然杂志(Nature)》292(5819):154-156;Klimanskaya等人,2005,“《在没有饲养细胞的情况下衍生的人类胚胎干细胞(Humanembryonic stem cells derived without feeder cells)》,”《柳叶刀(Lancet)》365(9471):1636-1641;所述文献中的每一个在此整体并入本文。
诱导的多能干细胞(iPSC)
在一些实施方案中,iPSC通过将某些干细胞相关基因转染到非多能细胞,诸如成人成纤维细胞,中来衍生。转染通常通过病毒载体,诸如逆转录酶病毒,来实现。转染的基因包括主转录调节物Oct-3/4(Pouf51)和Sox2,虽然提出其他基因增强诱导的效率。在3-4周之后,少量的转染细胞开始变得在形态学上和生物化学上与多能干细胞相似,并且通常通过形态学选择、倍增时间,或通过报道基因和抗生素选择来分离。如本文所用,iPSC包括但不限于小鼠中的第一代iPSC、第二代iPSC以及人类诱导的多能干细胞。在一些实施方案中,使用逆转录病毒***来使用以下四种关键基因将人类成纤维细胞转化为多能干细胞:Oct3/4、Sox2、Klf4以及c-Myc。在另选的实施方案中,使用慢病毒***,用OCT4、SOX2、NANOG以及LIN28来转化体细胞。在iPSC中诱导其表达的基因包括但不限于Oct-3/4(例如,Pou5fl);Sox基因家族的某些成员(例如,Sox1、Sox2、Sox3以及Sox15);Klf家族的某些成员(例如,Klf1、Klf2、Klf4以及Klf5)、Myc家族的某些成员(例如,C-myc、L-myc和N-myc)、Nanog以及LIN28。
在一些实施方案中,采用基于非病毒的技术来生成iPSC。在一些实施方案中,可使用腺病毒来将必需的四种基因运送到小鼠的皮肤和肝脏细胞的DNA中,从而产生与胚胎干细胞相同的细胞。因为腺病毒不将其自身基因中的任一种与靶向宿主组合,所以消除了产生肿瘤的危险。在一些实施方案中,重编程可通过质粒来实现而根本不需要任何病毒转染***,但效率非常低。在其他实施方案中,使用蛋白质的直接递送来生成iPSC,从而消除对于病毒或基因修饰的需要。在一些实施方案中,使用相似的方法可能生成小鼠iPSC:用通过聚精氨酸锚定剂引导到细胞中的某些蛋白质重复处理细胞足以诱导多能性。在一些实施方案中,诱导多能性的基因的表达还可通过在低氧条件下用FGF2处理体细胞来增加。
关于胚胎干细胞的更多细节可见于,例如,Kaji等人,2009,“《多能性的不含病毒的诱导和重编程因子的随后切除(Virus free induction of pluripotency andsubsequent excision of reprogramming factors)》,”《自然杂志(Nature)》458:771-775;Woltjen等人,2009,“《piggyBac转位将成纤维细胞重编程到诱导的多能干细胞(piggyBac transposition reprograms fibroblasts to induced pluripotent stemcells)》,”《自然杂志(Nature)》458:766-770;Okita等人,2008,“《生成小鼠诱导的多能干细胞而不用病毒载体(Generation of Mouse Induced Pluripotent Stem Cells WithoutViral Vectors)》,”《科学杂志(Science)》322(5903):949-953;Stadtfeld等人,2008,“《生成诱导的多能干细胞而不用病毒整合》,”《科学杂志(Science)》322(5903):945-949;以及Zhou等人,2009,“《使用重组蛋白生成诱导的多能干细胞(Generation of InducedPluripotent Stem Cells Using Recombinant Proteins)》,”《细胞干细胞(Cell StemCell)》4(5):381-384;所述文献中的每一个在此整体并入本文。
在一些实施方案中,示例性iPS细胞系包括但不限于iPS-DF19-9;iPS-DF19-9;iPS-DF4-3;iPS-DF6-9;iPS(***);iPS(IMR90);以及iPS(IMR90)。
关于涉及DE发育的信号传导途径的功能的更多细节可见于,例如,Zom和Wells,2009,“《脊椎动物内胚层发育与器官形成(Vertebrate endoderm development and organformation)》,”《细胞发育生物学年度综述(Annu Rev Cell Dev Biol)》25:221-251;Dessimoz等人,2006,“《FGF信号传导对于沿前后轴在体内建立肠管区域是必要的(FGFsignaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo)》,”《发育机制(Mech Dev)》123:42-55;McLin等人,2007,“《前内胚层中Wnt/β-连环蛋白信号传导的约束对于肝脏和胰腺发育是重要的。发育(Repressionof Wnt/β-catenin signaling in the anterior endoderm is essential for liverand pancreas development.Development)》,”134:2207-2217;Wells和Melton,2000,《发育(Development)》127:1563-1572;de Santa Barbara等人,2003,“《肠上皮的发育和分化(Development and differentiation of the intestinal epithelium)》,”《细胞和分子生命科学(Cell Mol Life Sci)》60(7):1322-1332;所述文献中的每一个在此整体并入本文。
用于从多能细胞(例如,iPSC或ESC)产生定形内胚层的任何方法可应用于本文所述的方法。在一些实施方案中,多能细胞衍生自桑椹胚。在一些实施方案中,多能干细胞是干细胞。用于这些方法中的干细胞可包括,但不限于,胚胎干细胞。胚胎干细胞可衍生自胚胎内细胞团或衍生自胚胎性腺嵴。胚胎干细胞或生殖细胞可来源于各种动物种类,包括,但不限于,各种哺乳动物种类,包括人类。在一些实施方案中,使用人类胚胎干细胞来产生定形内胚层。在一些实施方案中,使用人类胚胎生殖细胞来产生定形内胚层。在一些实施方案中,使用iPSC来产生定形内胚层。
本文公开了用于将多能干细胞(PSC)分化成含有胃底上皮组织的胃类器官的方法。申请人首先识别胚胎胃底发育中的关键事件,然后重演所述关键事件以产生所要求保护的组合物。申请人发现小鼠胚胎中Wnt/β-连环蛋白信号传导的破坏致使胃底上皮组织转化为胃窦上皮组织,而hPSC衍生的前肠祖细胞中的Wnt/β-连环蛋白活化促进人类胃底类型胃类器官(hFGO)的发育。申请人然后使用hFGO来暂时识别多种信号传导途径在胃底细胞类型(包括主细胞和功能性壁细胞)的上皮形态发生和分化中的不同作用。虽然hFGO是用于研究人类胃底及其谱系的发育的强力新模型,它们也代表用于研究人类胃生理学、病理生理学和药物发现的分子基础的重要的新模型***。
在一个方面,公开了一种诱导胃底组织形成的体外方法。所述方法可包括以下步骤:
a)将哺乳动物定形内胚层(DE)细胞与wnt途径活化剂、FGF信号传导途径活化剂(例如,FGF4)、BMP信号传导途径抑制剂(例如,头蛋白)以及视黄酸接触第一时间段。Wnt信号传导可用,例如,像Wnt3a的蛋白质,或,例如,通过像抑制GSK3J3的Chiron的化学品来活化。第一时间段可以是三天±24小时。视黄酸可在第一时间段±24小时的第三天添加。在一个方面,第一时间段可进行足以从定形内胚层形成三维后部前肠球形体的时间段。
b)将所述三维后部前肠球形体在具有生长因子、Wnt信号传导途径活化剂、EGF信号传导途径活化剂、BMP信号传导途径抑制剂以及视黄酸的基底膜基质中悬浮第二时间段。第二时间段可以是三天±24小时。第二时间段可进行足以诱导包括胃底hGO(hFGO)的胃底谱系的时间段。
c)将步骤b)的hFGO用wnt途径活化剂和EGF信号传导途径活化剂培养第三时间段。第三时间段可以是,例如,11天±24小时。
d)将步骤c的hFGO用wnt信号传导途径活化剂、EGF信号传导途径活化剂和FGF10培养第四时间段。第四时间段可以是,例如,10天±24小时。
e)将步骤d的hFGO与MEK抑制剂接触第五时间段。MEK抑制剂可以是例如PD0325901。第五时间段可以是两天时间段±24小时,或足以形成包含功能性胃底细胞类型的胃底组织的时间段。
在一个方面,步骤e)还可包括将胃底hGO与BMP4信号传导的活化剂接触的步骤。在某些方面,步骤e可进行足以发育SOX2+GATA+PDX1-上皮组织的时间段。
在一个方面,功能性胃底细胞类型可以是表达质子泵蛋白并且分泌酸的壁细胞。在一个方面,功能性胃底细胞类型可以是分泌胃蛋白酶原的主细胞。
在一个方面,步骤d和步骤e进行足以赋予谱系标记物MUC5AC、MUC6、PGC和GHRL的稳定表达的时间段。
在一个方面,定形内胚层可衍生自选自以下的前体细胞:胚胎干细胞、胚胎生殖细胞、诱导的多能干细胞、中胚层细胞、定形内胚层细胞、后内胚层细胞、后内胚层细胞以及后肠细胞;衍生自多能干细胞的定形内胚层;衍生自选自胚胎干细胞、成人干细胞或诱导的多能干细胞的多能干细胞的定形内胚层。
在一个方面,定形内胚层可由将多能干细胞与选自以下的一种或多种分子接触而衍生:活化素、生长因子的TGF-β超家族的BMP亚组;Nodal、活化素A、活化素B、BMP4、Wnt3a以及其组合。
存在许多方法来活化Wnt/β-连环蛋白途径(参见http://web.stanford.edu/group/nusselab/cgi-bin/wnt/)。合适的一些现有的wnt信号传导途径活化剂包括但不限于:
基于蛋白质的活化剂:Wnt配体,包括但不限于Wnt1、Wnt2、Wnt2b、Wnt3、Wnt3a、Wnt8等;Wnt配体活性的改性剂,包括但不限于活化的Wnt卷曲受体、(LRP)共受体、R-spondin蛋白、Dkk蛋白、Wnt配体分泌和运输(Wntless,Porcupine)、抑制β-连环蛋白降解APC和GSK3β抑制的调节剂、活化的β-连环蛋白、组成型活化的TCF/Lef蛋白。
化学活化剂:存在超过28种已知的活化或抑制Wnt/β-连环蛋白信号传导的化学品。一些活化剂包括但不限于GSK3-β抑制剂CHIR99021、BIO、LY2090314、SB-216763、锂、porcupine抑制剂IWP、LGK974、C59、SFRP抑制剂WAY-316606、β-连环蛋白活化剂DCA。
在一个方面,WNT途径活化剂可以是选自以下的一种或多种分子:Wnt1、Wnt2、Wnt2b、Wnt3、Wnt3a、Wnt4、Wnt5a、Wnt5b、Wnt6、Wnt7a、Wnt7b、Wnt8a、Wnt8b、Wnt9a、Wnt9b、Wnt10a、Wnt10b、Wnt11以及Wnt16,例如Wnt3a,或例如,浓度在约50至约1500ng/ml之间的Wnt3a。
合适的FGF信号传导途径活化剂包括:FGF配体FGF2、FGF4、FGF5、FGF8等。活化形式的FGF受体。刺激FGF受体和受体下游的信号传导组分的蛋白质和化学品包括调节其活性的MAPK、MEK、ERK蛋白质和化学品。FGF信号传导可通过抑制FGF信号传导途径的抑制剂(包括但不限于Sprouty蛋白家族成员)来活化。
在一个方面,BMP信号传导途径抑制剂可选自,例如,头蛋白、Dorsomorphin、LDN189、DMH-1以及其组合,其中所述前体细胞可与浓度在约50至约1500ng/ml之间的BMP抑制剂接触。
在一个方面,所述步骤在体外实施。
在一个方面,公开了一种包含根据前述方法产生的胃组织的组合物。胃组织的特征可在于,例如,不具有神经分布和/或血管。
在一个方面,公开了一种诱导胃底组织形成的体外方法。所述方法可包括以下步骤:将胃底hGO(hFGO)与wnt途径活化剂和EGF信号传导途径活化剂接触第一时间段,并且与MEK抑制剂接触第二时间段,(其中所述MEK抑制剂可以是PD0325901),其中所述第一和第二时间段进行足以形成功能性胃底细胞类型的时间段;
其中所述hFGO通过以下方式获得:将三维后部前肠球形体在基底膜基质中与生长因子、wnt途径活化剂、EGF信号传导途径活化剂、BMP信号传导途径抑制剂以及视黄酸接触足以将所述三维后部前肠球形体转化为所述hFGO的时间段;
其中所述三维后部前肠球形体通过以下方式获得:将哺乳动物定形内胚层(DE)细胞与wnt途径活化剂、FGF信号传导途径活化剂、BMP信号传导途径抑制剂以及视黄酸接触。
实施例
最近,在三维体外类器官***的发展方面已取得相当多的进展1,,2。已证明类器官是将构造复杂性和细胞多样性与传统细胞培养方法的易处理性和可扩展性组合的强力实验模型。通过多能干细胞(PSC;包括胚胎干细胞和诱导的PSC两者)的定向分化进行的类器官生成提供优于其他方法的若干优点,包括起始材料来源不受限制、不需要外科采集组织以及易于进行基因操纵。另外,基于PSC的方法允许直接研究正常和异常人类发育所基于的机制3。然而,将PSC分化为特定类器官类型依赖于正常器官发育的稳健分子知识。对于一些器官,诸如胃,在对于驱动胚胎发育的分子途径的理解方面存在较大的差距。
胃在哺乳动物中是在结构上最多样的器官之一4。在人类中,胃粘膜通常由两种类型的上皮腺体组成5,6。位于胃的较近解剖区域(原体和胃底)中的泌酸的腺体包括分泌酸的壁细胞、产生蛋白酶的主细胞、产生粘液的细胞以及内分泌细胞。位于较远的胃窦和幽门中的窦类型腺体主要含有粘液细胞和内分泌细胞。为了简化命名的特定解剖和种类的***,术语‘胃底’和‘胃窦’用于广泛描述这两种组织学类型的胃上皮。申请人先前已开发了一种引导以将hPSC分化为含有具有正常胃窦细胞类型的纯胃窦上皮组织的三维胃组织(人类胃类器官;hGO)的方法7。虽然胃窦hGO(hAGO)是用于研究胃窦谱系分配和宿主-微生物相互作用的稳健***,但是它们不允许研究胃底生物学和疾病。最近,Noguchi等人成功地将小鼠ESC分化为包括各种类型的小鼠胃组织的类器官8。然而,此方法使用小鼠ESC聚集和自发分化,从而产生异质的类器官,这通过复层上皮的存在来证明。此外,种类差异使得小鼠胃对于针对人类胃疾病进行建模不是最佳的9。因此,人类胃底上皮组织的稳健且高效的PSC衍生模型将代表胃生物学领域的显著进步。
胚胎器官发育通过相邻组织之间的指导性提示来引导10,11,并且hPSC分化为特定谱系主要依赖于使用这些信号来定向体外分化。申请人先前发现一种生成hAGO的步进分化方法,由此hPSC被分化为定形内胚层,模式化为后部前肠,然后规范化为推定的胃窦上皮组织7。申请人假设胃底和胃窦衍生自后部前肠祖细胞的同一群体,如果提供有适当的信号,所述群体可朝向胃底谱系定向。然而,鉴于驱动体内胃底发育的机制先前未知,申请人首先必须识别沿近侧-远侧轴使胚胎胃模式化的信号传导途径。
胚胎胃模式形成
为了帮助研究在胚胎发育期间调控胃底规范化的途径,申请人分析了小鼠胚胎来识别可在推定的胃底、胃窦和前胃之间进行区分的分子标记物。在E14.5时,申请人发现Sox2在所有的前肠器官谱系中表达,而Gata4受限于腺体胃上皮组织。在Gata4+区域内,Pdx1特定于推定的胃窦区域(图6,a);因此,相信胚胎胃底区域是Sox2+Gata+Pdx1-。
另外,申请人分析了公布的微阵列数据集(GSM326648-GSM32665012和GSM80809-GMS8081613)和E14.5前肠的解剖区域,从而证明了转录因子Irx2、Irx3和Irx5的表达在胚胎胃底中的富集于比在胃窦中大十倍(图6,b至c),从而表明它们的表达可在腺体胃上皮组织的区域之间进一步区分。
在分子水平下,胃的推定的胃底和胃窦区域已在E10.5时建立(图6,a)。在发育的此点,典型的Wnt信号传导途径在近侧胃中是活化的,但是在远侧胃中表现出很少活性或没有活性14,如使用Wnt报道基因小鼠品系Axin2-lacZ所示(图1b)。虽然已知Wnt/β-连环蛋白信号传导的调控在建立幽门-十二指肠边界中发挥作用14,15,但是未研究其在胃上皮模式化中的作用。为了确定Wnt/β-连环蛋白信号传导对于体内建立胃底是否在功能上是所需的,申请人使用Shh-cre(Shh-cre;β-连环蛋白fl/fl=cKO)在前肠上皮组织中去除β-连环蛋白(Ctnnb1)。Wnt/β-连环蛋白信号传导的破坏导致胃底特性的丧失,这通过在E10.5时胃底中的异常Pdx1表达来证明(图1,c)。异常Pdx1初始受限于胃底上皮组织的腹侧一半,这与先前报告的使用此Shh-cre系的重组活性一致16,但是它然后随时间扩展而包括近侧胃的大部分并且在E14.5前具有更大的曲率(图7,a)。另外,胃底标记物Irx2、Irx3和Irx5的表达在cKO胚胎中明显减少(图7,b)。总体上,这些数据支持以下结论:上皮Wnt/β-连环蛋白信号传导调控胃模式形成,因为它是胃底特性的初始规范化所需的,同时约束胚胎小鼠胃中的胃窦命运。
为了确定早期Wnt/β-连环蛋白介导的模式化异常对于随后的细胞分化的影响,申请人分析了E18.5时的cKO胚胎。cKO胚胎中的胃有缺陷并且在E18.5时尺寸减小(图1,d和图7,c至d),从而表明Wnt/β-连环蛋白在发育的后期阶段期间促进胃生长中的作用。此外,cKO胃完全错模式化,在上皮组织的整个最近侧区域中具有异常Pdx1表达(图1,d)。壁细胞(通过Atp4b的表达来标记的胃底细胞类型)在CKO胃中减少(图1,d)并且在β-连环蛋白缺陷上皮组织中完全不存在(图1,e)。相比之下,确实发育的壁细胞仅在表达β-连环蛋白的上皮组织中观察到(图1,e和图7,d至e)。综合考虑,这些体内数据支持通过其Wnt/β-连环蛋白信号传导诱导胃底规范化并抑制胃窦特性的模型。另外,此早期模式化的破坏与随后壁细胞的细胞自主丧失相一致,从而表明细胞分化在发育模式化缺陷之后受损。
从hPSC分化胃底hGO
申请人接下来研究了Wnt/β-连环蛋白信号传导在建立发育中的人类胃的胃底-胃窦模式中的作用。为了对胃分化的早期阶段进行建模,申请人以先前所述的用于将hPSC分化为类似于胃窦的胃类器官的方案开始,所述方案以高保真性重演早期胃发育的正常阶段7。以三维后部前肠球形体(S0X2+HNF1β+)开始,申请人测试Wnt/β-连环蛋白信号传导的刺激在胃规范化阶段期间是否将后部前肠上皮组织定向为胃底(SOX2+GATA+PDX1-)谱系而不是胃窦(SOX2+GATA+PDX1+)(图2,a)。实际上,用GSK3β抑制剂CHIR99021(CHIR)将β-连环蛋白活化三天,在第9天导致PDX1的几乎完全约束,伴随IRX2、IRX3和IRX5的显著增加的表达(图2,b至c)。重要的是,SOX2和GATA4水平不受CHIR处理的影响,从而确认球形体保持其胃特性。因此,暴露于CHIR使得SOX2+GATA+PDX1-上皮组织的形成具有增加的IRX表达,这是与推定的胃底上皮组织一致的特征。
申请人然后寻求确定CHIR处理的球形体是否进一步发育为含有类似于胃底的上皮组织的更成熟的hGO。有趣的是,从第6天至第9天的CHIR的三天脉冲不足以不可逆地规定胃底特性,这是因为hGO最终在后期阶段返回到PDX1+胃窦表型。然而,通过至少在第29天的CHIR处理进行的连续Wnt刺激产生胃底基因表达的稳定诱导(图8,a)。这与在体内胚胎胃发育期间的延长的Wnt/β-连环蛋白信号传导活性一致。虽然先前的研究表明胚胎胃中的异常Wnt活化促进肠命运14,15,但是CHIR处理的hGO未表现出肠标记物CDX2、MUC2、CCK或SCT的显著增加(图8,e和图9,a至b)。申请人还证明了虽然由于BMP信号传导的伴随抑制而活化Wnt/β-连环蛋白,CDX2仍保持抑制,这是因为用BMP4替换头蛋白产生了肠转录因子的稳健表达(图9,c)。
一旦部分区域在早期发育中建立,原始胃上皮组织就经历生长、腺体形态发生和定形细胞类型的分化的时期。申请人先前示出hAGO经历相似的形态学和细胞发育进程7。CHIR处理的hFGO与hAGO相比以相似的速率和效率生长,因为铺板的所有球形体中的75%至90%生长为类器官(图8,d)。在第20天,两种类型的hGO均含有在>90%的细胞中表达胃SOX2/GATA4标记的上皮,而PDX1受限于hAGO(在hAGO中87.1%±8.4%,并且在hFGO中3.9%±2.0%,p=3.07xl0-6;图8,e)。类器官在其整个发育期间维持其相应的胃特性(图8,b至c)。在第34天之前,hFGO和hAGO包含无所不在地表达胃特异性17密蛋白CLDN18的CDH1+CTNNB1+KRT8+极化柱状上皮(图2,e和图9,d)以及相当的未分化间充质细胞(图10,b)。一个值得注意的差异是hFGO具有携带从类器官上皮组织出芽的组织化腺体的不同构造(图2,d至e和图10,a),而hAGO具有复杂的折叠和原始的类似腺体的组织,但是很少有腺体芽7。因此,使胃底规范化的新型Wnt/β-连环蛋白依赖性机制在人类中是保守的并且可***纵以生成具有在分子上与发育中的胃底相似的腺体上皮组织的三维hFGO。
区域特异性胃细胞分化
分化的胃窦胃细胞类型首先在大约第27天时在hAGO中检测到,然后增加,直至第34天7,这与小鼠胃中的前几周产后发育类似18。在第34天,如所预期的,hFGO含有MUC5AC+表面粘液细胞和MUC6+颈粘液细胞两者,这与hAGO相似(图3,a至b和图11,a)。hFGO还形成各种内分泌细胞类型(图3,c),但是激素GAST的表达特定于hAGO,而GHRL在hFGO中10倍富集(图3,d),这与正常的胃内分泌模式一致19。为了在功能上定义hGO的区域特异性能力,申请人使用诱导型***来过表达前分泌转录因子NEUROG3。NEUROG3在两种hGO亚型中的表达产生泛内分泌标记物SYP以及常见的胃激素SST和GHRL的稳健表达(图11,c)。然而,仅hAGO能够产生表达GAST的G细胞,并且hFGO不能(图3,e和图11,c),这与G细胞在人类胃中的胃窦特异性分布一致19。
主细胞(胃底特异性分泌谱系)驻留在泌酸的腺体的基部中,并且被提出作为一类储备干细胞20。hFGO表现出主细胞特异性21转录因子MIST1的上皮表达(图4,a),具有酶原PGA5和PGC的转录物的100至1,000倍增加(图4,c),并且含有通过ELISA测量的显著增加的胃蛋白酶原含量。然而,转录物水平与在成人胃中存在的那些相比小于1%(图11,d),并且胃蛋白酶原阳性细胞通过免疫组织化学几乎不可检测(图4c,b至c)。与此一致的是,含有酶原颗粒的细胞22通过TEM而被识别到(图4,d),但是很少。相比之下,具有更不成熟的粘液颗粒模式的细胞是丰富的(图11,b)。因为体内主细胞在生命的前几周不表现出稳健的胃蛋白酶原表达(图12,a至b),所以申请人得出结论:主细胞存在于hFGO中,但不成熟。因此,hFGO代表用于剖析调控主细胞成熟的固有和非固有机制的稳健平台。
控制壁细胞分化的途径
在基线处,hFGO仅含有少量的壁细胞(PC;图5,a至b),这是胃底腺体的限定细胞类型,其通过质子泵(由ATP4A和ATP4B亚单元组成)使胃内腔酸化。对用于增加PC群体的高效方法的识别仍很困难,这是因为缺乏对于驱动其发育的信号传导机制的了解。因此,申请人使用PSC衍生的hFGO作为用于在功能上筛选在调控PC分化中发挥作用的候选信号传导途径的平台。针对筛选,申请人将第30天的hFGO暴露于信号传导激动剂或拮抗剂两天并且在第34天分析PC分化。虽然大部分信号传导操纵没有可观的作用,但是用PD0325901(PD03)进行的MEK途径的瞬时抑制使得ATP4A和ATB4B两者的实质性上调(图13,a)。另外,虽然BMP4单独不影响PC基因表达,但是它可增强PD03的作用(数据未示出)。因此,PD03/BMP4的两天脉冲足以诱导PC标记物ATP4A、ATP4B和GIF的快速且稳健的表达(图5,a至b和图13,d)。有趣的是,此作用未通过仅从培养基去除EGF或FGF而被观察到(图13,b),从而表明在MEK/ERK的上游可能具有负责限制hFGO培养中的PC分化的内源性信号传导相互作用。另外,PD03/BMP4处理仅影响PC谱系(图13,e),并且不能在hAGO中诱导PC(图13,c),从而进一步突出胃上皮组织的早期模式化限定其最终的分化潜力。
在第34天,hFGO上皮表现出与人类胃相当的组织化,其中粘液细胞沿表面区域排列并且PC集中在腺体部分中(图5,e)。此外,壁细胞形态与体内成熟的壁细胞高度相似(图5,c)。考虑到其与体内PC的相似性以及如在TEM上所见的其管泡状超微结构(图5,d),申请人假设hFGO中的PC将表现出响应于适当的刺激而分泌酸的能力。使用pH敏感染料(SNARF5F),用实时共焦显微镜法进行过测量(图14,a),hFGO响应于通过H2拮抗剂法莫替丁或H+K+-ATP酶拮抗剂奥美拉唑阻断的组胺而产生腔内pH的迅速且明显的降低(图5,f和图14,b)。为了使对于组胺的细胞应答可视化,将hGO用荧光染料吖啶橙(AO)培养,当被隔离在酸性隔室中时,所述吖啶橙变为橙色23。与分离的小鼠胃腺体相似,AO响应于组胺在hFGO腺体中的酸化细胞囊泡内积累(图5,g和图14,c至d)。这些数据表明PC响应于诱导酸的刺激物经历分泌性小管结构的适当变化。
认为体内分化的胃细胞谱系衍生自未分化干细胞或祖细胞的公共库。在此申请人已证明能够通过基因手段(对内分泌细胞进行NEUROG3介导的调控)或通过对非固有的信号传导途径的操纵(用于PC的PD03/BMP4)来改变hFGO中细胞类型的相对比例。由这些观察结果得出以下假设:hFGO可含有与从成人胃分离的那些类似的胃干细胞的群体。实际上,申请人发现解离的第34天的hFGO可连续传代以产生新的类器官(图15,a至b)。类器官从传代的hFGO再生长依赖于高Wnt和高FGF培养基,这与用于生长初级胃组织类器官的培养基相似24,25。在两轮传代之后,hFGO维持谱系标记物MUC5AC、MUC6、PGC以及GHRL的表达;然而,它们不含有PC并且对于壁细胞谱系的PD03/BMP4介导的诱导没有反应(图15,c至d)。此发现与在成人干细胞衍生的胃类器官中所观察到的相似,所述胃类器官不稳健地产生PC,虽然衍生自真实的泌酸的粘液20,26。因此,识别在hGO和成人胃类器官的长期培养中保护PC能力的条件是重要的。
概括而言,申请人将基于体内和体外的发现的研究直接应用于将hPSC分化为新组织类型。申请人已定义Wnt/β-连环蛋白信号传导在小鼠的胃发育期间使胃底区域规范化的新型功能,并且使用Wnt调节作为引导以将hPSC分化为三维人类胃底类器官的机械基础。在小鼠和人类两者中,将Wnt介导的胃底规范化引向随后的PC形成。在此定向分化方案的每个阶段,胃底特异性操纵产生稳健的PC诱导(图13,f)。先前的报告认识到间充质因子Barx1间接作用来约束Wnt信号传导并且帮助阻止胃中的肠基因表达14,15。鉴于当前的研究认识到上皮Wnt/β-连环蛋白功能,并且先前的工作认识到间充质途径,似乎相对于间充质,Wnt/β-连环蛋白在上皮组织中可具有不同作用。例如,Wnt/β-连环蛋白的间充质作用可调节其他信号传导途径,诸如BMP27,数据显示其与Wnt协同作用来促进从早期内胚层的肠规范化(图7和图9,c)。人类胃类器官***可与动物模型组合来用于剖析这些信号传导途径如何在间充质和上皮组织中相互作用,以协调早期胚胎胃肠发育。
还缺少控制将胃祖细胞分化为不同谱系的途径。申请人证明此新hGO平台用于识别MEK/ERK信号传导强力地约束壁细胞规范化的用途。与这些发现一致,MEK/MAPK依赖性途径的转基因活化导致体内壁细胞的丧失28,29。因此,hGO是用于识别并研究胃底和胃窦中正常细胞内环境平衡中涉及的信号传导机制的,新的且易处理的人类模型***。另外,发育程序的异常调控还可促成胃疾病,因为原体/胃底病变经常与壁细胞萎缩30-32、胃窦类型组织学33、甚至Pdx1的错误表达34相关联。因此,这些途径的靶向可具有临床用途,因为Choi等人最近证明MEK的病理学抑制足以恢复组织变形的小鼠模型中的正常壁细胞分化35。另外,既然现在已建立胃窦和胃底类型两者的hGO,可能研究这些人类胃组织如何在生理上相互作用,如何对感染和损伤有差异地做出反应,并且如何对药理学处理做出反应。
方法
小鼠实验
以下基因小鼠品系从Jackson实验室获得,收藏在辛辛那提儿童医院研究基金会动物中心(Cincinnati Children's Hospital Research Foundation animal facility),并且根据IACUC协议(0B09074)供养:Axin2:LacZ(贮存编号009120)、Shh:Cre(贮存编号005622)以及β-cateninfloxed(贮存编号004152)。使用定时交配来在各个阶段生成收获用于整体染色或组织解剖的胚胎,其中将观察到***栓的早晨标注为E0.5。在每个检查的发育阶段分析至少两窝胚胎。分析雄性和雌性胚胎两者。
多能干细胞培养
人类胚胎干细胞系WA01(H1;从WiCell获得)由在辛辛那提儿童医院医疗中心(Cincinnati Children's Hospital Medical Center)的多能干细胞中心(PluripotentStem Cell Facility)提供。细胞特性通过短串联重复序列分析(微卫星STR分析;应用生物***公司(Applied Biosystems))来确认,并且针对支原体污染常规性地测试细胞(MycoAlert支原体检测试剂盒;龙沙公司(Lonza))。在无饲养细胞的条件下,将多能细胞维持在mTesR1培养基(干细胞科技公司(Stem Cell Technologies))中的HESC合格的基质胶(BD生物科学公司(BD Biosciences))上。使用分散酶(英杰公司(Invitrogen))将菌落每四天传代一次。
后部前肠球形体的分化
用于胃球形体的定向分化的方案改编自先前的方案7,并且表1含有用于每个阶段的培养基和生长因子的完整列表。针对分化,使用细胞消化液(干细胞科技公司)将hPSC解离为单个细胞并且在具有Y-27632(10μM;Stemgent公司)的mTesR1中以大约200,000个细胞/孔的密度铺板到24孔板中。在第二天,通过在RPMI 1640培养基(英杰公司)中添加活化素A(100ng/ml;Cell Guidance Systems公司)来将细胞分化为定形内胚层(DE),持续三天。在第1天、第2天和第3天还为培养基增补分别在0%、0.2%和2.0%下的NEAA(1X;Gibco公司)和定义的FBS(dFBS;英杰公司)。另外,在第一天添加BMP4(50ng/ml;R&D Systems公司)。随后,通过在增补有NEAA和2.0%dFBS的RPMI 1640中将细胞暴露于CHIR99021(2μM;Stemgent公司)、FGF4(500ng/ml;R&D Systems公司)以及头蛋白(200ng/ml;R&D systems公司)三天来将DE分化为后部前肠内胚层。在最后一天添加视黄酸(2μM;西格玛奥德里奇公司(Sigma Aldrich))。培养基每天更换。此过程致三维后部前肠球形体自发形成。
表1.用于胃底hGO的分化方案。活化素A(100ng/ml;R&D Systems公司),CHIR99021(2uM;Stemgent公司),FGF4(500ng/ml;R&D systems公司),PD0325901(2uM;Stemgent公司),BMP4(50ng/ml;R&D Systems公司)。*BGM(基础肠培养基)=改进的DMEM/F12,N2(1X;英杰公司),B27(1X;英杰公司)L-谷氨酰胺,HEPES(10uM),以及青霉素/链霉素。**特定于胃底hGO方案。
前肠球形体-胃球形体的三维培养
采集后部前肠球形体并且转移到如先前所述的三维培养***36。简而言之,将球形体悬浮在50μl基质胶(BD生物科学公司)中并且作为液滴铺板到24孔板中。使基质胶在组织培养箱中固化10分钟,然后用含有生长因子和/或小分子激动剂的基础肠培养基(BGM)覆盖。BGM由改进的DMEM/F12培养基(Gibco公司)组成,增补有N2(1X;英杰公司)、B27(1X;英杰公司)、HEPES(10μM;Gibco公司)、L-谷氨酰胺、青霉素/链霉素、以及EGF(100ng/ml;R&DSystems公司)。在第6天至第9天期间,将球形体用RA和头蛋白培养以使胃窦谱系规范化。针对胃底规范化,在此阶段期间添加CHIR。随后将胃窦hGO在仅具有EGF的BGM中培养。从第6天至第30天连续将胃底hGO暴露于CHIR。另外,从第20天至第30天将FGF10(50ng/ml;R&DSystems公司)添加到胃底hGO,因为显示它增强由CHIR驱动的腺体形态发生。在第20天,采集球形体并且在1:10至1:20的稀释度下再铺板。
针对筛选用于识别增加壁细胞分化的因子的实验,使hFGO生长至第30天,然后暴露于个体信号传导途径激动剂和拮抗剂两天:DAPT(1μM;Stemgent公司)、SB431542(10μM;Stemgent公司)、BMP4(50ng/ml;R&D Systems公司)、PD0325901(2μM;Stemgent公司)、胃泌素(10nM;西格玛奥德里奇公司)、***(50nM;西格玛奥德里奇公司)、以及Wnt5a(50ng/ml;R&D Systems公司)。在处理之后,使hFGO再生长两天至第34天,然后通过qPCR对其进行分析。
RNA分离和qPCR
使用Nucleospin RNAII试剂盒(Machery Nagel公司)分离总RNA并且如先前所述转化为cDNA7。使用Quantitect SYBR-Green主混合物(凯杰公司(Qiagen))在Quantstudio6(应用生物***公司)上执行qPCR,并且引物序列在以下列出。
引物序列
用于qPCR的引物如下:
hATP4A,正向5-TGGTAGTAGCCAAAGCAGCC-3',反向5'-TGCCATCCAGGCTAGTGAG-3';
hATP4B,正向5-ACCACGTAGAAGGCCACGTA-3',反向5'-TGGAGGAGTTCCAGCGTTAC-3';
hAXIN2,正向5'-CTGGTGCAAAGACATAGCCA-3',反向5'-AGTGTGAGGTCCACGGAAAC-3';
hCCK,正向5'-CGGTCACTTATCCTGTGGCT-3',反向5'-CTGCGAAGATCAATCCAGCA-3';
hCDX2,正向5'-CTGGAGCTGGAGAAGGAGTTTC-3',反向5'-ATTTTAACCTGCCTCTCAGAGAGC-3';
hCHGA,正向5'-TGACCTCAACGATGCATTTC-3',反向5'-CTGTCCTGGCTCTTCTGCTC-3';
hGAPDH,正向5-CCCATCACCATCTTCCAGGAG-3',反向5'-CTTCTCCATGGTGGTGAAGACG-3';
hGAST,正向5'-CAGAGCCAGTGCAAAGATCA-3',反向5'-AGAGACCTGAGAGGCACCAG-3';
hGATA4,正向5'-TCCAAACCAGAAAACGGAAGC-3',反向5'-GCCCGTAGTGAGATGACAGG-3';
hGHRL,正向5-GCTGGTACTGAACCCCTGAC-3',反向5'-GATGGAGGTCAAGCAGAAGG-3';
hGIF,正向5'-CATTTTCCGCGATATTGTTG-3',反向5'-GCACAGCGCAAAAATCCTAT-3';
hIRX2,正向5'-GTGGTGTGCGCGTCGTA-3',反向5'-GGCGTTCAGCCCCTACC-3';
hIRX3,正向5'-GGAGAGAGCCGATAAGACCA-3',反向5'-AGTGCCTTGGAAGTGGAGAA-3';
hIRX5,正向5'-GGTGTGTGGTCGTAGGGAGA-3',反向5'-GCTACAACTCGCACCTCCA-3';
hMIST1,正向5'-TGCTGGACATGGTCAGGAT-3',反向5'-CGGACAAGAAGCTCTCCAAG-3';
hMUC2,正向5'-TGTAGGCATCGCTCTTCTCA-3',反向5'-GACACCATCTACCTCACCCG-3';
hMUC5AC,正向5'-CCAAGGAGAACCTCCCATAT-3',反向5'-CCAAGCGTCATTCCTGAG-3';
hMUC6,正向5'-CAGCAGGAGGAGATCACGTTCAAG-3',反向5'-GTGGGTGTTTTCCTGTCTGTCATC-3';
hPDX1,正向5'-CGTCCGCTTGTTCTCCTC-3',反向5'-CCTTTCCCATGGATGAAGTC-3';
hSCT,正向5'-GGTTCTGAAACCATAGGCCC-3',反向5'-GTCAGGGTCCAACATGCC-3';
hSOX2,正向5'-GCTTAGCCTCGTCGATGAAC-3',反向5'-AACCCCAAGATGCACAACTC-3';
mCdx2,正向5'-TCTGTGTACACCACCCGGTA-3',反向5'-GAAACCTGTGCGAGTGGATG-3';
mGata4,正向5-CCATCTCGCCTCCAGAGT-3',反向5'-CTGGAAGACACCCCAATCTC-3';
mGapdh,正向5'-TTGATGGCAACAATCTCCAC-3',反向5'-CGTCCCGTAGACAAAATGGT-3';
mIrx1,正向5'-AATAAGCAGGCGTTGTGTGG-3',反向5'-CTCAGCCTCTTCTCGCAGAT-3';
mIrx2,正向5'-AGCTGGTATGGATAGGCCG-3',反向5'-GGCTTCCCGTCCTACGTG-3';
mIrx3,正向5'-ATAAGACCAGAGCAGCGTCC-3',反向5'-GTGCCTTGGAAGTGGAGAAA-3';
mIrx5,正向5'-GGAGTGTGGTCGTAGGGAGA-3',反向5'-GCTACAACTCGCACCTCCA-3';
mPdx1,正向5'-ACGGGTCCTCTTGTTTTCCT-3',反向5'-TGGATGAAATCCACCAAAGC-3';
mPitx1,正向5'-GTCCATGGAGGTGGGGAC-3',反向5'-GCTTAGGCGCCACTCTCTT-3';
mSox2,正向5'-AAAGCGTTAATTTGGATGGG-3',反向5'-ACAAGAGAATTGGGAGGGGT-3';
mTrp63,正向5'-AGCTTCTTCAGTTCGGTGGA-3',反向5'-CCTCCAACACAGATTACCCG-3'。
免疫荧光染色
将组织在4℃下固定在4%低聚甲醛中过夜,然后在PBS中彻底洗涤。针对整体免疫荧光染色,将胚胎如先前所述进行处理37。简而言之,将它们在室温下在Dent漂白剂(Dent's Bleach)(4:1:1EtOH:DMSO:30% H202)中透化两小时,并且通过一系列甲醇洗涤再水合。然后将胚胎阻断一小时,在4℃下在初级抗体中孵育过夜,在PBS中洗涤,在4℃下在初级抗体中孵育过夜,并且彻底洗涤。针对石蜡包埋,将组织通过一系列乙醇洗涤再水合,在二甲苯中洗涤,然后包埋在石蜡中。针对染色,将玻片脱石蜡并且再水合。在蒸锅中在柠檬酸盐缓冲液中执行抗原回收45分钟。将初级抗体在4℃下孵育过夜。在初级抗体之后,将玻片在PBS中洗涤,然后在室温下与二级抗体(在1:500的稀释度下)一起孵育一小时。二级抗体(Jackson免疫研究实验室(Jackson ImmunoResearch Laboratories))在驴中制备并且缀合到Alexa Fluor 488、594或647。
初级抗体
用于免疫荧光染色的抗体与用于染色的抗原、宿主种类、制造商和目录号以及稀释度一起列出。Atp4b,兔,圣克鲁斯公司(Santa Cruz)sc84304,1:500;Cdh1,山羊,R&DSystems公司AF648,1:500;Cdh1,小鼠,BD生物科学公司610182,1:500;Cdx2,小鼠,Biogenex公司MU392A,1:500,Cldn18,兔,西格玛公司HPA018446,1:200;Ctnnb1,兔,圣克鲁斯公司sc7190,1:100;FoxF1,山羊,R&D Systems公司F4798,1:500,胃泌素,兔,Dako公司A0568,1:1,000;Gata4,山羊,圣克鲁斯公司scl237,1:200;Gif,兔,西格玛公司HPA040774,1:100;Ghrl,山羊,圣克鲁斯公司scl0368,1:200;组胺,兔,Immunostar公司22939,1:1,000;Krt8,大鼠,DSHB troma-1-s;1:100;Mist1,兔,西格玛公司HPA047834,1:200;Muc5ac,小鼠,Abcam公司ab3649,1:500;Muc6,小鼠,Abcam公司ab49462,1:100;Pdx1,山羊,Abcam公司ab47383,1:5,000;Pgc,绵羊,Abcam公司ab31464,1:10,000;Sst,山羊,圣克鲁斯公司sc7819,1:100;Syp,豚鼠,Synaptic Systems公司101004,1:1,000;波形蛋白,山羊,圣克鲁斯公司sc7557,1:200
成像
在尼康A1Rsi倒置共焦显微镜上执行共焦成像。针对整体成像,在即将成像之前,将胚胎在甲醇中脱水并且在Murray透明剂(Murray's clear)(2:1苯甲酸苄酯:苄醇)中进行透明化。在染色之后,将玻片用Fluoromount G(SouthrnBiotech公司)安装并且在室温下空气干燥过夜。
透射电子显微镜法
针对TEM,如先前所述对hGO进行处理7。简而言之,将类器官固定在3%戊二醛中,在0.1M二甲胂酸钠缓冲液中洗涤,并且在4%四氧化锇中孵育一小时。随后将它们洗涤,然后在乙醇系列中脱水,并且最后包埋在环氧丙烷/LX112中。然后对组织进行切片并且用2%醋酸铀酰染色,之后用柠檬酸铅染色。将图像在日立公司透射电子显微镜上可视化。
胃蛋白酶原ELISA
根据制造商的说明书使用人类胃蛋白酶原I(PGI)ELISA试剂盒(ThermoScientific公司,EHPGI)执行ELISA。简而言之,采集第34天的hGO并且在4℃下在细胞回收溶液(康宁公司(Corning))中孵育一小时,然后在PBS中洗涤。将类器官用RIPA缓冲液裂解,接着在室温下在高速度下剧烈涡旋30分钟。将裂解液压成丸,并且采集上清液并在-80℃下储存。针对ELISA,在技术性平行测定中执行样品和标准物。在μQuant微滴板读取仪(BioTek公司)上测量反应。测量在450nm下的吸光度,并且减去570nm吸光度。
酸分泌测定
如先前所述执行酸分泌测定(Schumacher等人,2015)。将hGO在腔室盖片(ThermoScientific公司)中生长,并且将所述腔室放置在倒置共焦显微镜(Zeiss LSM 710)上,并且在5% CO2和37℃条件下执行实验(孵育腔室,德国埃尔巴赫PeCon公司(PeCon,Erbach,Germany))。
将新鲜分离的小鼠胃底腺体或培养的hGO用吖啶橙(10μM)孵育,然后在458nm或488nm处激励吖啶橙荧光,并且分别在600至650nm(红色)或500至550nm(绿色)下采集图像。另一方面,为了监测hGO腔内pH,将比率式pH敏感染料5-(和-6)-羧基SNARF-5F(5mM原液:EX560nm,EM 565至605(绿色)和620至680(红色)nm:英杰公司)显微注射(46至92nl)到内腔中并且监测。还将荧光染料添加到培养基中。将组胺(100μM;西格玛公司)添加到培养基,同时在组胺之前预孵育法莫替丁(100μM;西格玛公司)或奥美拉唑(100μM;西格玛公司)至少30min。使用MetaMorph软件(Molecular Devices公司,宾夕法尼亚州唐宁敦(MolecularDevices,Downingtown,PA))分析图像。使用标准曲线将背景校正的620至680/565至605nm比值转化为pH。
统计分析
用Dunnett多重比较事后测试,使用未配对的学生T测试或单向ANOVA确定统计学显著性。<0.05的p值被认为是显著的。
统计学和实验再现性
不使用统计分析来确定实验样品大小,不使用特定随机化方法,并且在实验期间研究员是知情的。在图例中描述了统计方法和测量值。胃底hGO分化的方案被实验室中的七位独立用户成功地完成>20次。在所有情况下,所示的数据源于代表多个实验的单个实验。
示例性组合
以下实施例涉及本文的教导内容可组合或应用的各种非全面的方法。应理解,以下实施例不旨在限制可在本申请中或在本申请的随后提交中任何时间呈现的任何权利要求的覆盖范围。不旨在放弃权利要求。以下实施例仅仅出于例示性目的而提供。设想本文的各种教导内容可以多种其他方式布置和应用。还设想一些变型可省略以下实施例中提及的某些特征。因此,以下提及的方面或特征中的每一个均不应被认为是关键的,除非之后发明人或符合发明人利益的继承人以其他方式明确地这样指示。如果任何权利要求在本申请中或在涉及本申请的包括除以下提及的那些以外的另外特征的随后所提交的文档中呈现,这些另外的特征不应被假定出于有关专利性的任何原因而被添加。
f实施例1.体外生成一种胃底组织,其包括以下步骤:
a)将哺乳动物定形内胚层(DE)细胞与wnt途径活化剂、FGF信号传导途径活化剂(例如,FGF4)、BMP信号传导途径抑制剂(例如,头蛋白)以及视黄酸接触第一时间段,其中所述第一时间段足以从所述定形内胚层形成三维后部前肠球形体;
b)将所述三维后部前肠球形体在具有生长因子、Wnt信号传导途径活化剂、EGF信号传导途径活化剂、BMP信号传导途径抑制剂以及视黄酸的基底膜基质(例如,基质胶)中悬浮第二时间段,所述第二时间段足以诱导包括胃底hGO(hFGO)的胃底谱系;
c)将步骤b)的hFGO在wnt途径活化剂和EGF信号传导途径活化剂的存在下培养第三时间段,
d)将步骤c的hFGO用wnt信号传导途径活化剂、EGF信号传导途径活化剂和FGF10培养第四时间段;
e)将步骤d的hFGO与MEK抑制剂接触第五时间段(所述MEK抑制剂可以是,例如,PD0325901),持续足以形成包含功能性胃底细胞类型的胃底组织的时间段。
实施例2.根据实施例1所述的方法,其中所述第一时间段是三天±24小时,并且其中所述视黄酸在所述时间段±24小时的第三天添加
实施例3.根据任一项前述实施例所述的方法,其中所述第二时间段是三天±24小时
实施例4.根据任一项前述实施例所述的方法,其中所述第三时间段是11天±24小时
实施例5.根据任一项前述实施例所述的方法,其中所述第四时间段是10天±24小时
实施例6.根据任一项前述实施例所述的方法,其中所述第五时间段是两天时间段±24小时
实施例7.根据任一项前述实施例所述的方法,其中步骤e)还包括将所述胃底hGO与BMP4信号传导的活化剂接触的步骤。
实施例8.根据任一项前述实施例所述的方法,其中所述功能性胃底细胞类型是表达质子泵蛋白并且分泌酸的壁细胞。
实施例9.根据任一项前述实施例所述的方法,其中所述功能性胃底细胞类型是分泌胃蛋白酶原的主细胞。
实施例10.根据任一项前述实施例所述的方法,其中所述步骤e进行足以发育SOX2+GATA+PDX1-上皮组织的时间段。
实施例11.根据任一项前述实施例所述的方法,其中所述步骤d和所述步骤e进行足以赋予谱系标记物MUC5AC、MUC6、PGC和GHRL的稳定表达的时间段。
实施例12.根据任一项前述实施例所述的方法,其中所述定形内胚层衍生自选自以下的前体细胞:胚胎干细胞、胚胎生殖细胞、诱导的多能干细胞、中胚层细胞、定形内胚层细胞、后内胚层细胞、后内胚层细胞以及后肠细胞;衍生自多能干细胞的定形内胚层;衍生自选自胚胎干细胞、成人干细胞或诱导的多能干细胞的多能干细胞的定形内胚层。
实施例13.根据任一项前述实施例所述的方法,其中所述定形内胚层通过将多能干细胞与选自以下的一种或多种分子接触而衍生:活化素、生长因子的TGF-β超家族的BMP亚组;Nodal、活化素A、活化素B、BMP4、Wnt3a以及其组合。
实施例14.根据任一项前述实施例所述的方法,其中所述WNT途径活化剂是选自以下的一种或多种分子:Wnt1、Wnt2、Wnt2b、Wnt3、Wnt3a、Wnt4、Wnt5a、Wnt5b、Wnt6、Wnt7a、Wnt7b、Wnt8a、Wnt8b、Wnt9a、Wnt9b、Wnt10a、Wnt10b、Wnt11以及Wnt16,例如Wnt3a,或例如浓度在约50至约1500ng/ml之间的Wnt3a。
实施例15.根据任一项前述实施例所述的方法,其中所述BMP信号传导途径抑制剂选自例如头蛋白、Dorsomorphin、LDN189、DMH-1以及其组合,其中所述前体细胞与浓度在约50至约1500ng/ml之间的BMP抑制剂接触。所述BMP抑制剂可以是能够抑制BMP信号传导途径的蛋白质和/或化学品。
实施例16.根据任一项前述实施例所述的方法,其中所述步骤在体外实施。
实施例17.根据任一项前述实施例产生一种包含胃组织的组合物。所述胃组织的特征在于不具有神经分布和/或血管。
实施例18.通过以下步骤形成胃底组织:将胃底hGO(hFGO)与wnt途径活化剂和EGF信号传导途径活化剂接触第一时间段,并且与MEK抑制剂接触第二时间段,(其中所述MEK抑制剂可以是,例如,PD0325901),其中所述第一和第二时间段进行足以形成功能性胃底细胞类型的时间段;
其中所述hFGO通过将三维后部前肠球形体在基底膜基质中与生长因子、wnt途径活化剂、EGF信号传导途径活化剂、BMP信号传导途径抑制剂以及视黄酸接触足以将所述三维后部前肠球形体转化为所述hFGO的时间段来获得;
其中所述三维后部前肠球形体通过将哺乳动物定形内胚层(DE)细胞与wnt途径活化剂、FGF信号传导途径活化剂、BMP信号传导途径抑制剂以及视黄酸接触来获得。
参考文献
1.Lancaster,M.A.和Knoblich,J.A.《培养皿中的器官发生:
使用类器官技术对发育和疾病建模(Organogenesis in a dish:modelingdevelopment and disease using organoid technologies)》.《科学杂志(Science)》345,1247125-1247125(2014)。
2.Huch,M.和Koo,B.-K.《使用类器官培养对小鼠和人类发育建模(Modelingmouse and human development using organoid cultures)》.《发育杂志(Development)》142,3113-3125(2015)。
3.Zhu,Z.和Huangfu,D.《人类多能干细胞:发育生物学中的新兴模型(Humanpluripotent stem cells:an emerging model in developmental biology)》.《发育杂志(Development)》140,705-717(2013)。
4.Kim,T.-H.和Shivdasani,R.A.《胃发育、干细胞和疾病(Stomach development,stem cells and disease)》.《发育杂志(Development)》143,554-565(2016)。
5.Mills,J.C.和Shivdasani,R.A.《胃上皮干细胞》.《胃肠病学(Gastroenterology)》140,412-424(2011)。
6.Hoffmann,W.《干细胞和胃上皮组织癌症的现状(Current Status on StemCells and Cancers of the Gastric Epithelium)》.《国际分子科学杂志(IJMS)》16,19153-19169(2015)。
7.McCracken,K.W.等人《在多能干细胞衍生的胃类器官中对人类发育和疾病建模(Modelling human development and disease in pluripotent stem-cell-derivedgastric organoids)》.《自然杂志(Nature)》516,400-404(2014)。
8.Noguchi,T.-A.K.等人《从小鼠胚胎干细胞生成胃组织(Generation ofstomach tissue from mouse embryonic stem cells)》.《自然细胞生物学(Nature CellBiology)》17,984-993(2015)。
9.Peek,R.M.《幽门螺旋杆菌感染和疾病:从人类到动物模型(Helicobacterpylori infection and disease:from humans to animal models)》.《疾病模型和机制(Disease Models&Mechanisms)》1,50-55(2008)。
10.Zom,A.M.和Wells,J.M.《脊椎动物内胚层发育和器官形成(VertebrateEndoderm Development and Organ Formation)》.《细胞和发育生物学年度综述(Annu.Rev.Cell Dev.Biol.)》25,221-251(2009)。
11.Kraus,M.R.-C.和Grapin-Botton,A.《在体内和在培养物中使内胚层模式化并成形(Patterning and shaping the endoderm in vivo and in culture)》.《遗传学与发育新见(Curr.Opin.Genet.Dev.)》22,347-353(2012)。
12.Sherwood,R.I.、Chen,T.-Y.A.和Melton,D.A.《内胚层器官形成的转录动力学(Transcriptional dynamics of endodermal organ formation)》.238,29-42(2009)。
13.Roth,R.B.等人《基因表达分析揭示20个人类CNS区域之间的分子关系(Geneexpression analyses reveal molecular relationships among 20regions of thehuman CNS)》.《神经遗传学(Neurogenetics)》7,67-80(2006)。
14.Kim,B.-M.、Buchner,G.、Miletich,I.、Sharpe,P.T.和Shivdasani,R.A.《胃间充质转录因子Barx1通过抑制瞬时Wnt信号传导使胃上皮特性规范化(The stomachmesenchymal transcription factor Barx1 specifies gastric epithelial identitythrough inhibition of transient Wnt signaling)》.《发育细胞(DevelopmentalCell)》8,611-622(2005)。
15.Kim,B.-M.、Woo,J.、Kanellopoulou,C.和Shivdasani,R.A.《通过特异性微RNA调控小鼠胃发育和Barx1表达《Regulation of mouse stomach development and Barx1expression by specific microRNAs》》.《发育杂志(Development)》138,1081-1086(2011)。
16.Rodriguez,P.等人《小鼠食道和前胃的发育中的BMP信号传导(BMP signalingin the development of the mouse esophagus and forestomach)》.《发育杂志(Development)》137,4171-4176(2010)。
17.Lameris,A.L.等人《健康的和在炎性肠病期间的人类胃肠道中闭合蛋白的表达谱(Expression profiling of claudins in the human gastrointestinal tract inhealth and during inflammatory bowel disease)》.《斯堪的纳维亚胃肠病学杂志(Scand.J.Gastroenterol)》48,58-69(2013)。
18.Keeley,T.M.和Samuelson,L.C.《正常的和亨廷顿相互作用蛋白1相关缺陷小鼠的产后小鼠胃的细胞分化(Cytodifferentiation of the postnatal mouse stomachin normal and Huntingtin-interacting protein 1-related-deficient mice)》.《美国生理学胃肠和肝脏生理学杂志(Am.J.Physiol.Gastrointest.Liver Physiol.)》299,G1241-51(2010)。
19.Choi,E.等人《人类胃的细胞谱系分布图谱揭示胃窦中的异质腺体群体(Celllineage distribution atlas of the human stomach reveals heterogeneous glandpopulations in the gastric antrum)》.《肠杂志(Gut)》63,1711-1720(2014)。
20.Stange,D.E.等人《分化的Troy+主细胞充当补充干细胞来生成胃上皮组织的所有谱系(Differentiated Troy+chief cells act as reserve stem cells togenerate all lineages of the stomach epithelium)》.《细胞杂志(Cell)》155,357-368(2013)。
21.Lennerz,J.K.M.等人《转录因子MIST1是其表达在组织变形、发育不良和癌中丧失的新型人类胃主细胞标记物(The transcription factor MIST1is a novel humangastric chief cell marker whose expression is lost in metaplasia,dysplasia,and carcinoma)》.《美国生理学杂志(Am.J.Pathol.)》177,1514-1533(2010)。
22.Ramsey,V.G.等人《分泌粘液的胃上皮祖细胞成熟化为分泌消化酶的产酶细胞需要Mist1(The maturation of mucus-secreting gastric epithelial progenitorsinto digestive-enzyme secreting zymogenic cells requires Mist1)》.《发育杂志(Development)》134,211-222(2007)。
23.Lambrecht,N.W.G.、Yakubov,I.、Scott,D.和Sachs,G.《酸分泌期间连接到胃H-K-ATP酶的K外流通道的识别(Identification of the K efflux channel coupled tothe gastric H-K-ATPase during acid secretion)》.《生理基因组学(PhysiologicalGenomics)》21,81-91(2005)。
24.Schumacher,M.A.等人《胃生理学研究中鼠衍生的胃底类器官的使用(The useof murine-derived fundic organoids in studies of gastric physiology)》.《生理学杂志(J.Physiol.)》(伦敦)593,1809-1827(2015)。
25.Barker,N.等人《Lgr5(+ve)干细胞在胃中驱动自身更新并且在体外建立长寿命的胃单元(Lgr5(+ve)stem cells drive self-renewal in the stomach and buildlong-lived gastric units in vitro)》.《细胞干细胞(Cell Stem Cell)》6,25-36(2010)。
26.Bartfeld,S.等人《人类胃上皮干细胞的体外扩增及其对于细菌感染的应答(In vitro expansion of human gastric epithelial stem cells and theirresponses to bacterial infection)》.《胃肠病学(Gastroenterology)》148,126-136.e6(2015)。
27.Nielsen,C.、Murtaugh,L.C.、Chyung,J.C.、Lassar,A.和Roberts,D.J.《肌胃形成和Bapx1的作用(Gizzard Formation and the Role of Bapx1)》.《发育生物学(Developmental Biology)》231,164-174(2001)。
28.Goldenring,J.R.等人《转化生长因子-α的过表达改变胃细胞谱系的分化(Overexpression of transforming growth factor-alpha alters differentiation ofgastric cell lineages)》.《消化疾病与科学(Dig.Dis.Sci.)》41,773-784(1996)。
29.Speer,A.L.等人《成纤维细胞生长因子10-成纤维细胞生长因子受体2b介导的信号传导不是成人腺胃内环境平衡所需的(Fibroblast growth factor10-fibroblastgrowth factor receptor 2b mediated signaling is not required for adultglandular stomach homeostasis)》.《公共科学图书馆期刊(PLoS ONE)》7,e49127(2012)。
30.Goldenring,J.R.和Nomura,S.《胃粘膜III的分化(Differentiation of thegastric mucosa III)》,《泌酸萎缩和组织变形的动物模型(Animal models of oxynticatrophy and metaplasia)》.《美国病理学杂志:胃肠和肝脏生理学(AJP:Gastrointestinal and Liver Physiology)》291,G999-1004(2006)。
31.Huh,W.J.、Coffey,R.J.和Washington,M.K.《梅尼特里埃氏病:其模拟物和发病机理(Menetrier's Disease:Its Mimickers and Pathogenesis)》.《病理学与转化医学杂志(J Pathol Transl Med)》50,10-16(2016)。
32.Park,Y.H.和Kim,N.《作为胃癌的癌前病变的萎缩性胃炎和肠组织变形的综述(Review of atrophic gastritis and intestinal metaplasia as apremalignantlesion of gastric cancer)》.《癌症预防杂志(J Cancer Prev)》20,25-40(2015)。
33.Weis,V.G.和Goldenring,J.R.《对SPEM及其在肿瘤发生前的过程中的位置的当前理解(Current understanding of SPEM and its standing in the preneoplasticprocess)》.《胃癌(Gastric Cancer)》12,189-197(2009)。
34.Nomura,S.等人《与梅尼特里埃氏病和TGFα过表达相关联的胃底上皮组织的再模式化的证据(Evidence for Repatterning of the Gastric Fundic EpitheliumAssociated With Menetrier's Disease and TGFαOverexpression)》.《胃肠病学(Gastroenterology)》128,1292-1305(2005)。
35.Choi,E.、Hendley,A.M.、Bailey,J.M.、Leach,S.D.和Goldenring,J.R.《小鼠的胃主细胞中活化的Ras的表达导致完全范围的组织变形谱系转变(Expression ofActivated Ras in Gastric Chief Cells of Mice Leads to the Full Spectrum ofMetaplastic Lineage Transitions)》.《胃肠病学(Gastroenterology)》0,(2015)。
36.McCracken,KW、Howell JC、Wells,JM和Spence,JR,《在体外从多能干细胞生成人类肠组织(Generating human intestinal tissue from pluripotent stem cells invitro)》.《自然实验手册(Nat.Protocols)》6,1920-1928(2011)。
37.Ahnfelt-Ronne,J等人,《用于完整小鼠和鸡胚胎和器官中的蛋白质表达模式的三维重构的改进方法(An improved method for three-dimensional reconstructionof protein expression patterns in intact mouse and chicken embryos andorgans)》.《组织化学与细胞化学杂志(J.Histochem.Cytochem.)》55,925-930(2007)。
所有的百分比和比率按重量计算,除非另有说明。
所有的百分比和比率基于总组成计算,除非另有说明。
应理解,在整个本说明书中给出的每个最大数值限度包括每个较低的数值限度,就像此类较低数值限度在本文中明确地写出一样。在整个本说明书中给出的每个最小数值限度将包括较高的数值限度,就像此类较高的数值限度在本文中明确地写出一样。在整个本说明书中给出的每个数值范围将包括落在此类较宽的数值范围内的每个较窄的数值范围,就像此类较窄的数值范围全部在本文中明确地写出一样。
本文公开的尺寸和值不被理解为严格地限于所述的确切数值。实际上,除非另有规定,每个这种尺寸旨在意指所述的值以及围绕此值的功能上等同的范围两者。例如,公开为“20mm”的尺寸旨在意指“约20mm”。
本文引用的每个文献,包括任何交叉引用或相关的专利或申请,在此通过引用整体并入本文,除非明确地被排除或另外被限制。任何文献的引用不是对其作为本文所公开或要求保护的任何发明的现有技术的认可,或独立地或以与任何其他一个或多个参考文献的任何组合的方式提出、建议或公开任何此类发明的现有技术的认可。另外,当本文献中术语的任何含义或定义与通过引用并入的文献中同一术语的任何含义或定义冲突时,将以赋予本文献中术语的含义或定义为准。
虽然已示出并描述了本发明的特定实施方案,但是对本领域的技术人员将显而易见的是,可在不背离本发明的精神和范围的情况下做出各种其他改变和修改。因此,旨在于所附权利要求书中覆盖在本发明的范围内的所有此类改变和修改。
Claims (10)
1.一种诱导胃底组织形成的体外方法,其包括以下步骤:
a)将哺乳动物定形内胚层(DE)细胞与wnt途径活化剂、FGF信号传导途径活化剂、BMP信号传导途径抑制剂以及视黄酸接触第一时间段;
其中所述第一时间段持续足以从所述定形内胚层形成三维后部前肠球形体的时间长度;
b)将所述三维后部前肠球形体在具有生长因子、所述Wnt信号传导途径活化剂、所述EGF信号传导途径活化剂、所述BMP信号传导途径抑制剂以及视黄酸的基底膜基质中悬浮第二时间段,其中所述第二时间段持续足以诱导包括胃底hGO(hFGO)的胃底谱系的时间长度;
c)将步骤b)的所述hFGO用所述wnt途径活化剂和所述EGF信号传导途径活化剂培养第三时间段;
d)将步骤c的所述hFGO用所述wnt信号传导途径活化剂、所述EGF信号传导途径活化剂和FGF10培养第四时间段;
e)将步骤d的所述hFGO与MEK抑制剂接触第五时间段,其中所述第五时间段持续足以形成所述包含功能性胃底细胞类型的胃底组织的时间段。
2.根据权利要求1所述的方法,其中所述第一时间段是三天±24小时,并且其中所述视黄酸在所述时间段±24小时的第三天添加。
3.根据权利要求1所述的方法,其中所述第二时间段是三天±24小时。
4.根据权利要求1所述的方法,其中所述第三时间段是11天±24小时。
5.根据权利要求1所述的方法,其中所述第四时间段是10天±24小时。
6.根据权利要求1所述的方法,其中所述第五时间段是两天时间段±24小时。
7.根据权利要求1所述的方法,其中步骤e)还包括将所述胃底hGO与BMP4信号传导的活化剂接触的步骤。
8.根据权利要求1所述的方法,其中所述功能性胃底细胞类型是表达质子泵蛋白并且分泌酸的壁细胞。
9.根据权利要求1所述的方法,其中所述功能性胃底细胞类型是分泌胃蛋白酶原的主细胞。
10.根据权利要求1所述的方法,其中所述步骤e进行足以发育SOX2+GATA+PDX1-上皮组织的时间段。
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201662332194P | 2016-05-05 | 2016-05-05 | |
US62/332,194 | 2016-05-05 | ||
CN201780024903.5A CN109415685B (zh) | 2016-05-05 | 2017-05-05 | 用于体外制造胃底组织的方法和与其相关的组合物 |
PCT/US2017/031309 WO2017192997A1 (en) | 2016-05-05 | 2017-05-05 | Methods for the in vitro manufacture of gastric fundus tissue and compositions related to same |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201780024903.5A Division CN109415685B (zh) | 2016-05-05 | 2017-05-05 | 用于体外制造胃底组织的方法和与其相关的组合物 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116790476A true CN116790476A (zh) | 2023-09-22 |
Family
ID=60203655
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201780024903.5A Active CN109415685B (zh) | 2016-05-05 | 2017-05-05 | 用于体外制造胃底组织的方法和与其相关的组合物 |
CN202310699246.1A Pending CN116790476A (zh) | 2016-05-05 | 2017-05-05 | 用于体外制造胃底组织的方法和与其相关的组合物 |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201780024903.5A Active CN109415685B (zh) | 2016-05-05 | 2017-05-05 | 用于体外制造胃底组织的方法和与其相关的组合物 |
Country Status (7)
Country | Link |
---|---|
US (2) | US11066650B2 (zh) |
EP (2) | EP4177335A1 (zh) |
JP (2) | JP6963882B2 (zh) |
CN (2) | CN109415685B (zh) |
CA (1) | CA3016641A1 (zh) |
ES (1) | ES2929758T3 (zh) |
WO (1) | WO2017192997A1 (zh) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011140441A2 (en) | 2010-05-06 | 2011-11-10 | Children's Hospital Medical Center | Methods and systems for converting precursor cells into intestinal tissues through directed differentiation |
ES2860423T3 (es) | 2014-05-28 | 2021-10-05 | Childrens Hospital Med Ct | Métodos y sistemas para convertir células precursoras en tejidos gástricos mediante diferenciación dirigida |
AU2015331848B2 (en) | 2014-10-17 | 2022-03-03 | Children's Hospital Medical Center, D/B/A Cincinnati Children's Hospital Medical Center | In vivo model of human small intestine using pluripotent stem cells and methods of making and using same |
CA3016641A1 (en) | 2016-05-05 | 2017-11-09 | Children's Hospital Medical Center | Methods for the in vitro manufacture of gastric fundus tissue and compositions related to same |
US11767515B2 (en) | 2016-12-05 | 2023-09-26 | Children's Hospital Medical Center | Colonic organoids and methods of making and using same |
JP2020536529A (ja) * | 2017-10-10 | 2020-12-17 | チルドレンズ ホスピタル メディカル センター | 食道組織および/または臓器組成物およびそれを作製する方法 |
Family Cites Families (289)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE1095990B (de) | 1957-08-22 | 1960-12-29 | Chemiewerk Homburg Zweignieder | Verfahren zur Herstellung von wasserloeslichen Praeparaten und konzentrierten, bestaendigen waessrigen Loesungen von Digoxin |
WO1992007615A1 (en) | 1990-10-29 | 1992-05-14 | Regents Of The University Of Minnesota | A bioartificial liver |
US5523226A (en) | 1993-05-14 | 1996-06-04 | Biotechnology Research And Development Corp. | Transgenic swine compositions and methods |
US5912227A (en) | 1995-01-27 | 1999-06-15 | North Carolina State University | Method of enhancing nutrient uptake |
WO1998021312A1 (en) | 1996-11-08 | 1998-05-22 | Rpms Technology Limited | Human hepatocytes in three-dimensional support systems |
US7291626B1 (en) | 1998-04-09 | 2007-11-06 | John Hopkins University School Of Medicine | Inhibitors of hedgehog signaling pathways, compositions and uses related thereto |
US20020010550A1 (en) | 1998-09-14 | 2002-01-24 | George M. Grass | Pharmacokinetic-based drug design tool and method |
US7759113B2 (en) | 1999-04-30 | 2010-07-20 | The General Hospital Corporation | Fabrication of tissue lamina using microfabricated two-dimensional molds |
US6607501B2 (en) | 2001-05-14 | 2003-08-19 | Reynolds G. Gorsuch | Process and apparatus for utilization of in vivo extracted plasma with tissue engineering devices, bioreactors, artificial organs, and cell therapy applications |
WO2003076564A2 (en) | 2001-05-16 | 2003-09-18 | The General Hospital Corporation | Tissue-engineered organs |
CN101240262A (zh) | 2001-12-07 | 2008-08-13 | 杰龙公司 | 源自人胚胎干细胞的胰岛细胞 |
US20050170506A1 (en) | 2002-01-16 | 2005-08-04 | Primegen Biotech Llc | Therapeutic reprogramming, hybrid stem cells and maturation |
US20030187515A1 (en) | 2002-03-26 | 2003-10-02 | Hariri Robert J. | Collagen biofabric and methods of preparing and using the collagen biofabric |
US7160719B2 (en) | 2002-06-07 | 2007-01-09 | Mayo Foundation For Medical Education And Research | Bioartificial liver system |
US7695958B2 (en) | 2002-08-28 | 2010-04-13 | Asahi Kasei Kuraray Medical Co., Ltd. | Cell-filled hollow fiber membranes having modified cross-section |
TW571101B (en) | 2003-01-21 | 2004-01-11 | Ind Tech Res Inst | Fluid analysis apparatus |
EP1596926A2 (en) | 2003-02-07 | 2005-11-23 | The Johns Hopkins University | Hypoxia induced mitogenic factor |
US7727998B2 (en) | 2003-02-10 | 2010-06-01 | Banyu Pharmaceutical Co., Ltd. | Melanin-concentrating hormone receptor antagonists containing piperidine derivatives as the active ingredient |
DE10362002B4 (de) | 2003-06-23 | 2006-10-12 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Adulte pluripotente Stammzellen |
US8647873B2 (en) | 2004-04-27 | 2014-02-11 | Viacyte, Inc. | PDX1 expressing endoderm |
MX2009009225A (es) | 2003-12-23 | 2009-09-28 | Cythera Inc | Endodermo definitivo. |
US7541185B2 (en) | 2003-12-23 | 2009-06-02 | Cythera, Inc. | Methods for identifying factors for differentiating definitive endoderm |
US20050266554A1 (en) | 2004-04-27 | 2005-12-01 | D Amour Kevin A | PDX1 expressing endoderm |
US7985585B2 (en) | 2004-07-09 | 2011-07-26 | Viacyte, Inc. | Preprimitive streak and mesendoderm cells |
US7625753B2 (en) | 2003-12-23 | 2009-12-01 | Cythera, Inc. | Expansion of definitive endoderm cells |
US8586357B2 (en) | 2003-12-23 | 2013-11-19 | Viacyte, Inc. | Markers of definitive endoderm |
US8241905B2 (en) | 2004-02-24 | 2012-08-14 | The Curators Of The University Of Missouri | Self-assembling cell aggregates and methods of making engineered tissue using the same |
DE102004017476B4 (de) | 2004-04-08 | 2009-03-26 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Verfahren zur Herstellung einer Epithelzellen enthaltenden Zellzusammensetzung |
EP1740612B1 (en) | 2004-04-27 | 2019-08-07 | Viacyte, Inc. | Pdx1 expressing endoderm |
JP4650608B2 (ja) | 2004-05-18 | 2011-03-16 | 信越化学工業株式会社 | フォトマスクブランク及びフォトマスクの製造方法 |
WO2005113747A2 (de) | 2004-05-21 | 2005-12-01 | Fraunhofer Gesellschaft zur Förderung der angewandten Forschung e.V. | Multizelluläre gewebe- und organkultursysteme |
EP2319526A1 (en) | 2004-06-17 | 2011-05-11 | Thrasos Therapeutics, Inc. | Tdf-related compounds and analogs thereof |
CA2966883A1 (en) | 2004-07-09 | 2006-02-16 | Cythera, Inc. | Methods for identifying factors for differentiating definitive endoderm |
BRPI0513101A (pt) | 2004-07-12 | 2008-04-29 | Emisphere Tech Inc | forma unitária de dosagem para administração bucal, uso da forma unitária de dosagem e método para preparar a forma unitária de dosagem |
WO2006091231A2 (en) | 2004-07-21 | 2006-08-31 | Ambrx, Inc. | Biosynthetic polypeptides utilizing non-naturally encoded amino acids |
US8187878B2 (en) | 2004-08-13 | 2012-05-29 | University Of Georgia Research Foundation, Inc. | Methods for increasing definitive endoderm differentiation of pluripotent human embryonic stem cells with PI-3 kinase inhibitors |
US20060236415A1 (en) | 2005-03-09 | 2006-10-19 | Silversides David W | Neural crest cells specific promoters; isolated neural crest cells; and methods of isolating and of using same |
US7604929B2 (en) | 2005-04-21 | 2009-10-20 | In Vitro Technologies, Inc. | Cellular compositions and methods for their preparation |
WO2006126219A1 (en) | 2005-05-26 | 2006-11-30 | Fresenius Medical Care Deutschland G.M.B.H. | Liver progenitor cells |
GB0517382D0 (en) | 2005-08-26 | 2005-10-05 | Plasticell Ltd | Cell culture |
WO2007027905A2 (en) | 2005-08-31 | 2007-03-08 | Science And Technology Corporation @ Unm | Human renal stem cells |
ES2743202T3 (es) | 2005-10-27 | 2020-02-18 | Viacyte Inc | Endodermo de intestino proximal dorsal y ventral que expresa PDX1 |
WO2007065095A2 (en) | 2005-11-29 | 2007-06-07 | Spencer Rosero | System and method for supporting a biological chip device |
US20070239083A1 (en) | 2006-01-18 | 2007-10-11 | Axel Voss | Shock wave generators |
US20070238169A1 (en) | 2006-04-11 | 2007-10-11 | The Board Of Trustees Of The Leland Stanford Junior University | Cell sorter and culture system |
US8685730B2 (en) | 2006-05-02 | 2014-04-01 | Wisconsin Alumni Research Foundation | Methods and devices for differentiating pluripotent stem cells into cells of the pancreatic lineage |
WO2007136707A2 (en) | 2006-05-17 | 2007-11-29 | University Of Utah Research Foundation | Methods and compositions related to eosinophil regulation |
CA2656320C (en) | 2006-06-27 | 2015-04-28 | Intercept Pharmaceuticals, Inc. | Bile acid derivatives as fxr ligands for the prevention or treatment of fxr-mediated diseases or conditions |
US8071082B2 (en) | 2006-07-21 | 2011-12-06 | Massachusetts Institute Of Technology | End-modified poly(beta-amino esters) and uses thereof |
US8497240B2 (en) | 2006-08-17 | 2013-07-30 | Amylin Pharmaceuticals, Llc | DPP-IV resistant GIP hybrid polypeptides with selectable properties |
WO2008075339A2 (en) | 2006-12-18 | 2008-06-26 | Ben Gurion University Of The Negev | Scaffolding for tissue regeneration or repair |
FR2917425B1 (fr) | 2007-06-18 | 2010-11-19 | Univ Nancy 1 Henri Poincare | Procede de proliferation de cellules sur des multicouches de polyelectrolytes et son application, notamment a la preparation de biomateriaux cellularises |
EP2022848A1 (en) | 2007-08-10 | 2009-02-11 | Hubrecht Institut | A method for identifying, expanding, and removing adult stem cells and cancer stem cells |
US7695963B2 (en) | 2007-09-24 | 2010-04-13 | Cythera, Inc. | Methods for increasing definitive endoderm production |
CA2708780A1 (en) | 2007-12-11 | 2009-06-18 | Research Development Foundation | Small molecules for neuronal differentiation of embryonic stem cells |
EP2235162B1 (en) | 2008-01-08 | 2014-10-15 | The University Of Queensland | Method of producing a population of cells |
WO2009149179A2 (en) | 2008-06-03 | 2009-12-10 | Aethlon Medical Inc. | Enhanced antiviral therapy methods and devices |
EP2730645A3 (en) | 2008-06-04 | 2014-10-22 | TissUse GmbH | Organ-on-a-chip-device |
KR101612049B1 (ko) | 2008-06-24 | 2016-04-14 | 더 큐레이터스 오브 더 유니버시티 오브 미주리 | 자가-조립가능 다세포체 및 이들을 이용하여 3차원 생물학적 구조체를 생산하는 방법 |
US20130115673A1 (en) | 2008-07-16 | 2013-05-09 | Biotime, Inc. | Methods of Screening Embryonic Progenitor Cell Lines |
CA2741991A1 (en) | 2008-11-05 | 2010-05-14 | Merck Sharp & Dohme Corp. | Mechanism of neuromedin u action and uses thereof |
JP5351601B2 (ja) | 2008-12-26 | 2013-11-27 | 矢崎総業株式会社 | 絶縁キャップの製造方法及び絶縁キャップの製造装置 |
EP2412800A1 (en) | 2010-07-29 | 2012-02-01 | Koninklijke Nederlandse Akademie van Wetenschappen | Liver organoid, uses thereof and culture method for obtaining them |
KR101904224B1 (ko) | 2009-02-03 | 2018-10-04 | 코닌클리즈케 네덜란드세 아카데미 반 베텐샤펜 | 상피 줄기 세포용 배양 배지 및 상기 줄기 세포를 포함하는 오르가노이드 |
US9752124B2 (en) | 2009-02-03 | 2017-09-05 | Koninklijke Nederlandse Akademie Van Wetenschappen | Culture medium for epithelial stem cells and organoids comprising the stem cells |
GB201111244D0 (en) | 2011-06-30 | 2011-08-17 | Konink Nl Akademie Van Wetenschappen Knaw | Culture media for stem cells |
WO2010094694A1 (en) | 2009-02-23 | 2010-08-26 | F. Hoffmann-La Roche Ag | Assays to predict cardiotoxicity |
CN102421467B (zh) | 2009-03-13 | 2015-04-22 | 梅约医学教育与研究基金会 | 生物人工肝 |
RU2011139643A (ru) | 2009-03-17 | 2013-04-27 | Акскан Фарма, Инк. | Способ лечения неалкогольного стеатогепатита повышенными дозами урсодезоксихолевой кислоты |
WO2010127399A1 (en) | 2009-05-06 | 2010-11-11 | Walter And Eliza Hall Institute Of Medical Research | Gene expression profiles and uses thereof |
BRPI1012116A2 (pt) | 2009-05-20 | 2016-03-15 | Cardio3 Biosciences Sa | composição farmacêutica para o tratamento de doenças cardíacas |
CN102596989A (zh) | 2009-05-29 | 2012-07-18 | 诺沃-诺迪斯克有限公司 | 从hPS细胞来源的定形内胚层诱导获得特定内胚层 |
WO2010143747A1 (ja) | 2009-06-10 | 2010-12-16 | 公立大学法人奈良県立医科大学 | 人工腸管の作製法 |
CN102844036B (zh) | 2009-07-16 | 2016-01-20 | 生物时代股份有限公司 | 用于体外和体内软骨发生的方法和组合物 |
CA2921304C (en) | 2009-07-30 | 2018-06-05 | Tandem Diabetes Care, Inc. | Infusion pump system with disposable cartridge having pressure venting and pressure feedback |
US8501476B2 (en) | 2009-10-07 | 2013-08-06 | Brown University | Assays and methods for fusing cell aggregates to form proto-tissues |
EP2504424A1 (en) | 2009-11-25 | 2012-10-03 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Method for hepatic differentiation of definitive endoderm cells |
KR101923537B1 (ko) | 2009-12-23 | 2018-11-29 | 얀센 바이오테크 인코포레이티드 | 인간 배아 줄기 세포의 분화 |
JP5897543B2 (ja) | 2010-03-22 | 2016-03-30 | セルアーティス アーベー | Wntシグナル経路の調節による多能性細胞から肝細胞様細胞への分化誘導と成熟 |
EP2380920A1 (en) | 2010-04-22 | 2011-10-26 | QGel SA | Hydrogel precursor formulation and production process thereof |
WO2011139628A1 (en) | 2010-04-25 | 2011-11-10 | Mount Sinai School Of Medicine | Generation of anterior foregut endoderm from pluripotent cells |
WO2011140441A2 (en) | 2010-05-06 | 2011-11-10 | Children's Hospital Medical Center | Methods and systems for converting precursor cells into intestinal tissues through directed differentiation |
US8609406B2 (en) | 2010-08-24 | 2013-12-17 | Regents Of The University Of Minnesota | Non-static suspension culture of cell aggregates |
US9181528B2 (en) | 2010-08-31 | 2015-11-10 | Janssen Biotech, Inc. | Differentiation of pluripotent stem cells |
JP6163104B2 (ja) | 2010-11-15 | 2017-07-12 | アクセラレイテッド・バイオサイエンシズ・コーポレーション | ヒト・トロホブラスト幹細胞からの神経幹細胞の生成 |
BR112013016592B1 (pt) | 2010-12-31 | 2022-03-15 | Universitãt Für Bodenkultur Wien | Método de geração de células-tronco pluripotentes induzidas e células diferenciadas |
US8951781B2 (en) | 2011-01-10 | 2015-02-10 | Illumina, Inc. | Systems, methods, and apparatuses to image a sample for biological or chemical analysis |
US9200258B2 (en) | 2011-01-27 | 2015-12-01 | University Of Maryland, Baltimore | Multicellular organotypic model of human intestinal mucosa |
EP2484750A1 (en) | 2011-02-07 | 2012-08-08 | Ecole Polytechnique Fédérale de Lausanne (EPFL) | Monitoring system for cell culture |
SG10201601434RA (en) | 2011-02-28 | 2016-03-30 | Harvard College | Cell Culture System |
GB201106395D0 (en) | 2011-04-14 | 2011-06-01 | Hubrecht Inst | Compounds |
US20140158233A1 (en) | 2011-05-09 | 2014-06-12 | President And Fellows Of Harvard College | Aerosol delivery to a microfluidic device |
US10260039B2 (en) | 2011-05-11 | 2019-04-16 | Massachusetts Institute Of Technology | Microgels and microtissues for use in tissue engineering |
SG10201805199RA (en) | 2011-06-02 | 2018-07-30 | Harvard College | Methods and Uses for Ex Vivo Tissue Culture Systems |
EP2723851A4 (en) * | 2011-06-23 | 2015-05-20 | Philadelphia Children Hospital | SELF-RENEWING ENDODERMAL PRELIMINARY CELL LINES FROM HUMAN PLURIPOTENTIAL STEM CELLS AND METHOD OF USE THEREOF |
AU2012308724A1 (en) | 2011-09-12 | 2014-04-24 | Organovo, Inc. | Platform for engineered implantable tissues and organs and methods of making the same |
JP2013066414A (ja) | 2011-09-22 | 2013-04-18 | National Institute Of Advanced Industrial Science & Technology | 胃前駆細胞の表面マーカー |
US20190209690A9 (en) | 2011-10-12 | 2019-07-11 | The Johns Hopkins University | Bioreducible Poly (Beta-Amino Ester)s For siRNA Delivery |
WO2013063588A1 (en) * | 2011-10-28 | 2013-05-02 | The Board Of Trustees Of The Leland Stanford Junior University | Ex vivo culture, proliferation and expansion of primary tissue organoids |
EP3425391A1 (en) | 2011-11-04 | 2019-01-09 | inRegen | Drug screening and potency assays |
US9725687B2 (en) | 2011-12-09 | 2017-08-08 | President And Fellows Of Harvard College | Integrated human organ-on-chip microphysiological systems |
US10087422B2 (en) | 2011-12-09 | 2018-10-02 | President And Fellows Of Harvard College | Organ chips and uses thereof |
US10006904B2 (en) | 2011-12-19 | 2018-06-26 | Umc Utrecht Holding B.V. | Rapid quantitative assay to measure CFTR function in a primary intestinal culture model |
CA2859714C (en) | 2011-12-23 | 2023-10-17 | Anthrogenesis Corporation | Organoids comprising decellularized and repopulated placental vascular scaffold |
ES2833280T3 (es) | 2012-01-13 | 2021-06-14 | Massachusetts Gen Hospital | Células progenitoras pulmonares humanas aisladas y usos de las mismas |
WO2013116446A1 (en) | 2012-01-31 | 2013-08-08 | Wake Forest University Health Sciences | Tubular bioengineered smooth muscle structures |
EP2816893A1 (en) | 2012-02-22 | 2014-12-31 | Amgen Inc. | Autologous mammalian models derived from induced pluripotent stem cells and related methods |
EP2634251A1 (en) | 2012-02-29 | 2013-09-04 | Technische Universität Berlin | 3D in vitro bi-phasic cartilage-bone construct |
EP2836585A4 (en) | 2012-04-09 | 2015-12-23 | Thomas J Goodwin | ALTERNATIVE ION MAGNETIC RESONANCE MULTIPLE CHAMBER CULTURE APPARATUS (AIMR) AND METHODS OF USE |
BR112014028881A2 (pt) | 2012-05-23 | 2017-06-27 | Hoffmann La Roche | populações de células, banco de células, métodos de obtenção de uma população de células, métodos de identificação de um fator, métodos de seleção, métodos de fornecimento de terapia, populações de hepatócitos e método de obtenção de células |
US20140099709A1 (en) | 2012-06-19 | 2014-04-10 | Organovo, Inc. | Engineered three-dimensional connective tissue constructs and methods of making the same |
EP3789394A1 (en) | 2012-06-19 | 2021-03-10 | Intercept Pharmaceuticals, Inc. | Crystalline forms c of obeticholic acid |
DE102012105540A1 (de) | 2012-06-26 | 2014-04-24 | Karlsruher Institut für Technologie | Gefäßmodell, Verfahren zu seiner Herstellung und seine Verwendung |
WO2014013334A2 (en) | 2012-07-20 | 2014-01-23 | Agency For Science, Technology And Research | In vitro assay for predicting renal proximal tubular cell toxicity |
GB201216796D0 (en) | 2012-09-20 | 2012-11-07 | Cambridge Entpr Ltd | In vitro pancreatic differentiation |
DK2712918T3 (en) | 2012-09-28 | 2015-02-16 | Tissuse Gmbh | Multi-organ-chip with improved lifetime and homeostasis |
EP2716298A1 (en) | 2012-10-03 | 2014-04-09 | Institut Pasteur | A nod2-dependant pathway of cytoprotection of stem cells |
WO2014082096A1 (en) | 2012-11-26 | 2014-05-30 | The Trustees Of Columbia University In The City Of New York | Method for culture of human and mouse prostate organoids and uses thereof |
ES2625510T3 (es) | 2012-11-26 | 2017-07-19 | Gambro Lundia Ab | Sistema de soporte hepático |
AU2013359199C1 (en) | 2012-12-12 | 2021-06-17 | Massachusetts Institute Of Technology | Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications |
EP2743345A1 (en) | 2012-12-13 | 2014-06-18 | IMBA-Institut für Molekulare Biotechnologie GmbH | Three dimensional heterogeneously differentiated tissue culture |
US20150359849A1 (en) | 2013-01-31 | 2015-12-17 | President And Fellows Of Harvard College | Methods of increasing neuronal connectivity and/or treating a neurodegenerative condition |
WO2014127170A1 (en) | 2013-02-13 | 2014-08-21 | Wake Forest University Health Sciences | Bioengineered liver constructs and methods relating thereto |
GB201304245D0 (en) | 2013-03-08 | 2013-04-24 | Inst Quimic De Sarria | Chemical compounds |
WO2014165056A1 (en) | 2013-03-12 | 2014-10-09 | Board Of Regents, The University Of Texas System | High throughput mechanical strain generating system for cell cultures and applications thereof |
US9938499B2 (en) | 2013-03-13 | 2018-04-10 | Wisconsin Alumni Research Foundation | Methods and materials for hematoendothelial differentiation of human pluripotent stem cells under defined conditions |
US20160022873A1 (en) | 2013-03-14 | 2016-01-28 | Research Institute At Nationwide Children's Hospital, Inc. | Tissue engineered intestine |
DK3702443T3 (da) * | 2013-03-14 | 2022-04-04 | Brigham & Womens Hospital Inc | Sammensætninger og fremgangsmåder til opformering og dyrkning af epiteliale stamceller |
EP3656849A1 (en) | 2013-03-14 | 2020-05-27 | The Regents of The University of California | In vitro production of medial ganglionic eminence precursor cells |
US9442105B2 (en) | 2013-03-15 | 2016-09-13 | Organovo, Inc. | Engineered liver tissues, arrays thereof, and methods of making the same |
US20160237400A1 (en) | 2013-03-15 | 2016-08-18 | The Jackson Laboratory | Isolation of non-embryonic stem cells and uses thereof |
WO2014153294A1 (en) | 2013-03-17 | 2014-09-25 | The Regents Of The University Of California | Method to expand and transduce cultured human small and large intestinal stem cells |
US9677085B2 (en) | 2013-03-18 | 2017-06-13 | Massachusetts Institute Of Technology | Engineering a heterogeneous tissue from pluripotent stem cells |
EP2796873A1 (en) | 2013-04-25 | 2014-10-29 | QGel SA | Method for a cell-based drug screening assay and the use thereof |
US20160245653A1 (en) | 2013-04-30 | 2016-08-25 | Sangtae Park | Cylindrical resonator gyroscope |
JP6490669B2 (ja) | 2013-05-08 | 2019-03-27 | インリージェン | 単離腎細胞を含有するオルガノイド及びその使用 |
US10545133B2 (en) | 2013-05-13 | 2020-01-28 | The Johns Hopkins University | Molecular signatures of invasive cancer subpopulations |
US11060065B2 (en) | 2013-06-10 | 2021-07-13 | Corning Incorporated | Tissue structure and preparation method thereof |
CN105473706B (zh) | 2013-06-14 | 2021-10-26 | 昆士兰大学 | 肾祖细胞 |
KR102184473B1 (ko) | 2013-07-23 | 2020-11-30 | 고리츠다이가쿠호진 요코하마시리츠다이가쿠 | 생물학적 조직에 혈관계를 부여하는 방법 |
MX2016001714A (es) | 2013-08-09 | 2016-10-03 | Ardelyx Inc | Compuestos y metodos para inhibir el transporte de fosfato. |
PL3039123T3 (pl) | 2013-08-28 | 2021-07-19 | Promethera Biosciences S.A. / N.V. | Sposób wytwarzania komórek progenitorowych dorosłej wątroby |
GB201317869D0 (en) | 2013-10-09 | 2013-11-20 | Cambridge Entpr Ltd | In vitro production of foregut stem cells |
CN105849252B (zh) | 2013-10-25 | 2020-03-17 | 新加坡科技研究局 | 培养多能干细胞 |
JP6499084B2 (ja) | 2013-11-22 | 2019-04-10 | 国立研究開発法人理化学研究所 | 終脳又はその前駆組織の製造方法 |
CA2930784C (en) | 2013-11-22 | 2023-01-31 | Cellectis | Method for generating batches of allogeneic t cells with averaged potency |
EP2876441B1 (en) | 2013-11-26 | 2017-10-25 | Bergen Teknologioverforing AS | Quantitative analysis of contact-depending cell-to-cell transfer and disease transmission |
AU2014386207B2 (en) | 2013-12-20 | 2019-08-22 | President And Fellows Of Harvard College | Organomimetic devices and methods of use and manufacturing thereof |
EP3094720B1 (en) | 2014-01-14 | 2021-07-21 | Yale University | Compositions and methods of preparing airway cells |
US11648335B2 (en) | 2014-01-31 | 2023-05-16 | Wake Forest University Health Sciences | Organ/tissue decellularization, framework maintenance and recellularization |
AU2015217406A1 (en) | 2014-02-11 | 2016-08-25 | Celularity Inc. | Micro-organoids, and methods of making and using the same |
US10369254B2 (en) | 2014-02-26 | 2019-08-06 | The Regents Of The University Of California | Method and apparatus for in vitro kidney organogenesis |
CA2937882A1 (en) | 2014-02-27 | 2015-09-03 | Public University Corporation Yokohama City University | Method for generating cell condensate for self-organization |
DE102014003465A1 (de) | 2014-03-11 | 2015-09-17 | NeuroProof GmbH | Gewinnung von Gehirnregion-spezifischen neuronalen Kulturen aus dreidimensionalen Gewebekulturen von Stammzellen |
US11066649B2 (en) | 2014-03-19 | 2021-07-20 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Method for inducing human cholangiocyte differentiation |
EP3119401A4 (en) | 2014-03-21 | 2017-12-13 | Tobira Therapeutics, Inc. | Cenicriviroc for the treatment of fibrosis |
CN106536720B (zh) | 2014-04-04 | 2021-04-30 | 奥加诺沃公司 | 工程化的三维乳腺组织,脂肪组织和肿瘤疾病模型 |
WO2015157163A1 (en) | 2014-04-07 | 2015-10-15 | Rush University Medical Center | Screening assay for choice of prebiotic to prevent/treat gastrointestinal and systemic diseases |
WO2015156929A1 (en) | 2014-04-07 | 2015-10-15 | The Trustees Of Columbia University In The City Of New York | Method for culture of human bladder cell lines and organoids and uses thereof |
EA036704B1 (ru) | 2014-04-11 | 2020-12-10 | Саймабэй Терапьютикс, Инк. | Способ лечения неалкогольной жировой болезни печени и неалкогольного стеатогепатита |
CN106456673B (zh) | 2014-04-27 | 2021-04-16 | 纽约州立大学研究基金会 | 釉质产品和使用方法 |
WO2015173425A1 (en) | 2014-05-16 | 2015-11-19 | Koninklijke Nederlandse Akademie Van Wetenschappen | Improved culture method for organoids |
ES2860423T3 (es) | 2014-05-28 | 2021-10-05 | Childrens Hospital Med Ct | Métodos y sistemas para convertir células precursoras en tejidos gástricos mediante diferenciación dirigida |
WO2015184375A2 (en) | 2014-05-29 | 2015-12-03 | Whitehead Institute For Biomedical Research | Compositions and methods for promoting intestinal stem cell and/or non-stem progenitor cell function |
US10683476B2 (en) | 2014-05-29 | 2020-06-16 | Icahn School Of Medicine At Mount Sinai | Method and apparatus to prepare cardiac organoids in a bioreactor system |
EP3786285A1 (en) | 2014-06-05 | 2021-03-03 | Cedars-Sinai Medical Center | A novel and efficient method for reprogramming immortalized lymphoblastoid cell lines to induced pluripotent stem cells |
WO2015185714A1 (en) | 2014-06-06 | 2015-12-10 | Vrije Universiteit Brussel | Human hepatic 3d co-culture model and uses thereof |
FR3022110B1 (fr) | 2014-06-13 | 2016-07-01 | Liphatech Inc | Appat rodonticide et procede de lutte contre des rongeurs cibles nuisibles mettant en œuvre un tel appat |
CA2953122A1 (en) | 2014-06-20 | 2015-12-23 | Rutgers, The State University Of New Jersey | Single cell-derived organoids |
US10487314B2 (en) | 2014-06-26 | 2019-11-26 | The Trustees Of Columbia University In The City Of New York | Inhibition of serotonin expression in gut enteroendocrine cells results in conversion to insulin-positive cells |
EP3169811A4 (en) | 2014-07-17 | 2017-12-27 | Celmatix Inc. | Methods and systems for assessing infertility and related pathologies |
BR112017001623B1 (pt) | 2014-07-29 | 2022-11-29 | Shenzhen Hightide Biopharmaceutical, Ltd | Sal de adição ácido-base em uma forma substancialmente pura, composição farmacêutica compreendendo uma quantidade do referido sal e seus usos |
WO2016015158A1 (en) | 2014-07-30 | 2016-02-04 | University Health Network | Organoids for drug screening and personalized medicine |
WO2016027099A2 (en) | 2014-08-22 | 2016-02-25 | Cambridge Enterprise Limited | Resetting pluripotent stem cells |
JP2017530697A (ja) | 2014-08-28 | 2017-10-19 | プロメセラ バイオサイエンシーズ エス.アー./エンヌ.フェー. | 成体肝前駆細胞を作製する方法 |
EP3186365A4 (en) | 2014-08-29 | 2018-04-25 | Immunomedics, Inc. | Identification of cancer genes by in-vivo fusion of human cancer cells and animal cells |
JP2017526713A (ja) | 2014-09-12 | 2017-09-14 | トビラ セラピューティクス, インコーポレイテッド | 線維症を処置するためのセニクリビロック併用療法 |
EP3204488B1 (en) | 2014-10-06 | 2019-07-17 | Organovo, Inc. | Engineered renal tissues, arrays thereof, and methods of making the same |
US20170304369A1 (en) | 2014-10-08 | 2017-10-26 | Agency For Science, Technology And Research | Methods of differentiating stem cells into liver cell lineages |
JP6581655B2 (ja) | 2014-10-14 | 2019-09-25 | フジフィルム セルラー ダイナミクス,インコーポレイテッド | 多能性幹細胞由来ケラチノサイトの生成およびケラチノサイト培養の維持 |
AU2015331848B2 (en) | 2014-10-17 | 2022-03-03 | Children's Hospital Medical Center, D/B/A Cincinnati Children's Hospital Medical Center | In vivo model of human small intestine using pluripotent stem cells and methods of making and using same |
US20160121023A1 (en) | 2014-10-30 | 2016-05-05 | Massachusetts Institute Of Technology | Materials and Methods for Rescue of Ischemic Tissue and Regeneration of Tissue Integrity During Resection, Engraftment and Transplantation |
US10479977B2 (en) | 2014-11-07 | 2019-11-19 | The Trustees Of Columbia University In The City Of New York | Osteochondroreticular stem cells for bone and cartilage regeneration |
AU2015353853B2 (en) | 2014-11-25 | 2020-10-15 | President And Fellows Of Harvard College | Methods for generation of podocytes from pluripotent stem cells and cells produced by the same |
WO2016086052A1 (en) | 2014-11-25 | 2016-06-02 | International Stem Cell Corporation | Derivation of nueral crest stem cells and uses thereof |
GB201421094D0 (en) | 2014-11-27 | 2015-01-14 | Koninklijke Nederlandse Akademie Van Wetenschappen | Culture medium |
GB201421092D0 (en) | 2014-11-27 | 2015-01-14 | Koninklijke Nederlandse Akademie Van Wetenschappen | Culture medium |
AU2014277667B2 (en) | 2014-12-15 | 2022-07-14 | The University Of Queensland | Differentiation of pluripotent stem cells to form renal organoids |
CA2972057C (en) | 2014-12-22 | 2023-06-13 | Ecole Polytechnique Federale De Lausanne (Epfl) | Microstructured thin hydrogel films |
WO2016103269A1 (en) | 2014-12-23 | 2016-06-30 | Ramot At Tel-Aviv University Ltd. | Populations of neural progenitor cells and methods of producing and using same |
EA201791258A1 (ru) | 2015-01-09 | 2017-12-29 | Джилид Аполло, Ллс | Комбинированная терапия с применением ингибитора ацетил-коа-карбоксилазы (acc) для лечения неалкогольной жировой болезни печени |
EP3252144A4 (en) | 2015-01-28 | 2018-08-08 | Public University Corporation Yokohama City University | Method for preparing bone marrow cell aggregate |
US20160256672A1 (en) | 2015-02-10 | 2016-09-08 | Cedars-Sinai Medical Center | Induced pluripotent stem cell-derived hepatocyte based bioartificial liver device |
CN105985395A (zh) | 2015-02-13 | 2016-10-05 | 江苏奥赛康药业股份有限公司 | 一种奥贝胆酸化合物及含有该化合物的药物组合物 |
WO2016140716A1 (en) | 2015-03-02 | 2016-09-09 | The Trustees Of Columbia University In The City Of New York | Injectable microtissue systems, devices, and methods |
JP6807853B2 (ja) | 2015-03-03 | 2021-01-06 | プレジデント アンド フェローズ オブ ハーバード カレッジ | 機能的ヒト組織の作製方法 |
EP3265180A4 (en) | 2015-03-06 | 2019-04-24 | The University of North Carolina at Chapel Hill | HUMAN FIBROLAMELLAR HEPATOCELLULAR CARCINOMAS (HFL-HCCS) |
WO2016142690A1 (en) | 2015-03-06 | 2016-09-15 | Micromass Uk Limited | Inlet instrumentation for ion analyser coupled to rapid evaporative ionisation mass spectrometry ("reims") device |
WO2016144862A1 (en) | 2015-03-09 | 2016-09-15 | Intekrin Therapeutics, Inc. | Methods for the treatment of nonalcoholic fatty liver disease and/or lipodystrophy |
US10023922B2 (en) | 2015-03-23 | 2018-07-17 | Whitehead Institute For Biomedical Research | Reporter of genomic methylation and uses thereof |
MX2017012893A (es) | 2015-04-07 | 2018-01-15 | Intercept Pharmaceuticals Inc | Composiciones farmaceuticas para terapias combinadas. |
US10087417B2 (en) | 2015-04-22 | 2018-10-02 | William J. Freed | Three-dimensional model of human cortex |
US10557124B2 (en) | 2015-04-22 | 2020-02-11 | The Regents Of The University Of Michigan | Compositions and methods for obtaining stem cell derived lung tissue, and related uses thereof |
CN104877964A (zh) | 2015-04-24 | 2015-09-02 | 赵振民 | 一种唾液腺类器官和类腺泡的体外构建方法 |
PE20180690A1 (es) | 2015-04-27 | 2018-04-23 | Intercept Pharmaceuticals Inc | Composiciones de acido obeticolico y metodos de uso |
CA2980852A1 (en) | 2015-04-30 | 2016-11-03 | Helmholtz Zentrum Muenchen Deutsches Forschungszentrum Fuer Gesundheit Und Umwelt (Gmbh) | Means and methods for generation of breast stem cells |
US11299714B2 (en) | 2015-05-11 | 2022-04-12 | The Trustees Of Columbia University In The City Of New York | Engineered adult-like human heart tissue |
JP6847048B2 (ja) | 2015-06-03 | 2021-03-24 | タカラ バイオ ヨーロッパ アーベー | 哺乳動物肝細胞の成熟 |
GB201510950D0 (en) | 2015-06-22 | 2015-08-05 | Cambridge Entpr Ltd | In vitro Production of Cholangiocytes |
US11136548B2 (en) | 2015-06-24 | 2021-10-05 | Whitehead Institute For Biomedical Research | Culture medium for generating microglia from pluripotent stem cells and related methods |
EP3313416A4 (en) | 2015-06-26 | 2019-02-20 | The Trustees of Columbia University in the City of New York | GENETICALLY MODIFIED IPS CELLS HAVING A MARKER FOR SIGNALING THE EXPRESSION OF NEUROGENIN3, TPH2, FOXO1 AND / OR INSULIN GENES |
US20180216070A1 (en) | 2015-07-10 | 2018-08-02 | Etablissement Francais Du Sang | Method for obtaining human brown/beige adipocytes |
US10449221B2 (en) | 2015-07-29 | 2019-10-22 | Trustees Of Boston University | Differentiation of stem cells into thyroid tissue |
WO2017036533A1 (en) | 2015-09-03 | 2017-03-09 | Ecole Polytechnique Federale De Lausanne (Epfl) | Three-dimensional hydrogels for culturing adult epithelial stem cells and organoids |
EP3344264B1 (en) | 2015-09-03 | 2023-03-15 | The Brigham and Women's Hospital, Inc. | Three-dimensional differentiation of epiblast spheroids to kidney organoids models stage-specific epithelial physiology, morphogenesis, and disease |
WO2017048193A1 (en) | 2015-09-15 | 2017-03-23 | Agency For Science, Technology And Research (A*Star) | Derivation of liver organoids from human pluripotent stem cells |
MX2018003179A (es) | 2015-09-16 | 2018-08-21 | Tobira Therapeutics Inc | Terapia de combinación con cenicriviroc para el tratamiento de fibrosis. |
US20180258404A1 (en) | 2015-09-17 | 2018-09-13 | The Brigham And Women's Hospital, Inc. | Methods of generating nephrons from human pluripotent stem cells |
KR20180055894A (ko) | 2015-10-02 | 2018-05-25 | 웨이크 포리스트 유니버시티 헬스 사이언시즈 | 자발적으로 박동하는 심장 오르가노이드 구조체 및 그를 포함하는 통합된 바디-온-칩 장치 |
LU92845B1 (en) | 2015-10-08 | 2017-05-02 | Univ Du Luxembourg Campus Belval | Means and methods for generating midbrain organoids |
US10993433B2 (en) | 2015-10-15 | 2021-05-04 | Wake Forest University Health Sciences | Method of producing in vitro testicular constructs and uses thereof |
JP6873985B2 (ja) | 2015-10-15 | 2021-05-19 | ウェイク・フォレスト・ユニヴァーシティ・ヘルス・サイエンシズ | インビトロで肝臓構築物を産生する方法およびその使用 |
US11001811B2 (en) | 2015-10-16 | 2021-05-11 | Wake Forest University Health Sciences | Multi-layer airway organoids and methods of making and using the same |
WO2017066659A1 (en) | 2015-10-16 | 2017-04-20 | The Trustees Of Columbia University In The City Of New York | Jag1 expression predicts therapeutic response in nash |
US20180057788A1 (en) | 2016-08-29 | 2018-03-01 | EMULATE, Inc. | Development of spinal cord on a microfluidic chip |
SG11201803143YA (en) | 2015-10-19 | 2018-05-30 | Emulate Inc | Microfluidic model of the blood brain barrier |
WO2017070337A1 (en) | 2015-10-20 | 2017-04-27 | Cellular Dynamics International, Inc. | Methods for directed differentiation of pluripotent stem cells to immune cells |
JP6979946B2 (ja) | 2015-10-21 | 2021-12-15 | インディアナ ユニバーシティー リサーチ アンド テクノロジー コーポレーションIndiana University Research And Technology Corporation | ヒト内耳感覚上皮および感覚ニューロンを生成する方法 |
CA3002164A1 (en) | 2015-10-21 | 2017-04-27 | Indiana University Research And Technology Corporation | Derivation of human skin organoids from pluripotent stem cells |
JP6983771B2 (ja) | 2015-10-30 | 2021-12-17 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニアThe Regents Of The University Of California | 幹細胞からt細胞を作製する方法および該t細胞を用いた免疫療法的方法 |
US11866409B2 (en) | 2015-11-02 | 2024-01-09 | Carmel-Haifa University Economic Corporation Ltd. | Apoptosis related protein in the tgf-beta signaling pathway (ARTS) mimetic compounds, compositions, methods and uses thereof in induction of differentiation and/or apoptosis of premalignant and malignant cells, thereby restoring their normal-like phenotype |
WO2017079632A1 (en) | 2015-11-04 | 2017-05-11 | Cedars-Sinai Medical Center | Patient-derived ctc-xenograft models |
CN108366957A (zh) | 2015-11-06 | 2018-08-03 | 燿石治疗公司 | 用于治疗心血管疾病的吉卡宾组合 |
CN108601646B (zh) | 2015-11-12 | 2021-10-12 | 博奥司时代有限责任公司 | 用于产生胃肠道组织的***和方法 |
US10597623B2 (en) | 2015-11-13 | 2020-03-24 | The Johns Hopkins University | Multiwell cell culture system having rotating shafts for mixing culture media and method of use thereof |
WO2017096282A1 (en) | 2015-12-04 | 2017-06-08 | EMULATE, Inc. | Devices and methods for simulating a function of a liver tissue |
GB2562396B (en) | 2015-12-04 | 2022-04-13 | Harvard College | Devices for simulating a function of a liver tissue and methods of use and manufacturing thereof |
AU2016377701B2 (en) | 2015-12-23 | 2023-04-06 | Memorial Sloan-Kettering Cancer Center | Cell-based treatment and drug discovery in Hirschsprung's disease enabled by pluripotent stem cell-derived human enteric neural crest lineages |
WO2017117333A1 (en) | 2015-12-30 | 2017-07-06 | Cellular Dynamics International, Inc. | Microtissue formation using stem cell-derived human hepatocytes |
KR101733137B1 (ko) | 2015-12-30 | 2017-05-08 | (주)엑셀세라퓨틱스 | 연골조직 제조를 위한 3차원 오가노이드 블록 제작 방법 |
US20190367868A1 (en) | 2015-12-31 | 2019-12-05 | President And Fellows Of Harvard College | Neurons and compositions and methods for producing the same |
US20190002835A1 (en) | 2015-12-31 | 2019-01-03 | President And Fellows Of Harvard College | Methods for generating neural tissue and uses thereof |
JP2019506153A (ja) | 2016-01-08 | 2019-03-07 | マサチューセッツ インスティテュート オブ テクノロジー | 分化した腸内分泌細胞およびインスリン産生細胞の作製 |
EP3190176A1 (en) | 2016-01-11 | 2017-07-12 | IMBA-Institut für Molekulare Biotechnologie GmbH | Method for tissue culture development on scaffold and differentiated tissue culture |
JP2019502407A (ja) | 2016-01-14 | 2019-01-31 | オハイオ ステイト イノベイション ファウンデーション | 神経オルガノイド組成物および使用方法 |
US20170205396A1 (en) | 2016-01-15 | 2017-07-20 | Salk Institute For Biological Studies | Systems and methods for culturing nephron progenitor cells |
EP3411472A4 (en) | 2016-02-01 | 2020-01-22 | Cedars-Sinai Medical Center | SYSTEMS AND METHODS FOR GROWING INTESTINAL CELLS IN MICROFLUIDIC DEVICES |
AU2017217688B2 (en) | 2016-02-10 | 2023-01-19 | Wake Forest University Health Sciences | Model system of liver fibrosis and method of making and using the same |
US20190046583A1 (en) | 2016-02-11 | 2019-02-14 | Johns Hopkins Technology Ventures | Compositions and methods for neuralgenesis |
WO2017143100A1 (en) | 2016-02-16 | 2017-08-24 | The Board Of Trustees Of The Leland Stanford Junior University | Novel recombinant adeno-associated virus capsids resistant to pre-existing human neutralizing antibodies |
JP6376578B2 (ja) | 2016-02-18 | 2018-08-22 | 学校法人慶應義塾 | 細胞培養培地、培養方法、及びオルガノイド |
US10612094B2 (en) | 2016-02-19 | 2020-04-07 | Procella Therapeutics Ab | Genetic markers for engraftment of human cardiac ventricular progenitor cells |
US20170267970A1 (en) | 2016-02-29 | 2017-09-21 | Whitehead Institute For Biomedical Research | Three-Dimensional Hydrogels that Support Growth of Physiologically Relevant Tissue and Methods of Use Thereof |
GB201603569D0 (en) | 2016-03-01 | 2016-04-13 | Koninklijke Nederlandse Akademie Van Wetenschappen | Improved differentiation method |
US20190086391A1 (en) | 2016-03-08 | 2019-03-21 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Method and system for continuous biosensing |
EP3430132A4 (en) | 2016-03-14 | 2019-12-25 | Agency for Science, Technology and Research | GENERATION OF SPECIES-SPECIFIC ORGANOIDS FROM MENENCEPHALUS FROM HUMAN PLURIPOTENT STEM CELLS |
US20190076482A1 (en) | 2016-03-15 | 2019-03-14 | The Johns Hopkins University | Enhanced organoid formation and intestinal stem cell renewal |
US10772863B2 (en) | 2016-03-15 | 2020-09-15 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd | Methods of inducing metabolic maturation of human pluripotent stem cells— derived hepatocytes |
JP6960140B2 (ja) | 2016-03-16 | 2021-11-05 | 公立大学法人横浜市立大学 | 腫瘍組織再現法 |
US20200299649A1 (en) | 2016-03-29 | 2020-09-24 | Smsbiotech, Inc. | Compositions and methods for using small mobile stem cells |
EP3228306A1 (en) | 2016-04-04 | 2017-10-11 | ratiopharm GmbH | Complex compound comprising obeticholic acid and cyclodextrin and pharmaceutical formulation comprising the complex compound |
KR20190003549A (ko) | 2016-04-04 | 2019-01-09 | 휴멜티스 | 암 케어에서 환자 특이적 치료학적 판단을 위한 진단 방법 |
WO2017176810A1 (en) | 2016-04-04 | 2017-10-12 | Biotime, Inc. | Pluripotent stem cell-derived 3d retinal tissue and uses thereof |
JP6935101B2 (ja) | 2016-04-05 | 2021-09-15 | 学校法人自治医科大学 | 幹細胞を再樹立する方法 |
WO2017184586A1 (en) | 2016-04-18 | 2017-10-26 | The Trustees Of Columbia University In The City Of New York | Therapeutic targets involved in the progression of nonalcoholic steatohepatitis (nash) |
JP2019514872A (ja) | 2016-04-22 | 2019-06-06 | タイワンジェ ファーマシューティカルズ カンパニー リミテッドTaiwanj Pharmaceuticals Co., Ltd. | (非)アルコ−ル性脂肪肝炎又は非アルコ−ル性脂肪性肝疾患の治療のためのナルメフェン(NALMEFENE)、ナルトレキソン(naltrexone)及びその関連誘導体の応用 |
US20170321188A1 (en) | 2016-05-04 | 2017-11-09 | The Research Foundation For The State University Of New York | Methods of generating retinal progenitor cell preparations and uses thereof |
CA3016641A1 (en) | 2016-05-05 | 2017-11-09 | Children's Hospital Medical Center | Methods for the in vitro manufacture of gastric fundus tissue and compositions related to same |
EP3485001A4 (en) | 2016-05-25 | 2020-08-12 | Salk Institute for Biological Studies | COMPOSITIONS AND PROCESSES FOR THE PRODUCTION OF ORGANOIDS AND THE MODELING OF DISEASES |
US20170349659A1 (en) | 2016-06-03 | 2017-12-07 | The Board Of Trustees Of The Leland Stanford Junior University | Wnt signaling agonist molecules |
US20190136199A1 (en) | 2016-06-15 | 2019-05-09 | Children's Medical Center Corporation | Methods and compositions relating to lung cell differentiation |
GB201610748D0 (en) | 2016-06-20 | 2016-08-03 | Koninklijke Nederlandse Akademie Van Wetenschappen | Improved diffrentation method |
GB201611982D0 (en) | 2016-07-11 | 2016-08-24 | Cellesce Ltd | Cell culture |
EP3275997A1 (en) | 2016-07-28 | 2018-01-31 | QGel SA | Hydrogel precursor formulation and the use thereof |
AU2017306426A1 (en) | 2016-08-02 | 2019-01-24 | Memorial Sloan-Kettering Cancer Center | Treating metastatic cancer and model systems for metastatic disease |
WO2018027023A1 (en) | 2016-08-03 | 2018-02-08 | Wake Forest University Health Sciences | Cancer modeling platforms and methods of using the same |
EP3494213A4 (en) | 2016-08-04 | 2020-01-08 | Wake Forest University Health Sciences | MODEL FOR BLOOD BRAIN CABINETS AND METHOD FOR THE PRODUCTION AND USE THEREOF |
EP3505620B1 (en) | 2016-08-24 | 2023-07-26 | Keio University | 2d organoid for infection and culture of human diarrhea virus, and use of said 2d organoid |
KR102634265B1 (ko) | 2016-08-26 | 2024-02-08 | 더 유니버서티 어브 퀸슬랜드 | 심근세포 성숙 |
WO2018044685A1 (en) | 2016-08-28 | 2018-03-08 | Baylor College Of Medicine | A novel chicken egg-based metastasis model for cancer |
US20210301349A1 (en) | 2016-08-30 | 2021-09-30 | Beth Israel Deaconess Medical Center, Inc. | Compositions and methods for treating a tumor suppressor deficient cancer |
US20210277102A1 (en) | 2016-08-30 | 2021-09-09 | Beth Israel Deaconess Medical Center, Inc. | Compositions and methods for treating a tumor suppressor deficient cancer |
NZ753051A (en) | 2016-11-04 | 2023-03-31 | Children’S Hospital Medical Center | Liver organoid compositions and methods of making and using same |
AU2017363838B2 (en) | 2016-11-23 | 2023-06-29 | Morphocell Technologies Inc. | Encapsulated liver tissue |
US11767515B2 (en) | 2016-12-05 | 2023-09-26 | Children's Hospital Medical Center | Colonic organoids and methods of making and using same |
GB201622222D0 (en) | 2016-12-23 | 2017-02-08 | Cs Genetics Ltd | Reagents and methods for molecular barcoding of nucleic acids of single cells |
US20200040309A1 (en) | 2017-04-14 | 2020-02-06 | Children's Hospital Medical Center | Multi donor stem cell compositions and methods of making same |
EP3395942A1 (en) | 2017-04-25 | 2018-10-31 | IMBA-Institut für Molekulare Biotechnologie GmbH | Bi- or multi-differentiated organoid |
JP2020536529A (ja) | 2017-10-10 | 2020-12-17 | チルドレンズ ホスピタル メディカル センター | 食道組織および/または臓器組成物およびそれを作製する方法 |
-
2017
- 2017-05-05 CA CA3016641A patent/CA3016641A1/en active Pending
- 2017-05-05 JP JP2018550724A patent/JP6963882B2/ja active Active
- 2017-05-05 CN CN201780024903.5A patent/CN109415685B/zh active Active
- 2017-05-05 EP EP22189427.2A patent/EP4177335A1/en active Pending
- 2017-05-05 CN CN202310699246.1A patent/CN116790476A/zh active Pending
- 2017-05-05 US US16/084,599 patent/US11066650B2/en active Active
- 2017-05-05 ES ES17793451T patent/ES2929758T3/es active Active
- 2017-05-05 WO PCT/US2017/031309 patent/WO2017192997A1/en unknown
- 2017-05-05 EP EP17793451.0A patent/EP3452578B1/en active Active
-
2021
- 2021-07-14 US US17/375,293 patent/US20220064602A1/en active Pending
- 2021-10-15 JP JP2021169192A patent/JP7463326B2/ja active Active
Also Published As
Publication number | Publication date |
---|---|
JP2022025087A (ja) | 2022-02-09 |
US20220064602A1 (en) | 2022-03-03 |
EP4177335A1 (en) | 2023-05-10 |
US20190078055A1 (en) | 2019-03-14 |
JP2019514354A (ja) | 2019-06-06 |
WO2017192997A1 (en) | 2017-11-09 |
EP3452578A1 (en) | 2019-03-13 |
CA3016641A1 (en) | 2017-11-09 |
EP3452578B1 (en) | 2022-08-10 |
CN109415685A (zh) | 2019-03-01 |
CN109415685B (zh) | 2023-07-04 |
JP6963882B2 (ja) | 2021-11-10 |
US11066650B2 (en) | 2021-07-20 |
JP7463326B2 (ja) | 2024-04-08 |
EP3452578A4 (en) | 2019-12-04 |
ES2929758T3 (es) | 2022-12-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7059317B2 (ja) | 前駆細胞を指向性分化によって胃組織に変換するための方法及びシステム | |
CN109415685B (zh) | 用于体外制造胃底组织的方法和与其相关的组合物 | |
JP7464652B2 (ja) | 結腸オルガノイドならびにその作製方法および使用方法 | |
CN111565798A (zh) | 食道组织和/或类器官组合物及其制备方法 | |
KR20210040107A (ko) | 간-담도-췌장 조직 및 이를 제조하는 방법 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |