CN110494555B - 具有分支结构的肺芽类器官的生成和其用于肺疾病建模的用途 - Google Patents
具有分支结构的肺芽类器官的生成和其用于肺疾病建模的用途 Download PDFInfo
- Publication number
- CN110494555B CN110494555B CN201880020727.2A CN201880020727A CN110494555B CN 110494555 B CN110494555 B CN 110494555B CN 201880020727 A CN201880020727 A CN 201880020727A CN 110494555 B CN110494555 B CN 110494555B
- Authority
- CN
- China
- Prior art keywords
- lbo
- cells
- lung
- cell
- blbo
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000002220 organoid Anatomy 0.000 title claims abstract description 72
- 210000004072 lung Anatomy 0.000 title claims abstract description 71
- 208000019693 Lung disease Diseases 0.000 title claims description 17
- 230000035772 mutation Effects 0.000 claims abstract description 65
- 238000000034 method Methods 0.000 claims abstract description 49
- 210000001778 pluripotent stem cell Anatomy 0.000 claims abstract description 19
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 12
- 230000003176 fibrotic effect Effects 0.000 claims abstract description 11
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims abstract description 10
- 239000011159 matrix material Substances 0.000 claims abstract description 10
- 238000012258 culturing Methods 0.000 claims abstract description 9
- 210000004027 cell Anatomy 0.000 claims description 202
- 108010082117 matrigel Proteins 0.000 claims description 87
- 102000012804 EPCAM Human genes 0.000 claims description 44
- 101150084967 EPCAM gene Proteins 0.000 claims description 44
- 101150057140 TACSTD1 gene Proteins 0.000 claims description 44
- 230000014509 gene expression Effects 0.000 claims description 34
- 238000012360 testing method Methods 0.000 claims description 23
- 101000838926 Homo sapiens Hermansky-Pudlak syndrome 1 protein Proteins 0.000 claims description 21
- 108091033409 CRISPR Proteins 0.000 claims description 20
- 206010016654 Fibrosis Diseases 0.000 claims description 20
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 20
- 230000004761 fibrosis Effects 0.000 claims description 20
- 102100028902 Hermansky-Pudlak syndrome 1 protein Human genes 0.000 claims description 19
- 210000002438 upper gastrointestinal tract Anatomy 0.000 claims description 19
- 230000015572 biosynthetic process Effects 0.000 claims description 18
- 210000002242 embryoid body Anatomy 0.000 claims description 18
- 102100033936 AP-3 complex subunit beta-1 Human genes 0.000 claims description 17
- 101000779239 Homo sapiens AP-3 complex subunit beta-1 Proteins 0.000 claims description 17
- 101000612671 Homo sapiens Pulmonary surfactant-associated protein C Proteins 0.000 claims description 17
- 102100040971 Pulmonary surfactant-associated protein C Human genes 0.000 claims description 17
- 101000889443 Homo sapiens Trefoil factor 1 Proteins 0.000 claims description 14
- 238000012217 deletion Methods 0.000 claims description 13
- 230000037430 deletion Effects 0.000 claims description 13
- 102100024526 Biogenesis of lysosome-related organelles complex 1 subunit 3 Human genes 0.000 claims description 12
- 108010079245 Cystic Fibrosis Transmembrane Conductance Regulator Proteins 0.000 claims description 12
- 101000762344 Homo sapiens Biogenesis of lysosome-related organelles complex 1 subunit 3 Proteins 0.000 claims description 12
- 239000000523 sample Substances 0.000 claims description 12
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims description 10
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims description 10
- 206010064571 Gene mutation Diseases 0.000 claims description 9
- 201000005397 Hermansky-Pudlak syndrome 8 Diseases 0.000 claims description 9
- 101001086862 Homo sapiens Pulmonary surfactant-associated protein B Proteins 0.000 claims description 9
- 102100033472 Lysosomal-trafficking regulator Human genes 0.000 claims description 9
- 102100032617 Pulmonary surfactant-associated protein B Human genes 0.000 claims description 9
- 210000002744 extracellular matrix Anatomy 0.000 claims description 9
- -1 hTERT Proteins 0.000 claims description 9
- 108010057210 telomerase RNA Proteins 0.000 claims description 9
- 101000801640 Homo sapiens Phospholipid-transporting ATPase ABCA3 Proteins 0.000 claims description 8
- 102100033623 Phospholipid-transporting ATPase ABCA3 Human genes 0.000 claims description 8
- 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 8
- 230000002685 pulmonary effect Effects 0.000 claims description 8
- 229930002330 retinoic acid Natural products 0.000 claims description 8
- 230000028327 secretion Effects 0.000 claims description 8
- 239000004094 surface-active agent Substances 0.000 claims description 8
- 229960001727 tretinoin Drugs 0.000 claims description 8
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 claims description 7
- 101000762379 Homo sapiens Bone morphogenetic protein 4 Proteins 0.000 claims description 7
- 230000002159 abnormal effect Effects 0.000 claims description 7
- 210000005265 lung cell Anatomy 0.000 claims description 7
- 102100028412 Fibroblast growth factor 10 Human genes 0.000 claims description 6
- 102100028071 Fibroblast growth factor 7 Human genes 0.000 claims description 6
- 101000917237 Homo sapiens Fibroblast growth factor 10 Proteins 0.000 claims description 6
- 101001060261 Homo sapiens Fibroblast growth factor 7 Proteins 0.000 claims description 6
- 101001018064 Homo sapiens Lysosomal-trafficking regulator Proteins 0.000 claims description 6
- 102100028680 Protein patched homolog 1 Human genes 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- AQGNHMOJWBZFQQ-UHFFFAOYSA-N CT 99021 Chemical compound CC1=CNC(C=2C(=NC(NCCNC=3N=CC(=CC=3)C#N)=NC=2)C=2C(=CC(Cl)=CC=2)Cl)=N1 AQGNHMOJWBZFQQ-UHFFFAOYSA-N 0.000 claims description 5
- 102100028716 Hermansky-Pudlak syndrome 3 protein Human genes 0.000 claims description 5
- 101000985492 Homo sapiens Hermansky-Pudlak syndrome 3 protein Proteins 0.000 claims description 5
- 101000972276 Homo sapiens Mucin-5B Proteins 0.000 claims description 5
- 102100022494 Mucin-5B Human genes 0.000 claims description 5
- 102100027773 Pulmonary surfactant-associated protein A2 Human genes 0.000 claims description 5
- 230000004900 autophagic degradation Effects 0.000 claims description 5
- 230000008021 deposition Effects 0.000 claims description 5
- 230000035882 stress Effects 0.000 claims description 5
- 102100031249 H/ACA ribonucleoprotein complex subunit DKC1 Human genes 0.000 claims description 4
- 102100028715 Hermansky-Pudlak syndrome 4 protein Human genes 0.000 claims description 4
- 101000844866 Homo sapiens H/ACA ribonucleoprotein complex subunit DKC1 Proteins 0.000 claims description 4
- 101000985501 Homo sapiens Hermansky-Pudlak syndrome 4 protein Proteins 0.000 claims description 4
- 101000985516 Homo sapiens Hermansky-Pudlak syndrome 5 protein Proteins 0.000 claims description 4
- 101000695187 Homo sapiens Protein patched homolog 1 Proteins 0.000 claims description 4
- 101000864780 Homo sapiens Pulmonary surfactant-associated protein A1 Proteins 0.000 claims description 4
- 101000651017 Homo sapiens Pulmonary surfactant-associated protein A2 Proteins 0.000 claims description 4
- 102100030060 Pulmonary surfactant-associated protein A1 Human genes 0.000 claims description 4
- 239000013543 active substance Substances 0.000 claims description 4
- 210000001552 airway epithelial cell Anatomy 0.000 claims description 4
- 230000032258 transport Effects 0.000 claims description 4
- 108010076876 Keratins Proteins 0.000 claims description 3
- 102000011782 Keratins Human genes 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 3
- 230000002950 deficient Effects 0.000 claims description 3
- 210000004039 endoderm cell Anatomy 0.000 claims description 3
- 102000054765 polymorphisms of proteins Human genes 0.000 claims description 3
- 230000003612 virological effect Effects 0.000 claims description 3
- 101710098191 C-4 methylsterol oxidase ERG25 Proteins 0.000 claims description 2
- 101710090597 Smoothened homolog Proteins 0.000 claims description 2
- 208000035475 disorder Diseases 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims description 2
- 230000002438 mitochondrial effect Effects 0.000 claims description 2
- 230000004906 unfolded protein response Effects 0.000 claims description 2
- 102100028721 Hermansky-Pudlak syndrome 5 protein Human genes 0.000 claims 2
- 102100021796 Sonic hedgehog protein Human genes 0.000 claims 2
- 101710113849 Sonic hedgehog protein Proteins 0.000 claims 2
- 238000000423 cell based assay Methods 0.000 claims 2
- 102000008371 intracellularly ATP-gated chloride channel activity proteins Human genes 0.000 claims 2
- 101100310152 Homo sapiens SFTPA2 gene Proteins 0.000 claims 1
- 101150047113 SFTPA1 gene Proteins 0.000 claims 1
- 101150056682 Smo gene Proteins 0.000 claims 1
- 101150098162 abcA3 gene Proteins 0.000 claims 1
- 239000011149 active material Substances 0.000 claims 1
- 230000007960 cellular response to stress Effects 0.000 claims 1
- 239000013068 control sample Substances 0.000 claims 1
- 230000004060 metabolic process Effects 0.000 claims 1
- 208000023504 respiratory system disease Diseases 0.000 claims 1
- 210000001900 endoderm Anatomy 0.000 abstract description 42
- 230000007040 lung development Effects 0.000 abstract description 21
- 210000001671 embryonic stem cell Anatomy 0.000 abstract description 16
- 239000002775 capsule Substances 0.000 abstract description 15
- 210000003734 kidney Anatomy 0.000 abstract description 14
- 230000019552 anatomical structure morphogenesis Effects 0.000 abstract description 11
- 210000003716 mesoderm Anatomy 0.000 abstract description 9
- 208000005069 pulmonary fibrosis Diseases 0.000 abstract description 8
- 238000002360 preparation method Methods 0.000 abstract description 4
- 239000002609 medium Substances 0.000 description 52
- 238000002474 experimental method Methods 0.000 description 48
- 108090000623 proteins and genes Proteins 0.000 description 48
- 201000009794 Idiopathic Pulmonary Fibrosis Diseases 0.000 description 35
- 208000036971 interstitial lung disease 2 Diseases 0.000 description 32
- 210000002588 alveolar type II cell Anatomy 0.000 description 28
- 238000000338 in vitro Methods 0.000 description 25
- 241000699670 Mus sp. Species 0.000 description 24
- 210000001519 tissue Anatomy 0.000 description 21
- 102000004169 proteins and genes Human genes 0.000 description 19
- 201000010099 disease Diseases 0.000 description 18
- 102000003693 Hedgehog Proteins Human genes 0.000 description 16
- 108090000031 Hedgehog Proteins Proteins 0.000 description 16
- 150000001875 compounds Chemical class 0.000 description 16
- 239000012071 phase Substances 0.000 description 16
- 108020005004 Guide RNA Proteins 0.000 description 15
- 230000012010 growth Effects 0.000 description 15
- 230000006698 induction Effects 0.000 description 15
- 239000003550 marker Substances 0.000 description 15
- 230000004069 differentiation Effects 0.000 description 14
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 description 13
- 102100024270 Transcription factor SOX-2 Human genes 0.000 description 13
- 238000001727 in vivo Methods 0.000 description 13
- 108090000765 processed proteins & peptides Proteins 0.000 description 13
- 241000725643 Respiratory syncytial virus Species 0.000 description 12
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 11
- 101000711846 Homo sapiens Transcription factor SOX-9 Proteins 0.000 description 11
- 102100034204 Transcription factor SOX-9 Human genes 0.000 description 11
- 238000011161 development Methods 0.000 description 11
- 230000018109 developmental process Effects 0.000 description 11
- 210000000981 epithelium Anatomy 0.000 description 11
- 210000000056 organ Anatomy 0.000 description 11
- 238000010186 staining Methods 0.000 description 11
- 210000000130 stem cell Anatomy 0.000 description 11
- 238000002054 transplantation Methods 0.000 description 11
- 102000012605 Cystic Fibrosis Transmembrane Conductance Regulator Human genes 0.000 description 10
- 201000005402 Hermansky-Pudlak syndrome 2 Diseases 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 238000003559 RNA-seq method Methods 0.000 description 10
- 108020004999 messenger RNA Proteins 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- 108010067306 Fibronectins Proteins 0.000 description 9
- 102000016359 Fibronectins Human genes 0.000 description 9
- 208000029523 Interstitial Lung disease Diseases 0.000 description 9
- 230000006870 function Effects 0.000 description 9
- 101150111110 NKX2-1 gene Proteins 0.000 description 8
- 210000002950 fibroblast Anatomy 0.000 description 8
- 238000010166 immunofluorescence Methods 0.000 description 8
- 238000012423 maintenance Methods 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 8
- 108091035539 telomere Proteins 0.000 description 8
- 102000055501 telomere Human genes 0.000 description 8
- 210000003411 telomere Anatomy 0.000 description 8
- IMRYETFJNLKUHK-UHFFFAOYSA-N traseolide Chemical compound CC1=C(C(C)=O)C=C2C(C(C)C)C(C)C(C)(C)C2=C1 IMRYETFJNLKUHK-UHFFFAOYSA-N 0.000 description 8
- 210000005239 tubule Anatomy 0.000 description 8
- 238000010354 CRISPR gene editing Methods 0.000 description 7
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 7
- 108010017842 Telomerase Proteins 0.000 description 7
- 210000002821 alveolar epithelial cell Anatomy 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 7
- 230000002132 lysosomal effect Effects 0.000 description 7
- 102000004196 processed proteins & peptides Human genes 0.000 description 7
- 238000012216 screening Methods 0.000 description 7
- 238000013456 study Methods 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 238000004114 suspension culture Methods 0.000 description 7
- 108010035532 Collagen Proteins 0.000 description 6
- 102000008186 Collagen Human genes 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 108090000631 Trypsin Proteins 0.000 description 6
- 102000004142 Trypsin Human genes 0.000 description 6
- 229910002091 carbon monoxide Inorganic materials 0.000 description 6
- 229920001436 collagen Polymers 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 230000037433 frameshift Effects 0.000 description 6
- 238000002513 implantation Methods 0.000 description 6
- 230000024799 morphogenesis of a branching structure Effects 0.000 description 6
- 210000003463 organelle Anatomy 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 230000035935 pregnancy Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 230000008672 reprogramming Effects 0.000 description 6
- 208000011580 syndromic disease Diseases 0.000 description 6
- 239000012588 trypsin Substances 0.000 description 6
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 5
- 238000010453 CRISPR/Cas method Methods 0.000 description 5
- 102100029283 Hepatocyte nuclear factor 3-alpha Human genes 0.000 description 5
- 101001062353 Homo sapiens Hepatocyte nuclear factor 3-alpha Proteins 0.000 description 5
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 5
- 206010061603 Respiratory syncytial virus infection Diseases 0.000 description 5
- 241001093501 Rutaceae Species 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- 238000000692 Student's t-test Methods 0.000 description 5
- 210000005058 airway cell Anatomy 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 210000000254 ciliated cell Anatomy 0.000 description 5
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 5
- 210000002919 epithelial cell Anatomy 0.000 description 5
- 229960002591 hydroxyproline Drugs 0.000 description 5
- 238000003384 imaging method Methods 0.000 description 5
- 238000003125 immunofluorescent labeling Methods 0.000 description 5
- 230000001404 mediated effect Effects 0.000 description 5
- 239000008188 pellet Substances 0.000 description 5
- 230000035515 penetration Effects 0.000 description 5
- 238000001179 sorption measurement Methods 0.000 description 5
- 238000007619 statistical method Methods 0.000 description 5
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 5
- 108700028369 Alleles Proteins 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 4
- 208000031879 Chédiak-Higashi syndrome Diseases 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 4
- 102100029284 Hepatocyte nuclear factor 3-beta Human genes 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 description 4
- 101001062347 Homo sapiens Hepatocyte nuclear factor 3-beta Proteins 0.000 description 4
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 description 4
- 101001126417 Homo sapiens Platelet-derived growth factor receptor alpha Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 102100034256 Mucin-1 Human genes 0.000 description 4
- 102100030485 Platelet-derived growth factor receptor alpha Human genes 0.000 description 4
- 210000002383 alveolar type I cell Anatomy 0.000 description 4
- 150000001413 amino acids Chemical group 0.000 description 4
- 230000008436 biogenesis Effects 0.000 description 4
- 208000025517 cannabinoid hyperemesis syndrome Diseases 0.000 description 4
- 230000000875 corresponding effect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000001493 electron microscopy Methods 0.000 description 4
- 230000001973 epigenetic effect Effects 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 238000003205 genotyping method Methods 0.000 description 4
- 238000007901 in situ hybridization Methods 0.000 description 4
- 239000010410 layer Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 230000008506 pathogenesis Effects 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000012353 t test Methods 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- SVUOLADPCWQTTE-UHFFFAOYSA-N 1h-1,2-benzodiazepine Chemical class N1N=CC=CC2=CC=CC=C12 SVUOLADPCWQTTE-UHFFFAOYSA-N 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 3
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 201000003883 Cystic fibrosis Diseases 0.000 description 3
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 101000975496 Homo sapiens Keratin, type II cytoskeletal 8 Proteins 0.000 description 3
- 101000617738 Homo sapiens Survival motor neuron protein Proteins 0.000 description 3
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 3
- 102100023972 Keratin, type II cytoskeletal 8 Human genes 0.000 description 3
- 108010063954 Mucins Proteins 0.000 description 3
- 102000015728 Mucins Human genes 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 101000985497 Staphylococcus saprophyticus subsp. saprophyticus (strain ATCC 15305 / DSM 20229 / NCIMB 8711 / NCTC 7292 / S-41) 3-hexulose-6-phosphate synthase 1 Proteins 0.000 description 3
- 102100021947 Survival motor neuron protein Human genes 0.000 description 3
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 230000005856 abnormality Effects 0.000 description 3
- 239000012298 atmosphere Substances 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 230000034303 cell budding Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 210000002808 connective tissue Anatomy 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 description 3
- 229960005156 digoxin Drugs 0.000 description 3
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012894 fetal calf serum Substances 0.000 description 3
- 230000001605 fetal effect Effects 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 238000010362 genome editing Methods 0.000 description 3
- 210000002175 goblet cell Anatomy 0.000 description 3
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 3
- 239000011229 interlayer Substances 0.000 description 3
- 230000003902 lesion Effects 0.000 description 3
- 210000004379 membrane Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 108091070501 miRNA Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 210000001811 primitive streak Anatomy 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000002345 respiratory system Anatomy 0.000 description 3
- 229920002477 rna polymer Polymers 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 description 2
- 208000000884 Airway Obstruction Diseases 0.000 description 2
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 2
- 102100037242 Amiloride-sensitive sodium channel subunit alpha Human genes 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 108010006654 Bleomycin Proteins 0.000 description 2
- 206010006448 Bronchiolitis Diseases 0.000 description 2
- 102100035888 Caveolin-1 Human genes 0.000 description 2
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 description 2
- OHCQJHSOBUTRHG-KGGHGJDLSA-N FORSKOLIN Chemical compound O=C([C@@]12O)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)[C@@H](O)CCC1(C)C OHCQJHSOBUTRHG-KGGHGJDLSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 101710164669 Hedgehog-interacting protein Proteins 0.000 description 2
- 102100035960 Hedgehog-interacting protein Human genes 0.000 description 2
- 102100028798 Homeodomain-only protein Human genes 0.000 description 2
- 101000740448 Homo sapiens Amiloride-sensitive sodium channel subunit alpha Proteins 0.000 description 2
- 101000715467 Homo sapiens Caveolin-1 Proteins 0.000 description 2
- 101100395520 Homo sapiens HPS3 gene Proteins 0.000 description 2
- 101000839095 Homo sapiens Homeodomain-only protein Proteins 0.000 description 2
- 101000972282 Homo sapiens Mucin-5AC Proteins 0.000 description 2
- 101000625913 Homo sapiens T-box transcription factor TBX4 Proteins 0.000 description 2
- 101150009266 Hps1 gene Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 206010021143 Hypoxia Diseases 0.000 description 2
- 102000043138 IRF family Human genes 0.000 description 2
- 241000711408 Murine respirovirus Species 0.000 description 2
- 108020004485 Nonsense Codon Proteins 0.000 description 2
- 102100035423 POU domain, class 5, transcription factor 1 Human genes 0.000 description 2
- 101710126211 POU domain, class 5, transcription factor 1 Proteins 0.000 description 2
- 108010065129 Patched-1 Receptor Proteins 0.000 description 2
- 206010035148 Plague Diseases 0.000 description 2
- 108010014608 Proto-Oncogene Proteins c-kit Proteins 0.000 description 2
- 102000016971 Proto-Oncogene Proteins c-kit Human genes 0.000 description 2
- 108020004459 Small interfering RNA Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 101000985495 Staphylococcus saprophyticus subsp. saprophyticus (strain ATCC 15305 / DSM 20229 / NCIMB 8711 / NCTC 7292 / S-41) 3-hexulose-6-phosphate synthase 2 Proteins 0.000 description 2
- 101000985500 Staphylococcus saprophyticus subsp. saprophyticus (strain ATCC 15305 / DSM 20229 / NCIMB 8711 / NCTC 7292 / S-41) 3-hexulose-6-phosphate synthase 3 Proteins 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- 102100024754 T-box transcription factor TBX4 Human genes 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 102000013127 Vimentin Human genes 0.000 description 2
- 108010065472 Vimentin Proteins 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 102000013814 Wnt Human genes 0.000 description 2
- 108050003627 Wnt Proteins 0.000 description 2
- 108010016200 Zinc Finger Protein GLI1 Proteins 0.000 description 2
- 102100035535 Zinc finger protein GLI1 Human genes 0.000 description 2
- 108010076089 accutase Proteins 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 238000004458 analytical method Methods 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
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 210000000270 basal cell Anatomy 0.000 description 2
- 229960001561 bleomycin Drugs 0.000 description 2
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 2
- 210000001772 blood platelet Anatomy 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000022131 cell cycle Effects 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000004186 co-expression Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000003750 conditioning effect Effects 0.000 description 2
- 238000011257 definitive treatment Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000007877 drug screening Methods 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 230000029544 epithelial tube branching involved in lung morphogenesis Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 210000003754 fetus Anatomy 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000007614 genetic variation Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 102000049832 human HPS1 Human genes 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000007954 hypoxia Effects 0.000 description 2
- 238000009830 intercalation Methods 0.000 description 2
- 230000002687 intercalation Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 210000002780 melanosome Anatomy 0.000 description 2
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 238000000399 optical microscopy Methods 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 238000002823 phage display Methods 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 239000000419 plant extract Substances 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 108010054624 red fluorescent protein Proteins 0.000 description 2
- 238000007634 remodeling Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 238000004904 shortening Methods 0.000 description 2
- 210000001082 somatic cell Anatomy 0.000 description 2
- 230000002269 spontaneous effect Effects 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 210000004878 submucosal gland Anatomy 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 238000004627 transmission electron microscopy Methods 0.000 description 2
- 210000003501 vero cell Anatomy 0.000 description 2
- 210000005048 vimentin Anatomy 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 238000012800 visualization Methods 0.000 description 2
- MCSXGCZMEPXKIW-UHFFFAOYSA-N 3-hydroxy-4-[(4-methyl-2-nitrophenyl)diazenyl]-N-(3-nitrophenyl)naphthalene-2-carboxamide Chemical compound Cc1ccc(N=Nc2c(O)c(cc3ccccc23)C(=O)Nc2cccc(c2)[N+]([O-])=O)c(c1)[N+]([O-])=O MCSXGCZMEPXKIW-UHFFFAOYSA-N 0.000 description 1
- 102100040078 A-kinase anchor protein 5 Human genes 0.000 description 1
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 description 1
- 101150044980 Akap1 gene Proteins 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 101100189945 Arabidopsis thaliana PER63 gene Proteins 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 101000839068 Boana punctata Hylaseptin-P1 Proteins 0.000 description 1
- 108010083123 CDX2 Transcription Factor Proteins 0.000 description 1
- 102000006277 CDX2 Transcription Factor Human genes 0.000 description 1
- 108010040467 CRISPR-Associated Proteins Proteins 0.000 description 1
- 101100257359 Caenorhabditis elegans sox-2 gene Proteins 0.000 description 1
- 108090000932 Calcitonin Gene-Related Peptide Proteins 0.000 description 1
- 241000282836 Camelus dromedarius Species 0.000 description 1
- 102100023503 Chloride intracellular channel protein 5 Human genes 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- SUZLHDUTVMZSEV-UHFFFAOYSA-N Deoxycoleonol Natural products C12C(=O)CC(C)(C=C)OC2(C)C(OC(=O)C)C(O)C2C1(C)C(O)CCC2(C)C SUZLHDUTVMZSEV-UHFFFAOYSA-N 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 241000006867 Discosoma Species 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 102100041006 Forkhead box protein J1 Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 101150070312 HOX5 gene Proteins 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 101000890614 Homo sapiens A-kinase anchor protein 5 Proteins 0.000 description 1
- 101000906624 Homo sapiens Chloride intracellular channel protein 5 Proteins 0.000 description 1
- 101000906631 Homo sapiens Chloride intracellular channel protein 6 Proteins 0.000 description 1
- 101000892910 Homo sapiens Forkhead box protein J1 Proteins 0.000 description 1
- 101001032342 Homo sapiens Interferon regulatory factor 7 Proteins 0.000 description 1
- 101001139134 Homo sapiens Krueppel-like factor 4 Proteins 0.000 description 1
- 101001030211 Homo sapiens Myc proto-oncogene protein Proteins 0.000 description 1
- 101000600766 Homo sapiens Podoplanin Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102100038070 Interferon regulatory factor 7 Human genes 0.000 description 1
- 108010020437 Ki-67 Antigen Proteins 0.000 description 1
- 102100020677 Krueppel-like factor 4 Human genes 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 206010024971 Lower respiratory tract infections Diseases 0.000 description 1
- 208000032376 Lung infection Diseases 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 101000829705 Methanopyrus kandleri (strain AV19 / DSM 6324 / JCM 9639 / NBRC 100938) Thermosome subunit Proteins 0.000 description 1
- 102100022496 Mucin-5AC Human genes 0.000 description 1
- 102000007474 Multiprotein Complexes Human genes 0.000 description 1
- 108010085220 Multiprotein Complexes Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101100257363 Mus musculus Sox2 gene Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 101150038994 PDGFRA gene Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- 108010043958 Peptoids Proteins 0.000 description 1
- 208000012641 Pigmentation disease Diseases 0.000 description 1
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 1
- 108010051742 Platelet-Derived Growth Factor beta Receptor Proteins 0.000 description 1
- 102100026547 Platelet-derived growth factor receptor beta Human genes 0.000 description 1
- 102100037265 Podoplanin Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 102100034836 Proliferation marker protein Ki-67 Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 102100029812 Protein S100-A12 Human genes 0.000 description 1
- 208000008425 Protein deficiency Diseases 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 102000013380 Smoothened Receptor Human genes 0.000 description 1
- 108010052164 Sodium Channels Proteins 0.000 description 1
- 102000018674 Sodium Channels Human genes 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 239000004809 Teflon Substances 0.000 description 1
- 229920006362 Teflon® Polymers 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 101150071739 Tp63 gene Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 108010088412 Trefoil Factor-1 Proteins 0.000 description 1
- 102100039175 Trefoil factor 1 Human genes 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102100025038 Ubiquitin carboxyl-terminal hydrolase isozyme L1 Human genes 0.000 description 1
- 101710186825 Ubiquitin carboxyl-terminal hydrolase isozyme L1 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
- 102100031083 Uteroglobin Human genes 0.000 description 1
- 108090000203 Uteroglobin Proteins 0.000 description 1
- 102000008790 VE-cadherin Human genes 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 1
- 108091007916 Zinc finger transcription factors Proteins 0.000 description 1
- 102000038627 Zinc finger transcription factors Human genes 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000003838 adenosines Chemical class 0.000 description 1
- 201000000028 adult respiratory distress syndrome Diseases 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000008484 agonism Effects 0.000 description 1
- 101150118631 agrA gene Proteins 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229940049706 benzodiazepine Drugs 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000090 biomarker Substances 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
- 210000002459 blastocyst Anatomy 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 208000014759 blood platelet disease Diseases 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 108010018828 cadherin 5 Proteins 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000002612 cardiopulmonary effect Effects 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 101150038500 cas9 gene Proteins 0.000 description 1
- 150000003943 catecholamines Chemical class 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- VDQQXEISLMTGAB-UHFFFAOYSA-N chloramine T Chemical compound [Na+].CC1=CC=C(S(=O)(=O)[N-]Cl)C=C1 VDQQXEISLMTGAB-UHFFFAOYSA-N 0.000 description 1
- 210000004081 cilia Anatomy 0.000 description 1
- 239000013625 clathrin-independent carrier Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 208000035850 clinical syndrome Diseases 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- OHCQJHSOBUTRHG-UHFFFAOYSA-N colforsin Natural products OC12C(=O)CC(C)(C=C)OC1(C)C(OC(=O)C)C(O)C1C2(C)C(O)CCC1(C)C OHCQJHSOBUTRHG-UHFFFAOYSA-N 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000001143 conditioned effect Effects 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
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000003235 crystal violet staining Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000010339 dilation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229960003638 dopamine Drugs 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 230000008378 epithelial damage Effects 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000019688 fish Nutrition 0.000 description 1
- 231100000221 frame shift mutation induction Toxicity 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000007045 gastrulation Effects 0.000 description 1
- 231100000089 gene mutation induction Toxicity 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 230000004077 genetic alteration Effects 0.000 description 1
- 231100000118 genetic alteration Toxicity 0.000 description 1
- 210000001654 germ layer Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000031169 hemorrhagic disease Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 101150003074 hoxa5 gene Proteins 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 230000004941 influx Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000000867 larynx Anatomy 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 201000002364 leukopenia Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000010859 live-cell imaging Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 210000002752 melanocyte Anatomy 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 230000004065 mitochondrial dysfunction Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- PJUIMOJAAPLTRJ-UHFFFAOYSA-N monothioglycerol Chemical compound OCC(O)CS PJUIMOJAAPLTRJ-UHFFFAOYSA-N 0.000 description 1
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 1
- 229940051875 mucins Drugs 0.000 description 1
- 210000000651 myofibroblast Anatomy 0.000 description 1
- 210000004897 n-terminal region Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 210000000461 neuroepithelial cell Anatomy 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 238000012758 nuclear staining Methods 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000005868 ontogenesis Effects 0.000 description 1
- 229940127240 opiate Drugs 0.000 description 1
- 230000005305 organ development Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000003534 oscillatory effect Effects 0.000 description 1
- 229910000489 osmium tetroxide Inorganic materials 0.000 description 1
- 239000012285 osmium tetroxide Substances 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 1
- 210000002741 palatine tonsil Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000000849 parathyroid Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000000059 patterning Methods 0.000 description 1
- 239000000816 peptidomimetic Substances 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 210000003800 pharynx Anatomy 0.000 description 1
- 239000003016 pheromone Substances 0.000 description 1
- 150000003017 phosphorus Chemical class 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 238000011533 pre-incubation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 208000028529 primary immunodeficiency disease Diseases 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 229940048914 protamine Drugs 0.000 description 1
- 238000013138 pruning Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003235 pyrrolidines Chemical class 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 101150066583 rep gene Proteins 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 230000033458 reproduction Effects 0.000 description 1
- 230000003938 response to stress Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000037390 scarring Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 230000009291 secondary effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- IHQKEDIOMGYHEB-UHFFFAOYSA-M sodium dimethylarsinate Chemical compound [Na+].C[As](C)([O-])=O IHQKEDIOMGYHEB-UHFFFAOYSA-M 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 208000011317 telomere syndrome Diseases 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 229950003937 tolonium Drugs 0.000 description 1
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 230000032895 transmembrane transport Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- 210000003454 tympanic membrane Anatomy 0.000 description 1
- 108020005087 unfolded proteins Proteins 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
- A61L27/3882—Hollow organs, e.g. bladder, esophagus, urether, uterus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
-
- 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
- C12N5/0688—Cells from the lungs or the respiratory 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
- C12N5/0688—Cells from the lungs or the respiratory tract
- C12N5/0689—Stem cells; Progenitors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/22—Materials or treatment for tissue regeneration for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus
-
- 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
- C12N2501/117—Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
-
- 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
- C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
-
- 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
- C12N2503/00—Use of cells in diagnostics
- C12N2503/04—Screening or testing on artificial tissues
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Urology & Nephrology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Dermatology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Epidemiology (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Hematology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Developmental Biology & Embryology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- General Engineering & Computer Science (AREA)
- Reproductive Health (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Physics & Mathematics (AREA)
- Vascular Medicine (AREA)
- Food Science & Technology (AREA)
Abstract
本文描述了通过在3D基质中培养支化LBO或通过将所述LBO移植在免疫缺陷小鼠的肾包膜下由哺乳动物,优选人类多能干细胞,包括胚胎干细胞(ESC)和诱导性多能干细胞(IPSC)制备肺芽类器官(LBO)的新方法,所述肺芽类器官能够发育成分支气道和肺泡结构,至少部分地重现人类肺发育。支化LBO含有与肺间充质相容的肺内胚层和中胚层,且经历支化形态发生。还描述了携带某些诱导纤维化表型的突变的LBO,以及其制备方法。突变的(B)LBO可用于筛选可以治疗肺纤维化的药剂。
Description
相关申请的交叉引用
本申请要求2017年3月24日提交的美国临时申请序列号:62/476,335的优先权,该申请的内容并入本文。
政府利益声明
本发明是在政府资助下根据美国国立卫生研究院授予的授权号HL 134760进行。政府享有本发明的一定权利。
发明背景
特发性肺纤维化(IPF)是一种难以治疗的间质性肺病,中值存活期为3至4年,特征在于成纤维细胞灶以及肺泡的重塑和闭塞。1,2唯一的确定性治疗是肺移植,即一种干预措施,受供体器官的低可用性,以及严重的手术、药物和免疫并发症困扰。3因此迫切需要创新方法。开发这类方法需要非常缺乏了解这种毁灭性且日益流行的疾病的发病机制并为药物发现建立平台。
附图说明
以举例的方式且不以限制的方式在图中说明本发明。
图1A-1E.生成肺芽类器官。(图1A)在第6天和第8天之间在AFE的腹侧化期间使粘附结构发育(参见方案图6b),所述粘附结构可以在悬浮培养物中扩增(d10、d20)。代表>50次独立实验(ESC和iPSC)。比例尺250μm。(图1B)在生成LBO期间的细胞扩增。N=RUES2 ESC的3次独立的一式三份实验。(图1C)EPCAM、KRT8、NKX2.1、FOXA1和P63在d25 LBO中的表达。代表>10次ESC和iPSC的独立实验。比例尺100μm。(图1D)针对ECADH和PDGFRA对d25 LBO的染色。代表3次RUES2 ESC的独立实验。比例尺250μm。(图1E)通过RNAseq测定的内胚层和中胚层标记物在d25 LBO的EPCAM+和EPCAM-部分中的表达(3次独立的生物复制,RUES2 ESC)。
图2A-2G.LBO的体内潜能。(图2A)在将嵌入基质胶中的106个LBO细胞移植到NSG小鼠的肾包膜下1.5个月后生长的宏观方面。比例尺1cm。(图2B)在移植1.5个月后LBO来源的生长的HE染色。比例尺500μm。(图2C)在移植1.5个月后在LBO来源的生长中指定标记物的免疫荧光。比例尺100μm。(图2D)在移植5个月后LBO来源的生长的HE染色。比例尺250μm。(图2E)在移植5个月后在LBO来源的生长中指定标记物的免疫荧光。比例尺250μm。(图2F)在移植5个月后,在LBO来源的生长的小管抽吸液中左侧标记的蛋白质的斑点印迹。(图2G)在移植7个月后,LBO来源的生长中指定标记物的HE染色和免疫荧光。比例尺100μm。所有组都使用RUES2 ESC,代表4次独立实验。
图3A-3B.第70天时基质胶中的LBO分化。(图3A)在涂铺到基质胶中之后,LBO发育成分支结构的明场成像。RUES2 ESC。代表>50次独立实验。比例尺500μm。(图3B)在第25天针对涂铺在基质胶中的d70 RUES2来源的LBO中指定标记物的免疫荧光染色。代表4次独立实验。比例尺250μm。
图4A-4G.LBO在体外的长期发育。(图4A)在第25天嵌入基质胶中的d170 RUES2LBO的宏观外观。代表>50次独立实验。比例尺5mm。(图4B)在第25天嵌入基质胶中的d170RUES2和C12 LBO的明场成像。代表>50次独立实验。比例尺500μm。(图4C)在第25天嵌入基质胶中的d170 RUES2 LBO中指定标记物的免疫荧光。代表3次独立实验。MUC1+SFTPB和HT2-280的比例尺100μm。SFTPC的比例尺10μm。(图4D)在RUES2 ESC和HDF SV iPSC中,在第25天,嵌入基质胶中的d170 LBO的电子显微术。箭头指示LB。代表3次独立实验。(图4E)在第25天,在嵌入基质胶中的d170 LBO中SFTPB-BODIPY(绿色)的摄取。代表4次独立实验。比例尺100μm。(图4F)在第25天,在嵌入基质胶中的d170 LBO中SFTPB-BODIPY的摄取时程(平均值+s.e.m,n=RUES2 ESC的4次独立实验)。(图4G)在来源于hESC和hiPSC(12个生物独立样品)的d170 LBO中的全基因组表达与KeyGenes数据库的比较,显示与妊娠中期人肺的最佳匹配。
图5A-5D.LBO在建模人类疾病方面的潜在应用。(图5A)在用RSV感染且使用抗RSV(所有抗原)抗体染色1天和2天后全突变d170LBO的共聚焦图像。箭头:腔中的感染细胞。代表3次独立实验。比例尺100μm。(图5B)来自RUES2和RUES2-HPS1细胞的d50 LBO来源的基质胶群落的亮场成像。代表六次独立实验。比例尺500μm。(图5C)在RUES2和RUES2-HPS1细胞的3D基质胶培养物中,d50LBO来源的群落中EPCAM+和EPCAM-细胞的分率。(n=6,来自两次实验的3个技术重复的平均值±s.e.m;*P<0.0001;双尾学生t检验)。(图5D)RUES2和RUES2-HPS1的3D基质胶培养物中,间叶细胞标记物和ECM组分的免疫荧光染色。代表3次独立实验。比例尺500μm。
图6A-6E:肺芽类器官的表征。(图6A)用于生成肺和气道上皮细胞的已公开的2D引导的分化方案。(图6B)用于生成和分化LBO的方案的示意性概述。(图6C)由分离自d25RUES2 LBO的EPCAM+和EPCAM-细胞生成的RNAseq数据的非监测聚类(3个独立的生物复制)。(图6D)SHH和其转录目标GLI1、PTCH和HHIP,在AFE中、肺和气道中,以及d25LBO中其他AFE来源的谱系中表达的基因的表达(提取自图6(c)中所示的RNAseq数据;n=RUES2 ESC的3次独立实验)。(图6E)在d15、d20和d25,LBO中SHH的ISH。代表3次独立实验,RUES2 ESC。比例尺250μm。
图7A-7E.LBO在体内的潜能。(图7A)在移植到NSG小鼠的肾包膜下1.5个月之后,人类核的RUES2 ESC LBO来源的生长的染色。比例尺500μm。(图7B)对于鼠类CD31(mCD31)在移植5个月之后LBO来源的生长的染色。比例尺50μm。(图7C)对于SMA和EPCAM在移植5个月之后LBO来源的生长的染色。比例尺500μm。(图7D)在移植在NSG小鼠的肾包膜下5个月之后,显示纤毛细胞的LBO来源的生长的苏木素-伊红染色。比例尺25μm。(图7E)在移植在NSG小鼠的肾包膜下5个月之后,显示粘膜下腺的LBO来源的生长的苏木素-伊红染色。比例尺100μm。所有组都使用RUES2 ESC,代表4次独立实验。
图8A-8E.iPS和ES LBO的支化和支化的中胚层需求。(图8A)来源于在CHIR99021、BMP4、FGF7、FGF10和RA存在下在第25天涂铺在基质胶3D培养物中的RUES2和三种不同iPS系的LBO的d70培养物的支化群落。代表>10次独立实验。比例尺100μm。(图8B)以每6.4mm孔1个(顶部)或4个LBO(底部)将RUES2 LBO涂铺在基质胶中90天之后的支化群落。比例尺2.5mm。所有图像都代表>10次独立实验。(图8C)LBO中EPCAM-细胞的分率(n=3次独立实验,RUES2ESC)。(图8D)来自LBO的单一细胞,和来自从LBO分离的EPCAM+和EPCAM-细胞的群落。代表5次实验。比例尺500μm。(图8E)在基质胶3D培养物中由来源于LBO的单一细胞产生的群落的IF。代表5次实验。对于SFTPB和SFTPC,比例尺500μm,25μM。
图9A-9E.第170天在基质胶中的LBO突变。(图9A)来源于在CHIR99021、BMP4、FGF7、FGF10和RA存在下在第25天涂铺在基质胶中的三种iPS系的LBO的d170培养物的形态。代表>10次独立实验。比例尺250μm(图9B)指定标记物低放大率(平铺扫描)的免疫荧光图像。对来源于在CHIR99021、BMP4、FGF7、FGF10和RA存在下在第25天涂铺在基质胶中的C12 iPS系的LBO的d170培养物的连续切片进行染色。代表4次独立实验。比例尺1mm。(图9C)在HDFmRNAiPSC中,在第25天嵌入基质胶中的d170 LBO的电子显微术。箭头指示LB。代表3次独立实验。比例尺1μm。(图9D)在第14周远侧人类胎儿肺(HFL)中SOX2和SOX9的苏木素-伊红染色和表达。标记共表达SOX2和SOX9的试管(箭头)。代表3次独立实验。比例尺250μm。(图9E)来自KeyGenes数据库的具有妊娠中期人类器官和组织的全基因组表达谱的d170 LBO中全基因组表达谱的层次聚类。
图10A-10H.肺纤维化的建模。(图10A)HPS1基因,以及与gRNA互补的序列的位置的示意图(上部)。在由gRNA靶向的区域中,RUES2中的野生型等位基因和RUES2-HPS1细胞中的两种靶向等位基因的核苷酸序列(中部)。HPS1的外显子15和16的核苷酸和氨基酸序列,显示在RUES2-HPS1细胞的靶向等位基因中的缺失和提前终止密码子(下部)。(图10B)在RUES2和RUES2-HPS1细胞的3D基质胶培养物中,d50 LBO来源的群落中EPCAM+和EPCAM-细胞的流式细胞术分析的代表性实例。(n=6,来自两次实验的3个技术重复的平均值±s.e.m;*P<0.0001;双尾学生t检验)。(图10C)在3D基质胶培养物中,由亲代RUES2细胞和RUES2-HPS1细胞产生的LBO来源的支化群落的EPCAM和PDGFRA的免疫荧光染色的平铺扫描图像。代表五次独立实验。比例尺1mm。(图10D)来源于亲本RUES2和突变RUES2-HPS1细胞的d40 LBO的EPCAM+和EPCAM-细胞中,增殖抗原Ki67的表达的代表性实例。代表三个独立实验。(图10E)在RUES2和RUES2-HPS1细胞的3D基质胶培养物中LBO来源的群落中的羟基脯氨酸含量(平均值±s.e.m,n=3次独立实验,*P<0.05;双尾学生t检验)。(图10F)使用相对于DAPI的免疫荧光强度量化RUES2和RUES2-HPS1细胞的3D基质胶培养物中胶原I和III以及纤连蛋白(在每个实验中相对于1归一化RUES2对照之后,平均值±s.e.m,n=3次独立实验,对于纤连蛋白*P<0.05,对于胶原*P<0.01;双尾学生t检验)。(图10G)在图6B中所示的方案的第4天或第10天混合ZsGreen+和mCherry+RUES2来源的细胞之后,悬浮培养物在第25天时的LBO。代表>5次独立实验。(图10H)在培养方案的第4天或第10天混合细胞之后,来源于亲本RUES2或突变RUES2-HPS1细胞的EPCAM-细胞在d40培养物中的分率(平均值±s.e.m,n=3次独立实验;与亲本RUES2和在第10天与RUES2-HPS1混合的亲本RUES2相比*P<0.01;单因素ANOVA)。图10I为显示突变上皮细胞的照片并且图10J为显示具有较高百分比EPCAM细胞的HSP1细胞。
图11.肺发育。呼吸系统来源于在前部前肠内胚层(AFE)的腹面上出现的芽。这些通过模式化支化过程发育成近侧气道和远侧肺泡祖细胞(假腺管型期)。在小管期,细胞周期活性降低,且气道上皮在柄中发生特化,出现基细胞、杯状细胞、棒状细胞、纤毛细胞和其他细胞型(图11)。此期之后为囊状期,其中小管扩大成小囊,小囊将产生原始肺泡。95-97进一步通过不成熟小囊的分化、肺泡成熟和次级化分隔扩大肺泡数量主要在出生后继续。98
图12.第20天LBO的形态。左图显示具有折叠结构的类器官,其在基质胶中生成支化结构的可能性更高。右图显示在第4天以显著较低的细胞数起始的次优类器官(箭头),其在基质胶中生成支化结构的可能性较低。在RUES2、ESC的第20天类器官的代表性图像。比例尺:500微米。
图13A-13C.AP3B1的靶向突变。图13A显示使用Crispr/cas系统突变的标靶AP3B1(外显子4)序列。序列末端的暗灰色框表示基因型分型引物序列且序列内的中灰色序列表示gRNA序列。图13B显示选择HPS2克隆的所得突变序列。图13C表示蛋白质印迹,其显示突变的克隆不再产生HPS2。
图14A-14C.BLOC1S3的靶向突变。图14A显示使用Crispr/cas系统突变的标靶BLOC1S3(外显子2)序列。序列末端的暗灰色框表示基因型分型引物序列且序列内的中灰色序列表示gRNA序列。图14B显示选择HPS8的所得突变序列。图14C表示蛋白质印迹,其显示突变的克隆不再产生HPS8。
图15.显示使用Crispr/cas系统突变的标靶SFTPC(外显子2)序列。序列末端的暗灰色框表示基因型分型引物序列且序列内的中灰色序列表示gRNA序列。
图16A-16B.TERC的靶向突变。图16A显示使用Crispr/cas系统突变的标靶TERC(外显子1)序列。序列末端的暗灰色框表示基因型分型引物序列且序列内的中灰色序列表示gRNA序列。加框序列表示端粒模板序列。图16B显示选择TERC克隆的所得突变序列。
具体实施方式
我们已开发出通过在3D基质(LBO-3D)中培养LBO或通过将LBO异种移植(LBO-xeno)在诸如免疫缺陷小鼠的肾包膜下由哺乳动物,优选人类多能干细胞,包括胚胎干细胞(ESC)和诱导性多能干细胞(IPSC)制备肺芽类器官(LBO)的新方法,所述肺芽类器官能够发展成分支气道和肺泡结构,至少部分地重现人类肺发育。支化LBO(BLBO)含有与肺间充质相容的肺内胚层和中胚层,且经历支化形态发生。它们主要发育成与远侧肺相容的结构,即含有肺泡上皮细胞的肺泡结构,但还含有一些更近的细胞,即气道细胞。通过3D培养物制得的支化LBO有时称为BLBO-3D,且通过异种移植在体内制得的那些也称为BLBO-XENO。
如结果中所示和实例中所解释,LBO的发育基本呈三期进行:
1期:悬浮培养体外产生的前部前肠细胞以形成LBO,所述LBO为具有折叠上皮的球形结构(直到d25)。
2期:在大约在第25天开始的3D基质胶培养中,未支化的LBO球体在一周内开始支化。在异种移植到免疫缺陷小鼠的肾包膜下之后,支化花费更长时间且在接枝之后约2个月观察到。
3期:最后,在以异种移植物或3D基质胶培养物长期培养时,BLBO开始显示具有肺泡结构的形态发生的扩张尖端。
孵育LBO的时间越长(在3D或异种移植物中),支化形态发生的发育程度越高。BLBO-3D培养物已生长长达180天且BLBO-Xeno已进行多达7个月。BLBO生长的时间越长,存在的成熟肺泡细胞越多且类器官越大,但在第40天时HPS1细胞(LBO-HPS1DEL)中的纤维化表型已经很明显。
无论使用BLBO-3D还是BLBO-xeno,通常都将使用BLBO-3D在体外进行药物筛选,接着使用BLBO-xeno在体内验证。
定义
如本文所用的术语“人类多能干细胞(hPSC)”是指可包括诱导性多能干细胞(iPSC)的人类多能干细胞5iPSC通过将体细胞再编程为与ESC类似的多能性状态来生成,且因为对患者具有特异性。在具体实例中,iPS细胞是未分化的多能干细胞,其表达OCT4、SOX2、NANOG和SSEA4。
如本文所用,“前部前肠内胚层”(AFE)是指位于产生肝脏的内胚层的前部的内胚层。本领域普通技术人员将容易理解,“前部前肠内胚层”因此包括例如咽内胚层或肺内胚层,以及内胚层细胞其他高度分化的群体。因为胚胎组织表达分子标记物的特征组,所以术语“前部前肠内胚层”所涵盖的细胞类型可表现分子标记物的不同表达模式。本领域普通技术人员将理解,“前部前肠内胚层”产生各种组织,例如扁桃体、鼓膜、甲状腺、甲状旁腺、胸腺、气管、食管、胃、肺和喉/咽。前部前肠内胚层表达FOXA2、FOXA1、SOX2和EPCAM且对远侧内胚层标记物CDX2显阴性。
如本文所用,定型内胚层(DE)是在原肠胚形成之后出现的三种胚层中的一种,其产生肠道、肝脏、胰腺、胃和来源于AFE的所有其他器官,如上文所列举。DE表达标记物:FOXA2、FOXA1、cKIT、CXCR4、EPCAM。
肺芽类器官(LBO)来源于悬浮液中的人类多能干细胞且含有肺上皮(表达FOXA2、FOXA1、NKX2.1和EPCAM)和间充质祖细胞(表达PDGFRa、CD90、TBX4、HOXA5)。
肺芽类器官将在嵌入基质胶之后生成分支群落。在体外由悬浮培养物中的前部前肠细胞产生时,LBO通常为球状体。LBO通常在d20-d25之间形成且在类器官内部包括折叠结构(参见图12)。
如本文所用的术语“支化LBO”(BLBO)是指具有与支化形态发生相关的结构的LBO。随着BLBO进一步发育,它们开始展现具有胎儿肺泡结构的形态的扩张尖端。
如本文所用的术语“基质胶夹层”是指允许LBO 3维生长成BLBO的基质胶和LBO的排布结构。在一个具体实例中,排布结构涉及固化基质胶的底部部分、混合的基质胶/LBO中间部分以及固化的基质胶的顶部部分,进而类似于夹层构造。
本发明的实施方案
某些实施方案涉及新发现的肺芽类器官(LBO),其具有肺支化形态发生的特征。本文所公开的LBO由多能细胞(诸如胚胎干(ES)细胞或诱导性多能细胞(iPSC))发育而成,所述多能细胞经受一系列不同的孵育步骤以策划多能细胞分化成定型内胚层(DE)、前部前肠上皮(AFE)细胞,且随后最终成为具有折叠结构的LBO(LBOf)。LBOf(在悬浮培养物中至多约20-25天)在出芽上皮结构的尖端上表达音猬因子(SHH)但缺乏分支结构。随后将LBOf异种移植或嵌入3D基质胶。BLBO-3D具有如上所述的分支结构,所述分支结构形态上不如在BLBO-异种移植物中观察到的分支成熟,在BLBO-异种移植物中观察到的分支呈现分支气道和早期肺泡结构,包括迄今未在LGO-3D中体外观察到的I型肺泡上皮细胞和神经上皮小体。3D和异种移植BLBO都含有中胚层和肺内胚层。其他实施方案针对制备这些LBO的方法。
其他实施方案涉及制备LBO和BLBO以及筛选测试剂的方法,所述药剂例如可治疗使用LBO或BLBO建模的纤维化,所述LBO或BLBO具有已知引起纤维化的突变,诸如HPS1、HPS2SFTPC和TERC。具有突变HPS3、5、8和LYST的细胞系影响溶酶体相关的细胞器,但与临床纤维化无关;因此这些系可用作对照。
使用CRISPR/Cas系统生成或修正肺病相关的基因突变.
如本文所用的术语“CRISPR”作为规律成簇间隔短回文重复序列的缩写,是用于病原体防御的细菌基因组中的区域。如本文所用的术语“Cas”是指CRISPR相关蛋白的缩写;Cas9核酸酶是II型CRISPR系统的活性酶。如本文所用的术语“gRNA”是指引导RNA,其提供对Cas9核酸酶的靶向特异性和支架/结合能力。如本文所用的术语“gRNA序列”是指在靶向的基因组DNA中PAM序列之前的20个核苷酸。如本文所用的术语“PAM”是指前间区序列邻近基序,它是必须紧接在gRNA序列之后的所需序列。因此,如本文所用的术语“CRISPR/Cas系统”是指涉及使用RNA引导的核酸酶Cas(其由gRNA定向gRNA序列)编辑基因的系统。随后根据本文所述的技术使经基因修正或突变的细胞系发育成LBO。
BLBO也已由肺RUES2干细胞生成,所述干细胞用携带HPS1基因缺失,使用CRISPR/Cas9制得的突变工程改造(在文中称为“RUES2-HPS1DEL细胞)(图10),所述缺失使细胞易具有对IPF的高渗透,22,44且因此允许在hPSC中体外重演纤维化。使用基因组编辑的ESC避免了通常与iPSC相关的不完全再编程和背景遗传变异的问题。45在患有纤维化的具有HPS1突变的受试者中发现突变的LBO形式,其具有指示纤维化表型的异常形态。
制备其他突变的细胞系以研究肺病,包括纤维化、表面活性物质分泌疾病,例如ABCA3突变或囊性纤维化。下文描述用HPS2、HPS 3、HPS 5、HPS8和端粒酶突变的多能细胞制得细胞系。由这些细胞系生长的LBO也是本发明的实施方案。
HPS1(OMIM#604982):HPS1是BLOC3的一部分,且此突变对PF最具渗透性(目前为80%)。21已描述多种突变,其均消除BLOC3。在密码子321-322处存在移码热点。143,144我们已成功靶向此区域,且使用此系表明可在体外引发纤维化。
HPS2(OMIM#608233):HPS2突变使AP3复合物失去稳定,且还导致易患纤维化。因为AP3B1中的多种缺失和移码造成无义介导的mRNA降解,因此缺失整个蛋白质和AP3复合物,59,145我们在5’区中引入了缺失。通过光学显微术观察,HPS2突变的LBO-3D培养物呈现出纤维化,模拟预期的结果。
HPS8(OMIM#614077):BLOC1复合物的一部分BLOC1S3中的突变造成与IPF无关且充当对照的HPS形式。所描述的初始突变是1bp移码缺失,因为未观察到无义介导的mRNA降解,该1bp移码缺失理论上产生异常的244aa蛋白质。146然而,另一人类突变未显示无义介导的mRNA降解,mRNA不可检测。147因此通过靶向移码突变的5’区来使基因缺失。通过光学显微术,LBO-3D类器官呈现出产生扩张的分支尖端,这可能表明异常的表面活性物质分泌。所有HPS基因都在溶酶体相关细胞器(包括II型肺泡上皮细胞的板层小体)的生物发生方面起作用,且HPS8在体外可以具有表面活性物质分泌表型。
端粒酶(OMIM#614742)端粒酶组分中的突变造成iPS细胞中与细胞被再编程的患者的临床表型相关的端粒缩短。151,152重要的是,端粒的替代伸长似乎不在hPSC中发生。151因为IPF是端粒酶基因突变的最常见临床表现,133所以将端粒酶突变引入hESC是检验端粒病变对ATII细胞功能的影响的有效策略。hTERT和hTERC两者中的多种突变与临床上难以区分的短端粒综合征相关,临床表现的主要决定因素是实际的端粒长度。133我们已将杂合和双重杂合插入缺失引入hTERC的N末端区中。经由端粒FISH的连续传代验证端粒长度。使用来自早期和晚期传代的细胞(>15),其显示显著缩短端粒。152已制得TERC缺失系。它们形成非常小的LBO,如所预期,通过光学显微术看起来呈纤维化。
还根据本文的教示产生以下系:
HPS5(OMIM#607521).HPS5与间质性肺病无关且将充当对照并类似于PIPS3编码BLOC2复合物的蛋白质。人类中已知已知的唯一突变是在密码子leu675至val676处的纯合4-bp缺失(AGTT)。突变导致移码,密码子682处的无义多肽截短,造成C端处的蛋白质损失40%。
HPS3(OMIM#060118):HPS3与间质性肺病无关,且将充当对照。除了其他以外,HPS3由HPS3基因中的大缺失造成,所述HPS3基因为BLOC2复合物的一部分。57因为缺失相应的mRNA和BLOC2复合物,57所以进行5’区中的完全缺失。
LYST:(OMIM#606897):已描述多种移码,其引起严重的儿童期发作CHS,在白血细胞和黑素细胞中具有证实的巨大颗粒。64,148-150我们将在Lys633/Lys634处创建插入缺失,这导致密码子提前终止。
SFTPC(OMIM#178620):我们将使用引导RNA和含有点突变的同源单链80bp DNA片段将杂合T->A颠换引入外显子5的核苷酸128。此杂合突变产生Dutch家族中的高度渗透性IPF。11对于SFTPC而言,尽管更具挑战性且效率不高,但必须引入在患者身上观察到的特异性突变,因为由异常折叠蛋白而不是蛋白质缺乏引起的蛋白质毒性会导致疾病。4,5,67
相反地,可以使用Crispr/cas系统在体外修正来源于携带肺病相关基因突变的上文论述的iPSC,诸如C12系,以制备经基因修正的细胞系。使用出于修正基因缺陷的预期目的经过遗传改变的细胞生产LBO提供一种测试这类遗传改变修正疾病表型的能力的可行方法。
如本文所用的术语“肺病相关突变”是指已知引起肺病表型的基因突变或多态性。例如,某些肺病由以下基因的不完全列表中的基因突变引起:HPS1、2、4、hTERT、hTERC、角化不良蛋白、CFTR、DKC1、SFPTB、SFTPC、SFTPA1、SFTPA2、MUC5B、SHH、PTCH、SMO、ABCA3。下文提供这些基因的基因ID编号:
基因名称 | 基因ID | 替代名称 |
CFTR | 1080 | |
HPS1 | 3257 | |
HPS2 | 7031 | TFF1 |
HPS4 | 89781 | |
TERT | 7015 | |
TERC | 7012 | |
DKC1 | 1736 | |
SFTPB | 6439 | |
SFTPC | 6440 | |
SFTPA1 | 653509 | |
SFTPA2 | 729238 | |
MUC5B | 727897 | |
SHH | 6469 | |
PTCH1 | 5727 | |
SMO | 6608 | |
ABCA3 | 21 |
另外,囊性纤维化与囊性纤维化跨膜转运调节因子(CFTR)和多态性相关的钠通道上皮1α(SCNN1A)基因中的基因突变相关,且这类突变/多态性高度可变。参考这类基因的表达蛋白,突变包括CFTR蛋白中的F508、CFTR蛋白中的G551、CFTR蛋白中的G542、CFTR蛋白中的N1303、CFTR蛋白中的R117、CFTR蛋白中的W1282、CFTR蛋白中的R553、CFTR蛋白中的c.3849+10kb、CFTR蛋白中的c.2657+5、CFTR蛋白中的c.3140-26以及SCNN1A蛋白中的VI14。另外,Anjali Jacob等人,Cell Stem Cell 21,1-17,2017年10月5日的题为Differentiation of Human Pluripotent Stem Cells into Functional Lung AlveolarEpithelial Cells的公开使用这类Crispr/cas系统修正纯合表面活性物质突变(SFTPB121ins2)以恢复肺泡上皮2型细胞中的表面活性物质处理。另一KatherineB.McCauley等人,2017,Cell Stem Cell 20,844-857的题为Efficient Derivation ofFunctional Human Airway Epithelium from Pluripotent Stem Cells via TemporalRegulation of Wnt Signaling的公开使用CRISPR修正毛喉素诱发的肿胀中的缺陷,所述肿胀通过基因编辑以修正与复合型杂合CFTR表型DF508/DF508相关的疾病突变来救援。
可根据本领域已知(参见例如美国专利公开20170022507)和本文所述的技术使携带突变基因的细胞(包括上文描述的那些)进行CRISPR/Cas系统。通常,使细胞在生长和扩增期(诸如多能期)进行CRISPR/Cas诱导的基因突变。这些细胞随后将发育成如本文教示的LBO且观察这些细胞的表型和/或生物标记物表达的变化。
概述
II型肺泡上皮细胞在IPF和家族PF中的核心作用。
肺纤维化是过量纤维结缔组织(纤维化)在肺中的形成或发育,也称为“肺的瘢痕形成”。肺纤维化可以是其他疾病的继发效应。这些疾病中的大多数分类为间质性肺病。实例包括自身免疫性疾病、病毒感染或肺的其他纤维损伤。然而,肺纤维化也会在没有任何已知病因的情况下出现(称为“突发性”),且与其他形式的纤维化的不同之处在于它对任何免疫抑制性治疗都不起反应。
特发性肺纤维化(IPF)是一种频率增加的难治疗间质性肺病,中值存活期为3至4年,特征在于成纤维细胞灶以及肺泡的重塑和闭塞。1,2唯一的确定性治疗是肺移植,一种干预措施,受供体器官的低可用性,以及严重的手术、药物和免疫并发症困扰。3
ATII细胞在赫曼斯基-普德拉克综合征(Hermansky-Pudlak Syndrome;HPS)中的
作用
IPF的原因是ATII细胞中的缺陷这一观点进一步得到以下事实的支持:患有赫曼斯基-普德拉克综合征(HPS)的患者子组显示高IPF发病率,也称为HPS相关的间质性肺炎(HPSIP)。56HPS由异常的生物发生和溶酶体相关细胞器(LRO)的转运引起且特征在于分别与黑素体和血小板δ颗粒(都为LRO)的功能障碍相关的色素异常和出血素质。存储、分泌和循环表面活性物质的ATII细胞的板层小体(LB)也是LRO。21,22引起HPS的突变影响四种不同的蛋白质复合物:溶酶体相关细胞器(BLOC)1(HPS7、8、9)、BLOC2(HPS3、5、6)、BLOC3(HPS 1、4)和AP3(HPS2)的生物发生。尽管这些复合物的功能尚不清楚,但它们都参与LRO的蛋白质转运和生物发生。21,22在九种已知突变中,三种(HPS1和HPS4,影响BLOC3和HPS2,使AP3失能)与30岁后的IPF相关,该IPF在临床上、预后上、放射学上和组织学上非常类似于IPF。21,22在HPS1中,IPF的发病率>80%,这使其成为最具渗透性的IPF突变。21若干种具有自发突变的小鼠品系拟表型人类HPS的各种子组的色素缺陷和血小板异常,且在鉴别人类中的缺陷基因方面起作用。57-62尽管没有任何一者呈现自发性IPF,但博来霉素(bleomycin)诱发的纤维化与人类HPS子组中IPF的发病率分离。19在HPS2mt小鼠中,ATII细胞中的转基因修正拯救纤维化易感性,从而表明ATII技能失调在发病机制中的关键作用。PF在老龄ep/pe小鼠中发生,所述小鼠具有HPS1和HPS2的突变,因此可以提供IPF的最佳小鼠模型。18,63白细胞异常色素减退综合征(Chediak-Higashi syndrome;CHS)也是LRO的疾病,其由患者中LYST以及小鼠中beige(be)的突变引起,64其中先天性免疫缺陷和神经退行性病变是主要表现。4LYST参与囊泡融合或分裂,但其确切功能尚不了解。65在beige小鼠和CHS患者中,LB增大,4,19,33,34,与死于HPSIP66的患者和ep/pe小鼠类似,但CHS与PF无关。18,58这些发现表明并非每一种ATII损伤都沉淀纤维化。
进一步支持ATII细胞作用的是在ep/pe小鼠的ATII细胞中观察到的增加的细胞凋亡以及溶酶体和ER应激,这些发现在人类HSPIP样品的有限组中得以证实。18已经在散发性IPF中的ATII细胞中观察到相似类型的应激,包括内质网中的未折叠蛋白(UPRER,也与SFTPC突变相关)、4,5,67低自体吞噬、6,8,9,68线粒体机能失调7和细胞凋亡。ATII细胞在IPF中的作用最可能与其最特异性的功能相关:表面活性物质的生产、分泌和循环。溶酶体基础设施对于细胞质量控制机制而言至关重要,包括回应于应激的自体吞噬和线粒体自噬。35-38
然而,人类ATII细胞的分离和维持颇具挑战性。重要的是,在诊断后分离自患者的ATII细胞的特征可以没有关于疾病倾向的信息,因为许多观察到的变化可能是次要的。此外,据信疾病起始发生在临床症状之前许多年。1,2通过定向分化人类多能干细胞(hPSC)产生的ATII细胞将促进在ATII细胞中由导致纤维化的损伤或突变诱导的功能和转录组共性的发现。来源于哺乳动物胚泡的内细胞群,胚胎干细胞(ESC)可以在体外维持多能性状态,且在生物体内生成每种细胞型。80诱导性多能干细胞(iPSC)通过经由OCT4、KLF4、MYC和SOX2的短暂表达将体细胞再编程为与ESC类似或相同的多能性状态来生成。80-89CRIPSR/Ca9介导的基因组编辑现在允许工程改造hPSC中的所需突变。90-94hPSC来源的ATII细胞处于疾病前状态,因此允许检测预先存在的异常。
不存在关于IPF的基于hPSC的模型公开的研究。小鼠模型一直不能从根本上阐明此高度流行的毁灭性疾病的发病机制。如本文呈现的结果所示,已经组合若干种技术上和概念上创新和独特的方法,包括由hPSC产生远侧肺细胞和间充质;对hESC基因组修饰以引入与IPF相关的突变;以及使用影响ATII细胞但与IPF无关的突变作为对照以寻找在细胞中与IPF倾向突变共有的功能或表达特征从而获得迫切需要的对IPF发病机制的了解。
呼吸系统来源于在前部前肠内胚层(AFE)的腹面上出现的芽,所述芽通过模式化支化过程发育成近侧气道和远侧肺泡祖细胞(假腺管型期)。在小管期,细胞周期活性降低,且气道上皮在柄中发生特化,出现基细胞、杯状细胞、棒状细胞、纤毛细胞和其他细胞型。此期之后为囊状期,其中小管扩大成将产生原始肺泡的小囊6,7。(图11)
我们先前报导了在2D中通过顺序发育步骤由定型内胚层(DE)将hPSC(胚胎干细胞(ESC)和经再编程的诱导性多能干细胞(iPSC))分化成AFE、肺野祖细胞,以及最终肺和气道上皮细胞。这些发育公开于美国专利号US20160168535和US20150247124。(图6A)10-12
如上文提及,已开发出用于制备LBO的新方法的实施方案,所述LBO含有与肺间充质相容的肺内胚层和中胚层,且在3D培养物中经历支化形态发生和远侧肺发育。还描述通过将LBO异种移植到诸如免疫缺陷小鼠的肾包膜下来制备具有最发达形态发生的LBO的新方法的实施方案。经过异种移植的LBO至少部分地重现人类肺发育且因此可用于建模和评估影响肺发育的因素,包括IPF样表型是否在体内出现。在其他实施方案中,BLBO-3D和BLBO-xeno由RUES2-HPS1DEL细胞制得,所述细胞具有使用CRISPR/Cas9在hESCsh中工程改造的HPS1突变,所述突变使得易具有对IPF的高渗透22,44;这些突变LBO允许体外重演纤维化且预期通过异种移植在体内同样如此。基因组编辑的ESC避免了与iPSC相关的不完全再编程和背景遗传变异的问题。45
在疾病建模方面的大多数工作使用iPS细胞。45,134,135在iPS生成期间,清除成熟细胞的表观遗传标记,且建立多能性网络和表观遗传标记,其维持细胞呈对应于ESC的多能性状态的多能性状态。45,136,137尽管iPSC非常类似于ESC(如果不相同的话),但已经描述不完全再编程和表观遗传记忆的持续性,这有利于iPSC分化成它们最初来源于的细胞类型,尽管该问题存在争议。45,138,139此外,遗传背景是iPS系之间变异的最重要影响因素,45,140需要来自足够数量的患者的多种克隆以实现统计学强度。CRISPR/Cas9介导的基因组编辑技术的可用性允许将患者突变引入ESC。91-93这很大程度上消除了遗传背景变异,以及由不完全再编程和表观遗传记忆引起的偏倚和变异性(如果这些存在的话)。然而,iPSC的使用在分散性IPF中是优选地,其中可以存在家族倾向但其中尚未鉴别出相关的突变,或其中可以涉及多种基因座。
尽管一个组报导了人类肺类器官的生成,8,9这些小结构含有表达肺和气道8细胞的标记物的细胞且在异种移植入小鼠中后具有一定的气道潜能9,但它们不满足前述类器官的标准,因为既未观察到肺发育,尤其支化形态发生和靠近远端特化的特征,也未观察到功能。因此,至今为止,尚不存在可用于建模正常或异常(诸如纤维化)肺的肺类器官培养物。
结果
A.在中胚层存在下的3D肺发育
因为IPF是一种具有主要间充质组分的纤维化疾病,所以我们旨在建立一种存在间充质的模型。在培养物中由人类多能干细胞生成LBO。下文所述的策略可应用于ES细胞(例如RUES2细胞)或iPS细胞,其例如使用来自健康人类皮肤成纤维细胞7,9(第16-25段)和IRF7缺陷型C12 hiPSC细胞系的仙台病毒或被修饰mRNA生成。28将细胞维持在以15,000-18,000个细胞/cm2涂铺的小鼠胚胎成纤维细胞(MEF)上。
在图1-4中所述的结果中,用正常RUES2胚胎干细胞(ESC)进行实验,但用iPSC获得相似的结果。通过以下方式诱导内胚层:耗尽小鼠胚胎成纤维细胞(mfe)且随后在类胚体/原条形成培养基中培养hPSC,之后换到内胚层诱导培养基。如前文所述诱导前部前肠内胚层9,且随后用腹侧化培养基(支化培养基)处理AFE 48h且观察三维团块形成。随后通过在孔周围轻缓吹吸使所述团块悬浮。在腹侧化培养基/支化培养基存在下由前部前肠上皮(AFE)诱导腹侧肺命运早期,在悬浮培养物中易于脱离和扩增的粘附结构在BMP4、FGF10、FGF7、视黄酸(RA)和GSK3β拮抗剂CHIR99201(图6B)(前文显示为肺发育所需的因子6,7)存在下形成为细胞团块(图1A-1B)。由7.5x105个定型内胚层(DE)细胞生成2490±129个团块(n=3;RUES2 ESC)。将AFE的悬浮团块(下文称为肺芽类器官(LBO))维持于浸入支化培养基中的非组织培养物处理的多孔板且每隔一天给料直到d20-d25,这时将其嵌入100%基质胶中或植入NOD.Cg-Prkdcscid.I12rgtmlWjl/SzJ(NSG)小鼠的肾包膜下。
在LBO的悬浮培养期,所述结构形成EPCAM+KRT8+ECAD+FOXA1/2+AFE细胞的折叠片(FOXA2:89.07%±3.36%,EPCAM+:92.08%±1.88%,n=3;RUES2 ESC)(图1C)。到第25天,51.26±4.37%(n=3;RUES2 ESC)的细胞表达肺标记物NKX2.1+(图1C)。除了上皮祖细胞标记物p63(18.59%±1.49%,n=3;RUES2 ESCs,图1C)之外,不存在成熟肺和气道细胞的标记物(未显示ECRt)。所述细胞被中胚层PDGFRA+ECAD-包围(图1D)。RNAseq(图6C)证实内胚层/肺基因(FOXA2、SOX2、NKX2.1)在EPCAM+细胞中的强烈富集(图1E)。EPCAM-细胞表达中胚层基因(图1e),其中一些基因(诸如TBX4和HOX5旁系同源基因)在肺中胚层内表达13,14。在成熟肺和气道中以及在其他AFE来源的谱系中表达的基因在EPCAM+部分中几乎检测不到(图6D)。音猬因子(SHH)在内胚层细胞中表达,且其转录标靶15PTCH1、GLI1和HHIP在中胚层中表达(图6D)。原位杂交证实第15天在内胚层部分中的SHH表达。在第25天,SHH在出芽上皮结构的尖端中表达最强烈(图6E)。这些发现与早期发育小鼠肺一致,其中SHH最初在整个肺内胚层中表达,但在支化形态发生期间逐渐变得限于分支尖端15-17。因为它们含有多种空间组织上与体内发育肺芽相似的细胞类型,我们将这些结构称为肺芽类器官(LBO)。
B.在异种移植之后人类肺芽类器官的体内潜能。
在大约第20-25天,在手术之前使大约一百万d20-d25 LBO细胞与5μl基质胶混合且移植到NOD.Cg-Prkdcscid.I12rgtm1Wjl/SzJ(NSG)小鼠的肾下。在移植到免疫缺陷型NSG小鼠的肾包膜下时,来自人类RUE2细胞的LBO-xeno产生生长物(图2A),所述生长物具有大量间充质组织,包括平滑肌、结缔组织和疏松结缔组织和罕见软骨。LBO-xeno在大约1.5个月之后含有被间充质组织包围的管状结构(图2B)。所述管均匀内衬有与气道上皮相容的FOXA2+NKX2.1+SOX2+上皮,所述上皮在基底层含有MUC5AC+(杯状体)细胞和p63+细胞(图2C)。所有细胞都是人类细胞(图7a),上皮细胞除外,其属于小鼠来源(图7B)。在5个月之后,出现被SMA+中胚层细胞(图7C)包围的分支结构(图2D、图7C)。所有上皮细胞都是NKX2.1+,但肺发育后期的近侧标记物SOX218,19从分支尖端排除,该SOX2表达产生表面活性物质的II型肺泡上皮(ATII)细胞的标记物SFTPB和SFTPC(图2E)20。柄和中央小管表达近侧(气道)标记物FOXJ1(纤毛细胞)、CC10(棒状细胞)和黏蛋白(杯状细胞)(图2E)。苏木素-伊红染色显示大量多纤毛细胞(图2D),而活成像记录摆动纤毛。此外,在较大管状结构附件观察到粘膜下腺(图7E)。总体而言,生长物内的形态和表达模式与肺支化形态发生期间的靠近远端特化一致6,7。管状结构中的流体含有所有测试的肺和气道的分泌产物但对细胞表面黏蛋白21MUC1显阴性,表明检测到分泌蛋白且未检测到与脱落细胞相关的蛋白质,并提供关于功能的证据(图2F)。在7个月后,与神经上皮小体22相容的CGRP+PGP9.5+细胞的圆顶状组存在于气道样结构中。(图2G)此外,生长物区域发展成薄细胞层网络(图2G),其含有表达ATII细胞标记物(SFTPC、ABCA3、HT2-280)23的细胞和携带I型肺泡上皮细胞(ATI)标记物(HT1-56、HOPX、PDPN、CAV1、SCNN1A、AKAP5、CLIC5)20的细胞,但未检测到成熟ATI细胞的其他标记物(RAGE、AQP5)20(图2G)。然而,未观察到肺泡毛细管网和支气管肺泡管。我们得出结论:尽管未实现完全的表型和结构突变,这可能至少部分是因为异位定位,但LBO重现在异种移植之后肺发育的许多基本特征,包括支化形态发生和靠近远端特化。
C.体外长期BLBO-3D发育和ATII功能:3D基质中的LBO能够生成支化群落,且在这些培养条件中支化不需要间充质细胞
在CHIR99021、FGF10、FGF7、BMP4和RA存在下将来自RUES2的d25 LBO嵌入基质胶中之后(参见图6B),获得>95%迅速扩增的支化结构(对应于RUES2的图3A且还可参见对应于iPSC的图8A,包括来自具有突变IRF7的患者的系C12,所述突变造成流感感染后的急性呼吸窘迫综合征)24。RUES2细胞表达肺内胚层的标记物(在第70天FOXA2+:95.17%±1.54%,NKX2.1+:74.97%±4.37%,EPCAM+:96.83%±0.62%,SOX9+:92.42%±3.81%n=3;RUES2ESC)(图3B)。MUC1的均匀腔内表达表明极化(图3B)。表达ATII标记物SFTPC、SFTPB和ABCA3的细胞存在于所有结构中(图3B)。气道杯状细胞(MUC5B或MUC5AC)较稀少而其他气道细胞(棒状细胞(SCGB3A2)、纤毛细胞(FOXJ1)和基细胞(KRT5和P63))未检测到(图中未示)。尽管单独涂铺的RUES2 LBO在每个方向随机分支且在90天内填充6.4mm孔,但它们在近距离涂铺在一起时形成仅占据孔的一部分的分支树(图8B)。这些发现表明,可在体外操纵正常RUES2细胞的支化结构,且支化结构之间的互相推斥作用可在确定其结构方面起一定作用。
表达VIMENTIN和CD90的RUES2中的间充质细胞围绕LBO-3D结构存在(图3B)。然而,如通过针对EPCAM-细胞的流式细胞术所测定,其比例在基质胶培养期间下降到总群体的2%(图8C)。自d25 LBO纯化获得的EPCAM+,而非EPCAM-细胞在涂铺到基质胶中之后产生支化群落,但效率较低(0.30±0.0316%)(图8D)。这些支化群落呈现出与由完整LBO产生的基质胶群落相似的标记物表达模式(图8E)。这些发现表明,LBO中的稀少祖细胞能够生成支化群落,且在这些培养条件中支化不需要间充质细胞。
D.RUES2 LBO体外重现直到人类妊娠的倒数第二个三月的人类肺发育
在RUES2 LBO-3D培养>170天之后,已出现由具有扩张尖端的支化小管组成的宏观组织(图4A),该宏观组织令人想起在肺发育的囊状期期间形成的小囊(图4B、图9A),84.86%±5.21%细胞是NKX2.1+,而大多数细胞是SOX9+(76.75±6.89%)且少量(23.78±5.21%)是SOX2+(图9B)(n=4,一次是ESC且三次是iPS系)。大多数腔内细胞表达HT2-280、MUC1、SFTPB、SFTPC和ABCA3(RUES2图4C,iPSC图9B),鉴别出这些细胞为ATII细胞。电子显微术显示大量板层小体(LB),其为存储表面活性物质的细胞器25(图4D、图9C)。
为检查ATII细胞功能,添加共价连接于荧光脂质BODIPY的SFTPB。在数分钟内,SFTPB-BODIPY被RUES2 LGO细胞吸收且分泌在腔中(图4E、图4F)。尽管小鼠中ATII细胞和推定的双潜能肺泡祖细胞的标记物HOPX20,26广泛表达(图9B),但未检测到其他ATI标记物(AQP5、CLIC5、AKAP5、CAV1、AGER)。SOX9是随着肺泡成熟下调且产后变得不可检测的远侧尖端的标记物,其在体外大多数在分支结构的尖端和外缘处表达,与小鼠发育一致,其中SOX9为远侧肺标记物27-29(图9B)。
气道标记物(MUC5AC、SCGB3A2)在基质胶LBO-3D RUES2群落中的表达受限于共表达SOX2和SOX9的结构(图9B),且因此可能更近。尽管SOX2和SOX9的共表达在小鼠中不常见19,但在人类妊娠中期胎儿远侧肺中鉴别出大量较大的SOX2+SOX9+结构(图9D),,从而表明LBO-3D重现人类肺发育。SOX9的表达、主要表达ATII标记物的囊状结构的形成以及成熟ATI细胞的缺失与肺发育的小管期一致,这在小鼠妊娠结束时出现,但在人类中处于倒数第二个三月期间。为进一步验证d170基质胶LBO-3D培养物的发育期,我们对来自RUES2细胞和三种iPSC系的12个独立样品进行RNAseq。交叉参考KeyGenes数据库,该数据库含有妊娠早期和中期以及成年期期间人体器官的表达谱30,显示出与妊娠中期胎儿肺的最佳匹配,而与其他器官没有任何匹配(图4G、图9E)。综合而言,结构化蛋白质表达和转录组学数据表明d170基质胶RUES2 LBO类器官达到人类妊娠的倒数第二个三月。
E.LBO-3D在建模人类疾病方便的潜在应用
1.LBO模型复制远侧肺中RSV感染的形态特征。
我们接着探索选择的感染和纤维化肺病可以被重现。我们对感染呼吸道合胞体病毒(RSV)的LBO-3D是否呈现人类肺感染的特征有疑问。RSV是婴儿下呼吸道感染的主要原因,且造成毛细支气管炎以及小气道堵塞2,31。当前不存在获得许可的疫苗或有效的抗病毒药,且在感染后免疫力短暂32。人类中的RSV向性包括纤毛细胞和肺泡上皮细胞2,3。对人类气道上皮细胞系的先前研究表明,感染RSV的细胞溶胀且脱离上皮33,该发现与档案病理标本中感染细胞所引起的小气道堵塞,以及与毛细支气管炎的临床综合征一致3。在用RSV感染d170基质胶LBO-3D培养物2天之后,共聚焦显微术揭示溶胀的感染细胞流入支化结构的腔中(图5A,箭头)。在第1天未发现流入,尽管有病毒感染的迹象。LBO中的RSV感染因此重现人类中感染的重要特征。
2.RUES2-HPS1DEL LBO-xeno模型显示间充质细胞在使用CRISPR-CAS9制备的HPS1突变型细胞系中积聚
用CRIPSR/Cas9构建体转染RUES2系,已在成神经细胞瘤系中筛选所述构建体以诱导合适突变。挑选克隆,并将其通过使用跨越CRISPR同源区的引物的PCR分析,之后进行质粒克隆和测序以检测具有所需突变或插入缺失的系。除了验证缺失的经典方法(PCR,测序)之外,对于HPS突变而言,通过WB验证所涉及复合物的缺失(BLOC1-3和AP3复合物广泛表达)21,60,141,如已展示每个突变使所编码蛋白所属的整个复合物失去稳定。141,142突变的靶向序列提供于图13-16。
F.RUES2细胞(RUES2-HPS1DEL)和纤维化迹象
接着,我们试图建模与赫曼斯基-普德拉克综合征(HPS)的一些形式相关的肺纤维化5。HPS的特征在于由异常生物发生和溶酶体相关细胞器(LRO)的转运引起的色素和出血异常,所述溶酶体相关细胞器包括血小板致密颗粒和黑素体。34一些形式,尤其是HPS1与早期发作的难治性肺纤维化(HPS间质性肺炎(赫曼斯基-普德拉克综合征相关的间质性肺炎或HPSIP))相关,该肺纤维化临床上与特发性肺纤维化(IPF)相似5,特征在于肺泡的纤维性闭塞以及具有3-4年的中值存活期35。ATII细胞的LB也是LRO34,该事实可以解释IPF与造成HPS的一些突变的相关性5。
由具有CRISPR-CAS9诱导的HPS1缺失的RUES2细胞(下文称为RUES2-HPS1DEL)产生的来源于LBO-3D的基质胶群落(图10A)展现在基质胶培养物中轮廓界限不如来自亲代RUES2系的LBO清晰的支化结构(图5B),EPCAM-间充质细胞的分率增加(图5C、图10B),其异质表达间充质标记物PDGFRA、PDGFRB、SMA、VIMENTIN和CD90(图5D,图10C中低放大率平铺扫描)。与亲代细胞相比,EPCAM-展示(但EPCAM+群体并未展示)在RUES2-HPS1DEL细胞的培养物中显著增强的增殖(图10D、图E),从而表明间充质细胞的表达解释EPCAM-细胞的分率增加。然而,令人惊讶的是,早在悬浮培养的第15天,甚至在检测到任何ATII标记物之前EPCAM-细胞的过度增殖在RUES2-HPS1DEL LBO中就已经显著。此外,在RUES2-HPS1DEL细胞中,增加的羟脯氨酸含量(图10F)以及增强的细胞外基质(ECM)自发荧光(图10C)和对胶原1和3的免疫荧光染色以及纤连蛋白(图5D、图10G)表明增加的ECM沉积。混合实验(图10H-图J)与以下观点一致:间充质细胞的积聚受突变的上皮细胞驱动,且不受突变的间充质细胞的细胞内在特性驱动,该发现与以下观点一致:HPSIP36和可能的其他形式IPF4可由上皮损伤引起。综合而言,这些发现表明,有可能使用LBO至少建模一些纤维化肺病。
关于携带某些突变(HPS2、HPS8、SFPTC和端粒酶)的其他细胞系的发育的信息和数据提供于下文实施例3中。用于制备携带突变的每种细胞系的技术类似于如实施例1和3中所阐述的针对HPS1细胞系的技术。将每种基因突变的序列以及将用于将每种突变插入细胞基因组的gRNA标靶序列提供于图13-16。携带HPS2和端粒酶的LBO表明与针对HPS1-LBO观测到的类似的纤维化异常。
讨论
体外基质胶中LBO和LBO来源的支化群体以及在异种移植到小鼠肾包膜下之后的生长物满足真正类器官的定义1且因此这些群体适合称为肺芽类器官(LBO)。先前报道的“人类肺类器官”不能完全称为类器官,因为它们在体外或在异种移植后都未展示支化8,9。此外,相比于本发明LBO,先前描述的肺类器官是在血清存在下,以及BMP4、RA和Wnt激动不存在下产生的,而我们已证实BMP4、RA和Wnt激动对体外肺特化至关重要10。最后,这些结构在接枝到免疫缺陷型小鼠的肾包膜下之后未体内发育,但在皮下移植之后需要在生物生成的支架上预孵育以生成气道上皮细胞9。相比之下,首次在感染RSV的RUES2细胞的LBO-3D模型中复制远侧肺中RSV感染的形态特征,目前不存在复制人类感染的模型。RUES2-HPS1DELLBO-3D模型也展示缺乏HPS1的细胞中纤维化(赫曼斯基-普德拉克综合征相关的间质性肺炎或HPSIP)的证据,该HPS1是造成临床上、预后上、放射学上和组织学上与IPF无法区分的肺纤维化的最具渗透性形式的突变4,5,36。然而,值得注意的是,虽然疾病HPSIP通常在生命的第30年或40年出现,但可以在定向分化的40天内在RUES2-HPS1DEL LBO模型中体外复制纤维化表型。不受理论束缚,体外培养的应激可以重现由衰老诱导的变化并导致表型非常快速的出现,特别是因为年龄和端粒功能障碍是IPF的主要危险因素35,37。LBO模型的局限性在于在基质胶中培养6个月后,就结构、标记物表达和全基因组表达标记而言,类器官匹配人类妊娠的妊娠中期。这些发现表明,如在LBO系统中模拟的肺发育与子宫内人类肺发育保持同步。因此,完整的终末成熟仍然是类器官领域的挑战1。第二个限制在于支化呈现随机性,该发现与目前尚未证明的支化形态发生的‘空间填充’模型一致38。然而,已经证实LBO-3D支化可以通过在基质胶中彼此接近地涂铺若干个LBO来引导,在这种情况下,类生物远离彼此支化,表明可以在体外操纵支化。第三个限制在于尚不清楚存在于LBO中的间充质的确切性质和图式形成。体内异种移植显示LBO-xeno相关的中胚层细胞不可能产生内皮细胞、骨骼或骨骼肌,从而表明间充质在某种程度上是特定的。肺中各种间充质谱系及其个体发育仍然很难表征。6然而,肺血管系统可能并非来源于肺间充质。近侧肺血管来源于常见的心肺间充质祖细胞,而肺泡毛细管网的发育起源可能源于先前存在的躯干血管中出现的VE-钙粘蛋白+祖细胞6,39。第四个限制在于体外培养物强烈偏向远侧肺,并且尽管共表达SOX2和SOX9的一些区域表达了杯状细胞和棒状细胞前体的更近侧标记物,但没有观查到成熟的棒状细胞、纤毛细胞或基细胞。我们也可能无法在体外实现ATI标记物的诱导,尽管在体内植入后存在ATI可能性。
总之,这项工作表明,尽管存在某些局限性,LBO(3D和xeno)将成为研究人类肺发育、建模肺病和针对药物对正常LBO和LBO(模拟肺病,诸如RSV感染和纤维化)的效果对其进行筛选的有用工具。
药物筛选
本发明提供一种筛选测试剂的方法,所述测试剂尤其防止或减少肺和气道细胞球体中胶原的形成,如本文所述。然而,它不限于防止或减少胶原,因为纤连蛋白和任何其他细胞外基质蛋白,以及所有类型的间充质细胞(成纤维细胞、脂成纤维细胞、肌成纤维细胞等)也可以减少。
另一筛选实施方案鉴定了增加或减少通过本发明方法制备的细胞群中表面活性物质产生的测试剂。
药剂的实例包括蛋白质、肽、非肽化合物、合成化合物、发酵产物、细胞提取物、植物提取物、动物组织提取物等、核酸、肽、蛋白质、非肽化合物、合成化合物、发酵产物、细胞提取物、细胞培养上清液、植物提取物、哺乳动物组织提取物、血浆等。测试物质可以是新物质或已知物质。测试物质可以呈盐形式,这类盐可以是具有生理学上可接受的酸或碱的盐。这些物质可以是新物质或已知物质。另外,使用组合化学技术产生的化合物文库,通过固相合成或噬菌体展示产生的随机肽文库等也是测试物质的优选实例。
如本文所用,术语“测试剂”非常广泛(如下所述),并且可以指代医药或非医药化合物或底物,评估其阻止如本文所述的肺和气道细胞球体中胶原形成,或防止胶原蛋白表达球体崩溃的能力。
在一个实施方案中,用可以通过细胞膜转运的小分子量测试试剂处理BLBO。这类药剂的量可以由本领域技术人员确定,但通常可以在约0.01微摩尔(0.01μM)至1mM之间。培养的球体或其他测试细胞与测试化合物的接触持续时间可以变化。测定化合物减少或防止在BLBO中纤维化的能力可以在任何时间进行,只要它是在开始施用测试物质之后即可。
使用本发明的方法筛选的文库可包含多种类型的化合物。在一些实施方案中,所述化合物是肽分子。在非限制性实例中,肽分子可存在于噬菌体展示文库中。在其他实施方案中,化合物的类型包括但不限于肽类似物,包括包含非天然存在的氨基酸(例如D-氨基酸)的肽;氨基酸的磷类似物,诸如α-氨基磷酸,或具有非肽键的氨基酸;核酸类似物,诸如硫代磷酸酯和PNA、激素、抗原、合成或天然存在的药物、鸦片制剂、多巴胺、5-羟色胺、儿茶酚胺、凝血酶、乙酰胆碱、前列腺素、有机分子、信息素、腺苷,蔗糖、葡萄糖、乳糖和半乳糖。还可以使用多肽或蛋白质的文库。
在一个实施方案中,组合文库是有机小分子文库,诸如但不限于苯并二氮杂类异戊二烯、噻唑啉酮、偏噻嗪烷酮、吡咯烷、吗啉代化合物和二氮杂蒎二酮。在另一个实施方案中,组合文库包含类肽;无规生物低聚物;苯二氮/>多样体,诸如苯乙烯、苯并二氮杂/>和二肽;插烯多肽;非肽类肽模拟物;寡聚氨基甲酸酯;肽基膦酸酯;肽核酸文库;抗体文库;或碳水化合物文库。组合文库本身为可商购的(参见例如Advanced ChemTech EuropeLtd.,Cambridgeshire,UK;ASINEX,Moscow Russia;BioFocus pic,Sittingboume,UK;Bionet Research(Key Organics Limited的分公司),Camelford,UK;ChemBridgeCorporation,San Diego,Calif.;ChemDiv Inc,San Diego,Calif.;ChemRx AdvancedTechnologies,South San Francisco,Calif.;ComGenex Inc.,Budapest,Hungary;EvotecOAI Ltd,Abingdon,UK;IF LAB Ltd.,Kiev,Ukraine;Maybridge plc,Cornwall,UK;PharmaCore,Inc.,North.Carolina;SIDDCO Inc,Tucson,Ariz.;TimTec Inc,Newark,Del.;Tripos Receptor Research Ltd,Bude,UK;Toslab,Ekaterinburg,Russia)。/>
在一个实施方案中,可以合成用于本发明方法的组合化合物文库。
预期用于本文的示例性合成低分子量生物活性分子包括来自分子多样性文库(MolBio)的MaxiVerse.TM.、LOPAC1280(来自Sigma)、药物样筛选化合物的MyriaScreenDiversity Collection(来自Sigma),可从www.biofocus.com/offerings/compound-libraries.htm?gclid=CMXYzorejp4CFSZdagodh-ktmsw获得的化合物文库,以及其任何两个或更多个的组合。
预期用于本文的示例性抗体包括可以与人类细胞类型功能性相互作用的任何抗体(或其片段),无论所述抗体是单克隆还是多克隆。示例性抗体包括免疫球蛋白亚型、Fab片段等的抗体,例如以下抗体:识别标靶LBO特有的细胞表面标记物;识别其表达由暴露于多因子培养基诱导的任何细胞表面蛋白质;或抑制抑制的细胞信号传导通路等;以及它们的任何两者或更多者的组合。
考虑用于本文的示例性核酸包括寡核苷酸、DNA分子、RNA分子等,以及它们的任何两种或更多种的组合。
预期用于本文的示例性DNA分子包括编码锌指核酸酶、锌指转录因子、cDNA过表达文库等的DNA-质粒/载体,以及它们的任何两种或更多种的组合。
预期用于本文的示例性RNA分子包括siRNA(参见例如www.sigmaaldrich.com/life-science/functional-genomics-and-mai/sima.html)、sh RNA(参见例如(如可在www.sigmaaldrich.com/life-science/functional-genomics-and-mai.html和openbiosystems.com/RNAi/shrnaLibraries/获得)、微RNA(参见例如,如可在www.mirbase.org/index.shtml获得)等,以及它们的任何两种或更多种的组合。如本领域的技术人员容易认识到,可以直接(例如使用siRNA或微RNA)或以含有病毒表达载体的病毒形式将RNA分子点在阵列上,所述病毒表达载体含有所关注RNA分子(例如微RNA或shRNA)。
用于筛选测试化合物文库的本发明筛选方法优选包括优选地在生理条件下,使测试化合物与标靶LBO接触。
具有支化形态发生的肺组织的形成
根据如下文实施例2所述的技术制备肺芽类器官。方案涉及三期。第一,在诱导多能细胞向定型内胚层(DE)分化的条件下,使人类多能细胞(诸如诱导性多能干细胞或胚胎干细胞)经受类胚体/原条形成培养基。该第一期通常需要4天(d0-d4)并形成具有内胚层的类胚体,如通过CXCR4和c-kit的表达所确定的。第二,(d5-d6)使类胚体在用于使类胚体形成前部前肠图式形成的条件下经受前部化培养基。第三,(d6-d20-25)随后在诱导腹侧化和肺芽类器官(LBO)的最终产生的条件下使细胞经受腹侧化/支化培养基。LBO形成通过在出芽上皮结构的尖端上发生音猬因子(SHH)表达确定(参见图6E)。
在培养过程的d20-d25之间产生LBO后,然后选择具有折叠结构的类器官并以夹层构造嵌入基质胶中。折叠结构包括EPCAM+KRT8+ECAD+FOXAl/2+AFE细胞的折叠片(FOXA2:89.07%±3.36%,EPCAM+:92.08%±1.88%,n=3;RUES2 ESC)(图1C)。形成夹层包括在孔或其他合适的容器中加入第一量的基质胶,并使其固化以形成夹层的底部部分。使所选择的具有折叠结构的类器官与基质胶混合并置于底部部分的顶部并使其固化以形成中心细胞层。将不含细胞的另一量的基质胶置于嵌入细胞层的顶部并使其固化以形成夹层的顶部部分。将腹侧化培养基/支化培养基置于孔中且定期补充。在嵌入基质胶中一周之后从类器官中产生分支芽。在嵌入后2-3周观察到大量支化类器官。
作为上文论述的基质胶的替代方案,可实施其他凝胶基质,诸如Gjorevsky等人,Nature.2016年11月24日;539(7630):560-564.doi:10.1038/nature20168和DiMarco等人,Biomater Sci.2015年10月15日;3(10):1376-85中所描述的那些。
实施例
实施例1:方法
试剂
所使用的试剂列于下表1中。
人类样品
对由哥伦比亚转化免疫学中心的人类研究中心获得的人类胎儿组织的使用获得了哥伦比亚大学医学中心(CUMC)人类研究评审委员会的批准,并根据批准的方案进行实验。
培养基
hPSC维持培养基由补充有以下各者的DMEM/F12(1:1)组成:20%敲除血清替代物、0.1mMβ-巯基乙醇、1ml Primocin、5ml非必需氨基酸、5ml GlutaMax和20ng/ml FGF-2。无血清分化(SFD)培养基由补充有以下各者的IMDM/Ham氏F12(3:1)组成:N2、B27、0.05%牛血清白蛋白、1%青霉素-链霉素、50μg/ml抗坏血酸、2mM Glutamax、0.4μM单硫代甘油和不同的生长因子混合物,如表2中所示。
hPSC维持
将洛克菲勒大学胚胎干细胞系2(RUES2,NIH批准号NIHhESC-09-0013,注册号0013,第17-28代)、仙台病毒和来自健康人类真皮成纤维细胞的经修饰的mRNA生成hiPSC系7,9(第16-25代)和IRF7缺陷型C12 hiPSC系28维持在以15,000-18,000个细胞/cm2涂铺的小鼠胚胎成纤维细胞(MEF)上。将细胞在hPSC维持培养基中培养,每天更换培养基。将hPSC用Accutase/EDTA传代,洗涤并以1:48的稀释度重新涂铺。将培养物保持在37℃下的潮湿的5% CO2气氛中。对各系进行核型测定,并每6个月使用PCR验证支原体污染。
内胚层诱导
如前所述进行内胚层的诱导9。简单来说,将MEF通过传代到提供有hPSC维持培养基并保持在37℃下的潮湿的5% CO2气氛中的基质胶上24小时来耗尽。在MEF耗尽后,在低吸附板中的类胚体/原条形成培养基(表2)中进行原条和类胚体诱导,持续12-16小时,之后换到内胚层诱导培养基(表2),持续36-40小时。每天对类胚体给料且将其维持在37℃下的潮湿的5% CO2/5% O2气氛中。通过CXCR4和c-KIT的表达测定内胚层产率。对于iPS系,使用人类CD 184(CXCR4)微珠试剂盒纯化内胚层细胞。在所有实验中使用的细胞具有>90%内胚层产率。
前部前肠内胚层诱导
如前所述诱导前部前肠内胚层9。在第4天,用0.05%胰蛋白酶/EDTA使类胚体解离且将其以80,000-105,000个细胞/cm2的密度涂铺在纤连蛋白涂布的多孔板上。将细胞在前部化培养基-1中孵育24小时,之后转到前部化培养基-2中再孵育24小时。
肺芽类器官的形成
在前部前肠内胚层诱导结束时,用腹侧化培养基(支化培养基)处理细胞48小时并观察到三维团块形成。随后通过在孔周围轻缓吹吸使所述团块悬浮。悬浮团块在下文中称为肺芽类器官(LBO)。将LBO维持在浸没在支化培养基中的非组织培养物处理的多孔板中,并且每隔一天给料直至d20-d25。
基质胶中的支化形态发生
将d20-d25 LBO嵌入24孔穿透小室(transwell insert)中的100%基质胶中且在孵育箱中孵育直到基质胶固化。将支化培养基添加到孔,之后插入穿透小室,也将支化培养基添加到穿透小室中。每隔一天更换培养基。描述在基质胶中LBO和LBO来源的支化群落的生成的逐步方案可见于实施例2中。
免疫荧光染色
将LBO和支化基质胶培养物新鲜嵌入最佳切割温度(OCT)中。将样品切成5-8μm,然后空气干燥2小时。将切片用4%多聚甲醛在室温(RT)下固定20分钟,并用DPBS洗涤5分钟。将切片用0.3% Triton X-100/PBS透化30分钟,然后在5%驴血清中封闭1小时。将一级抗体(表3)在4℃下孵育过夜。第二天,用DPBS洗涤切片3x5分钟,然后在室温下用二级抗体(表3)孵育2小时,用DPBS洗涤3x10分钟,然后用含有DAPI的荧光封固剂固定。为了3D成像,如上所述对D25LBO染色,但以完整类器官染色。
从LBO分离EPCAM+和EPCAM-群体
通过0.05%胰蛋白酶/EDTA解离LBO。在4℃下用APC-缀合的EPCAM对细胞染色。使用BD流式细胞分选仪(San Jose,CA),通过荧光激活细胞分选(FACS)分离EPCAM+和EPCAM-细胞。
RNAseq
使用Direct-zolTMRNA MicroPrep试剂盒纯化来自LBO的总RNA。使用Agilent微流体RNA 6000纳米芯片试剂盒(Agilent Technologies,Santa Clara,CA)在2100生物分析仪(Agilent Technologies,Santa Clara,CA)上测定RNA浓度和RNA完整数(RIN)。将RIN大于9的那些样品用于RNAseq。使用聚-A-拉下从总RNA样品富集mRNA。使用Illumina TruSeq RNA制备试剂盒(Illumina,San Diego,CA)制备文库。随后使用哥伦比亚基因组中心的Illumina HiSeq2000(Illumina,San Diego,CA)对文库测序。使样品在每个泳道中多路复用,使得每个样品产生目标数量的单端/双端100bp读段,作为整个泳道1.8亿读段的一部分。将RTA(Illumina,San Diego,CA)用于碱基识别且bc12fastq(版本1.8.4)用于将BCL转化为fastq格式,加上衔接子修剪。使用具有4个错配和10个最大多次命中的Tophat(版本2.0.4)将读段作图到参考基因组(NCBI/build37.2)。为了解决来自外显子-外显子连接的读段的作图,Tophat从头开始推断新的外显子-外显子连接,并将它们与来自已知mRNA序列的连接结合作为参考注释。我们使用具有默认设置的cufflinks(版本2.0.2)评估基因和剪接同种型的相对丰度。我们使用DEseq测试了在不同条件下差异表达的基因,DEseq是基于负二项分布的R包,其对来自RNAseq实验和差异表达测试的数字读数进行建模。
原位杂交
使用地高辛(digoxigenin;DIG)-UTP标记的SHH核糖核酸探针在冷冻切片(5-8μm)上进行原位杂交。简单来说,将人类成人肺组织cDNA用作模板以产生含有T7或T3启动子序列的SHH PCR产物(正向:AATTAACCCTCACTAAAGGGACAGCTCGGAAGTCATCAGTT;反向:TAATACGACTCACTATAGGGGCCTCTGAGTGGTGGCCATCTT)。使用PCR产物作为模板以使用T7MAXIscript试剂盒(Ambion)产生SHH核糖核酸探针,然后使用RNeasy微试剂盒(Qiagen)来清洁核糖核酸探针。将不同期的LBO新鲜嵌入OCT。将样品切成5-8μm,然后在室温下用4%多聚甲醛固定20分钟。将切片用DEPC-DPBS洗涤3x5分钟并在乙酰化缓冲液(584μl三乙醇胺/50ml DEPC-H2O/125μl乙酸酐)中乙酰化10分钟。在0.1% Triton X-100/PBS中在室温下进行透化30分钟,然后用DEPC-DPBS洗涤3x5分钟。将切片与杂交缓冲液(5%硫酸葡聚糖/4x SSC/50%甲酰胺/1x Denhardt氏/5%鱼精DNA)一起在室温下孵育至少2小时,然后与200ng/ml DIG标记的SHH探针一起在杂交缓冲液中在72℃下孵育过夜。第二天,将切片与预先温热至72℃的0.2x SSC一起孵育2小时,然后冷却至室温持续30分钟。将切片用新鲜的0.2x SSC洗涤5分钟,然后用PBS洗涤另外5分钟。将切片与封闭溶液(2%绵羊血清/TBST)一起孵育1小时,然后在4℃下与抗DIG-AP Ig一起孵育过夜。将切片用TBST洗涤3x10分钟,并在有色反应缓冲液(100mM Tris,pH 9.5/0.1% Tween-20/100mM NaCl/50mM MgCl2)中冲洗10分钟。通过将切片与BM-紫一起孵育来使颜色显色。
小鼠肾包膜移植
将NOD.Cg-Prkdescid.I12rgtm1Wjl/SzJ(NSG)小鼠圈养在专门的无病原体小鼠设施中。所有小鼠在10-13周龄时使用,且未选择性别。设置实验以在每个时间点使用5-7只小鼠。未使用统计学方法预先确定样品大小。未对实验进行随机化。根据哥伦比亚大学机构动物管理及使用委员会批准的方案进行实验和动物护理。在手术前将一百万个d20-d25 LBO细胞与5μl基质胶混合并植入肾包膜下。切除生长物,将其新鲜嵌入OCT中以用于免疫荧光或固定在4%多聚甲醛中以用于石蜡包埋。使用苏木精/伊红染色分析组织学。
斑点印迹
将从5个月移植物的管状结构吸出的3微升流体沉积在硝酸纤维素印迹膜(GEHealthcare Life Sciences)上。将斑点印迹膜空气干燥5分钟,并在5%牛奶/PBS中封闭1小时,且然后用指定的一级抗体(表3)在4℃下探测过夜。将HRP缀合的二级抗体施加至膜,然后用ECL蛋白质印迹检测药剂进行信号检测并暴露于X射线胶片。
成像
使用机动化Leica DMI6000 B(Leica Microsystems,Buffalo Grove,IL)或DMi8(Leica Microsystems,Buffalo Grove,IL)倒置显微镜或双光子共聚焦激光扫描显微镜Leica TCS SP8(Leica Microsystems,Buffalo Grove,IL)对样品成像。使用iPhone 6(型号:MG5A2LL/A,Apple,Cupertino,CA)拍摄宏观图像。
活LBO中SPB-BODIPY的摄入和量化
将d170 LBO用CellMaskTM深红质膜染色剂染色10分钟并洗涤5次,然后在加载SPB-BODIPY之前成像以获得本底荧光水平(0分钟)。然后用位于基质胶正上方的20ng/ml纯化的人类SPB-BODIPY蛋白装载培养物(每种培养物总共10ng)。使用双光子共聚焦激光扫描显微镜(Leica TCS SP8)每2分钟拍摄图像,并使用Leica Application Suite X定量荧光强度。在统计分析之前从所有测量值中减去本底荧光值。
免疫荧光的量化
使用ImageJ定量每个核标记物的图像。简单来说,将图像转换为8位图像,并调整阈值以与核染色相对应,这允许测量总面积。通过ImageJ的“分析颗粒”功能分析总面积。通过将阳性细胞的总面积除以DAPI的总面积来计算阳性细胞的百分比。为了细胞外基质定量,使用Leica Application Suite X定量荧光强度。在统计分析之前,将所述值归一化至每个单独实验的RUES2对照。
透射电子显微术
透射电子显微术(TEM)在纽约大学朗格尼医学中心显微术中心进行。将LBO用0.1M二甲胂酸钠缓冲液(pH 7.2)中的2.5%戊二醛固定2小时,并在室温下用1%四氧化锇后固定1.5小时,然后以标准方式处理并嵌入EMbed 812(Electron Microscopy Sciences,Hatfield,PA)。将半薄切片切成1mm并用1%甲苯胺蓝染色以评价保存质量并找到所关注区域。切割超薄切片(60nm),将其固定在铜网上,并通过标准方法用乙酸铀酰和柠檬酸铅染色。将染色网在Philips CM-12电子显微镜下检查,并用Gatan(4kx2.7k)数码相机(Gatan,Inc.,Pleasanton,CA)拍照。
呼吸道合胞体病毒制备和感染
通过用来自pDsRed的野生型Discosoma REP基因替换增强的绿色荧光蛋白基因,由全长RSV质粒1,MP224产生表达重组红色荧光蛋白(RFP)的RSV A2(rrRSV)。对于细胞维持,使HEp-2细胞(ATCC编号CCL-23)和Vero细胞(ATCC编号CCL-81)在单层培养物中生长,并在37℃下的5% CO2的潮湿气氛中,维持在补充有10%胎牛血清(FCS)和2mM L-谷氨酰胺的DMEM中。在HEp-2细胞(ATCC编号CCL-23)中制备病毒原液。简单来说,使HEp-2细胞生长过夜,用OptiMEM洗涤,且用rrRSV接种。在2.5小时的吸附期后,将细胞在补充有1% FCS的DMEM中孵育3天。通过一次冻融循环收获病毒,然后在3,500r.p.m.下进行澄清离心并储存在-80℃下。使用2%甲基纤维素覆盖物、5%(v/v)甲醛固定和第5天结晶紫染色(0.015%w/v),通过Vero细胞中的噬斑测定测定病毒滴度。对于d170 LBO的RSV感染,将1ml中107个噬斑形成单位(PFU)的RSV直接添加到各孔中每种基质胶培养物上,并在37℃下孵育3小时。然后移除RSV接种物,并用SFD培养基洗涤培养物5次持续5分钟,并维持在支化培养基中。在指定的时间点收集培养物,以使用抗RSV(所有抗原)抗体(Meridian Life Science,B65890G)进行全标本染色。使用倒置显微镜或双光子共聚焦激光扫描显微镜Leica TCS SP8(LeicaMicrosystems,Buffalo Grove,IL)拍摄图像。
数据可用性
支持本研究发现的RNA测序数据集可从Sequence Read Archive(SRA)获得。d25LBO测序的SRA登录号为SRP073749且d170 LBO的为SRR4295269。
统计学数据和再现性
使用未配对的双尾学生t检验或单因素ANOVA进行统计学分析,适当时使用Prism7。结果显示为平均值±s.e.m,p值<0.05视为具有统计学显著性。除非另有说明,否则N值是指生物学上独立的重复。研究人员在动物研究中的实验和结果评估期间对分配不知情,因为没有进行统计学分析。
实施例1的其他参考文献
1.Hallak LK,Spillmann D,Collins PL,Peeples ME.Glycosaminoglycansulfation requirements for respiratory syncytial virus infection.J Virol2000;74:10508-10513.
实施例2:用于生成三维肺芽类器官和其衍生的支化群落的详细方案.
该方案描述了将人类多能干细胞(hPSC)定向分化为能够支化形态发生的三维肺芽类器官(LBO)。基于我们组先前公布的2D方案^1-3^,我们设计了一个3D系统,其中hPSC在粘附2D培养物中依序分化为定型内胚层(DE)、前部前肠内胚层(AFE)和腹侧AFE,然后进行悬浮培养以允许LBO形成。当在第25天涂铺在基质胶中时,LBO经历了大量向外支化并最终形成扩张尖端,使人联想到在肺发育的囊状期形成的小囊。这些培养物可用于研究人类肺发育和支化形态发生。
类器官是由多种细胞类型构成的结构,其在空间组织上与器官类似并且重现至少一些特定的器官功能^4^。已经描述了几种类型的类器官,其衍生自成人组织和多能干细胞。该技术可能将对发育生物学、器官生理学和功能以及疾病建模的研究产生重大影响^5,6^。然而,真正的人类肺类器官模型目前尚未实现。呼吸系统由逐渐更小的气道的复杂分支系统组成,所述气道在发生气体交换的肺泡中终止^7,8^。先前已经报道了人类肺类器官的生成^9,10^。然而,所描述的类器官未显示支化形态发生或靠近远端特化,而未记录功能。当前方案中描述的肺芽类器官(LBO)模型显示支化形态发生、靠近远端特化以及早期肺泡发生的证据(体内和体外)。它们的发育达到等效于人类发育的妊娠中期的时期。基质胶中LBO衍生的支化结构含有2型肺泡上皮细胞(AT2),其具有大量板层小体并且能够在体外摄取和释放表面活性蛋白。此外,在异种移植后证实了黏蛋白和表面活性蛋白的分泌以及纤毛运动。因此,该方案产生的LBO满足真正类器官的定义,并且可用于研究人类肺部发育并可能用于对人类肺病进行建模。
试剂:
/>
/>
装置:
常氧孵育箱(95%空气/5% CO2)
低氧孵育箱(5% O2/5% CO2)
离心机
血球计
采摘罩
程序:
*在基质胶上的MEF耗尽(d-1)*
1.在冰上解冻基质胶,并将冰桶与基质胶一起放置在4℃过夜。
2.在冰冷的IMDM中稀释基质胶(1:30)。
3.向每个10cm2组织培养处理的培养皿中加入6ml稀释的基质胶溶液,且使其在室温下放置至少3小时或在4℃下放置过夜。
4.为了制备一个6孔板类胚体(EB),使用1ml/孔的Accutase解离人类多能干细胞(hPSC)的两个汇合孔(来自6孔板),并在含常氧孵育箱中孵育2-3分钟。
5.抽吸Accutase。
6.通过终止培养基中和酶。
7.通过在1,400r.p.m.下离心4分钟使解离的细胞成团粒。
8.尽可能吸出酶和终止培养基。
9.用10-12ml hPSC维持培养基再悬浮细胞。
10.在从培养皿中吸出上清液后,将细胞涂铺在基质胶-涂布的培养皿中(参见步骤3)。
11.将细胞在常氧孵育箱中孵育过夜。
内胚层诱导(d0-d4)
1.在第0天,从基质胶涂布的培养皿中移除hPSC维持培养基并加入3ml胰蛋白酶。将该培养皿在常氧孵育箱中孵育1-1.5分钟。
2.吸出胰蛋白酶溶液,并通过加入10ml终止培养基终止剩余的酶。
3.收集脱落的细胞并通过在1,400r.p.m.下离心4分钟使其成团粒。
4.吸出酶和终止培养基。
5.用12ml类胚体/原条形成培养基再悬浮细胞,并分配到6孔低吸附板(2ml/孔)。
6.将低吸附板置于低氧孵育箱中以允许类胚体(EB)形成。
7.在12-16小时后,将所有EB收集在15ml管中,并在800r.p.m.下离心1分钟。
8.吸出类胚体/原条形成培养基。
9.用12ml内胚层诱导培养基轻缓再悬浮EB,并将它们均匀地分配回低吸附板(2ml/孔)。
10.将板返回到低氧孵育箱。
11.在第2天,向每个孔中加入1ml新鲜的内胚层诱导培养基。
12.在第3天,向每个孔中加入2ml新鲜的内胚层诱导培养基。
13.在第4.1天-第4.3天,通过CXCR4和c-kit表达的流式细胞术分析检查内胚层产率。如果内胚层产率>90%,则继续分化。
前部化(d5-d6)
1.通过在DPBS中将纤连蛋白稀释至0.2%(体积/体积,1:500,4μg/ml)来制备纤连蛋白-涂布的6孔板。向每个孔中加入2ml纤连蛋白/DPBS溶液,并将板在常氧孵育箱中孵育至少30分钟或4℃过夜。
2.用胰蛋白酶将EB解离成单细胞(每个6孔EB板3ml胰蛋白酶,最多4分钟消化)。
3.通过终止培养基中和酶。
4.使用血球计对细胞计数。
5.通过在1,400r.p.m.下离心4分钟使解离的细胞成团粒。
6.吸出终止培养基。
7.用7.5x 10^5^个细胞/2ml的前部化培养基-1再悬浮细胞。
8.向每个孔中加入2ml细胞混合物(6孔板,纤连蛋白涂布,参见步骤1)。
9.在常氧孵育箱中孵育板。
10.在24小时(±1小时)后,用前部化培养基-2(2ml/孔)替换前部化培养基-1。
11.将培养板返回到常氧孵育箱。
腹侧化和肺芽类器官(LBO)形成(d6-d25)
1.在24小时(±1小时)之后,用腹侧化培养基/支化培养基置换前部化培养基-2(2ml/孔)。
2.将培养板返回到常氧孵育箱。
3.48小时后,吸出所有腹侧化培养基/支化培养基,并向每个孔中加入2ml新鲜的腹侧化培养基/支化培养基。
4.使用P1000吸头轻缓地在整个孔中上下吹吸使类器官悬浮。
5.将悬浮的类器官转移到非组织培养物处理的板。
6.将板返回到常氧孵育箱。
7.每隔一天通过倾斜板并使类器官下沉到底部边缘来对类器官给料。在避免接触类器官的同时移除旧培养基。向每个孔中加入2ml新鲜的腹侧化培养基/支化培养基。
支化类器官(第20天-实验结束)
1.在d20-d25之间,在采摘罩下选择具有折叠结构的类器官。
2.将每个小室中所需数量的类器官放入每个孔中(96孔U形底板,每孔含有100μl新鲜的腹侧化培养基/支化培养基)。通常,每个小室(24孔小室)涂铺一至四个类器官。
3.将24孔小室置于非组织培养物处理的板中。
4.将50μl 100%冷基质胶放入每个小室的底部。
5.等待5分钟或直到基质胶固化。
6.一次一个孔地移除腹侧化培养基/支化培养基。
7.使类器官与30μl 100%冷基质胶轻缓混合,以避免产生气泡。
8.立即将类器官-基质胶混合物置于小室的中心。
9.等待5分钟以使基质胶固化以将类器官固定在小室的中心。
10.在小室中再加入50μl 100%冷基质胶,制成基质胶夹层。
11.将板置于常氧孵育箱中10分钟,以确保所有基质胶固化。
12.向小室中加入500μl的腹侧化培养基/支化培养基,并将另外500μl的腹侧化培养基/支化培养基加入孔中。
13.在常氧孵育箱中孵育培养物,并每2-3天更换培养基。
定时:
每个步骤的动手时间:
基质胶上的MEF耗尽(d-1):20分钟
内胚层诱导(d0-d4):2小时
前部化(d5-d6):1小时
腹侧化和肺芽类器官(LBO)形成:30分钟加类器官悬浮:5分钟/板
支化类器官:大约2小时完成嵌入24个小室并为其提供培养基。
预期结果:
使用该分化方案,在换到腹侧化培养基/支化培养基后2天(方案的第8天)将形成将成为类器官的粘附团块。早在d10-d12就在悬浮类器官内出现折叠结构。在嵌入基质胶后一周,从类器官产生分支芽。在嵌入后2-3周观察到大量支化类器官。在早期类器官形成期间,不同的细胞系表现不同。在类器官悬浮之前,几种iPS系在第8天倾向于没有明显粘附团块。然而,它们在悬浮后形成了类器官,并且它们在基质胶中支化。
实施例2的另外参考文献:
1 Green,M.D.et al.Generation of anterior foregut endoderm from humanembryonic and induced pluripotent stem cells.Nat Biotechnol 29,267-272,doi:10.1038/nbt.1788(2011).
2 Huang,S.X.et al.The in vitro generation of lung and airwayprogenitor cells from human pluripotent stern cells.Nat Protoc 10,413-425,doi:10.1038/nprot.2015.023(2015).
3 Huang,S.X.et al.Efficient generation of lung and airway epithelialcells from human pluripotent stem cells.Nat Biotechnol 32,84-91,doi:10.1038/nbt.2754(2014).
4 Lancaster,M.A.&Knoblich,J.A.Organogenesis in a dish:modelingdevelopment and disease using organoid technologies.Science 345,1247125,doi:10.1126/science.1247125(2014).
5 Fatehullah,A.,Tan,S.H.&Barker,N.Organoids as an in vitro model ofhuman development and disease.Nat Cell Biol 18,246-254,doi:10.1038/ncb3312(2016).
6 Clevers,H.Modeling Development and Disease with Organoids.Cell 165,1586-1597,doi:10.1016/j.cell.2016.05.082(2016).
7 Herriges,M.&Morrisey,E.E.Lung development:orchestrating thegeneration and regeneration of a complex organ.Development 141,502-513,doi:10.1242/dev.098186(2014).
8 Morrisey,E.E.&Hogan,B.L.Preparing for the first breath:genetic andcellular mechanisms in lung development.Dev Cell 18,8-23,doi:10.1016/j.devcel.2009.12.010(2010).
9 Dye,B.R.et al.A bioengineered niche promotes in vivo engraftmentand maturation of pluripotent stem cell derived human lung organoids.Elife 5,doi:10.7554/eLife.19732(2016).
10 Dye,B.R.et al.In vitro generation or human pluripotent stem cellderived lung organoids.Elife4,doi:10.7554/eLife.05098(2015).
实施例3:RUES2-HPS1系的生成和表征
在哥伦比亚大学医学中心的干细胞核心设施中生成RUES2-HPS1系。简单来说,将RUES2细胞(第25代)在涂有基质胶的六孔板中培养至70-80%汇合度。使用Nucleofector4D,用每个6孔板的孔7.5μg HPS1引导RNA质粒(pX330,Addgene Plasmid#42230)加上2.5μgCas9mCherry对细胞进行电穿孔。Cas9mCherry衍生的mCherry用作荧光标记物以分选转染的细胞。在转染后24小时,使用具有Bio-Rad S3e细胞分选仪的FACS分选细胞,并以约2,000个细胞/6cm培养皿接种在MEF进料机上。在分选后7-10天挑选群落。分离来自单独克隆的基因组DNA,并使用HPS1特异性PCR引物(HPS1-F-1(GTAGAGGCAGCAGATCCAAGAGG)和HPS1-R-1(GAACAAGGTGGTCCACACA)。预期为420bp条带)对其进行基因分型。使用In-Fusion反应(Clontech,Mountain View,CA)将PCR产物克隆到质粒中以获得合适的序列。测序揭示每个等位基因中的提前终止密码子(图10)。实施上述技术以产生携带HPS2(图13,AP3B1,外显子4))、HPS8(图14,BLOC1S3外显子2)、SFPTC(图15,SFTPC外显子2)或端粒酶(图16,TERC,外显子1)基因中的突变的细胞系。鉴于本文的教示,本领域技术人员将理解,可以产生许多携带所需突变的其他细胞系,然后将其生长成LBO以供进一步研究、评价和筛选。
羟脯氨酸含量
根据制造商的方案(Sigma,MAK008-1KT)测量羟脯氨酸含量。简单来说,用组织玻璃Teflon dounce匀化器(含10mg样品的100μl水)均匀化来自RUES2或RUES2-HPS1培养物的样品,并将其转移至压力密封小瓶,之后每10毫克样品加入100μl浓盐酸(约12M)。将混合物在120℃下水解3小时。将样品在96孔板中在60℃下干燥,然后在室温下用氯胺T/氧化缓冲液混合物干燥5分钟,并在60℃下用DMAB试剂再干燥90分钟。在560nm下测量羟脯氨酸含量。将相同量的基质胶用作对照。
使用KeyGenes比较分析
使用KeyGenes将从来自RUES2、C12、HDF SV和HDF mRNA系的d170 LBO获得的RNAseq数据与不同的妊娠早期和妊娠中期以及成体器官(包括肺)进行比较。使用Cluster3.0进行d170 LBO的12个样品和来自妊娠中期的19个器官的75个样品的分层聚类,并通过TreeView对其进行观察。通过KeyGenes计算87个分类基因。
表1.试剂
/>
表2.培养基
表3.抗体和稀释液
用于免疫荧光染色的抗体
/>
用于蛋白质印迹的抗体
参考文献
上标中的参考文献列在参考文献列表1中,并且下标中的参考文献列在参考文献列表2中。
参考文献列表1:
1 Lancaster,M.A.&Knoblich,J.A.Organogenesis in a dish:modelingdevelopment and disease using organoid technologies.Science 345,1247125,doi:10.1126/science.1247125(2014).
2 Collins,P.L.,Fearns,R.&Graham,B.S.Respiratory syncytial virus:virology,reverse genetics,and pathogenesis of disease.Current topics inmicrobiology and immunology 372,3-38,doi:10.1007/978-3-642-38919-1_1(2013).
3 Johnson,J.E.,Gonzales,R.A.,Olson,S.J.,Wright,P.F.&Graham,B.S.Thehistopathology of fatal untreated human respiratory syncytial virusinfection.Mod Pathol 20,108-119,doi:10.1038/modpathol.3800725(2007).
4 Mulugeta,S.,Nureki,S.&Beers,M.F.Lost aftertranslation:insights frompulmonary surfactant for understanding the role of alveolar epithelialdysrunction and cellular quality control in fibrotc lung disease.Americanjournal of physiology .Lung cellular and molecular physiology 309,L507-525,doi:10.1152/ajplung.00139.2015(2015).
5 Vicary,G.W.,Vergne,Y.,Santiago-Cornier,A.,Young,L.R.&Roman,J.Pulmonary Fibrosis in Hermansky-Pudlak Syndrome.Ann Am Thorac Soc,doi:10.1513/AnnalsATS.201603-186FR(2016).
6 Herriges,M.&Morrisey,E.E.Lung development:orchestrating thegeneration and regeneration of a complex organ.Development 141,502-513,doi:10.1242/dev.098186(2014).
7 Morrisey,E.E.&Hogan,B.L.Preparing for the first breath:genetic andcellular mechanisms in lung development.Dev Cell 18,8-23,doi:S1534-5807(09)00527-9[pii]10.1016/j.devcel.2009.12.010(2010).
8 Dye,B.R.etal.In vitro generation of human pluripotent stem cellderived lung organoids.eLife 4,doi:10.7554/eLife.05098(2015).
9 Dye,B.R.et al.A bioengineered niche promotes in vivo engraftmentand maturation of pluripotent stem cell derived human lung organoids.eLife 5,doi:10.7554/eLife.19732(2016).
10 Huang,S.X.et al.Efficient generation of lung and airway epithelialcells from human pluripotent stem cells.Nature biotechnology 32,84-91,doi:10.1038/nbt.2754(2014).
11 Green,M.D.et al.Generation of anterior foregut endoderm from humanembryonic and induced pluripotent stem cells.Nature biotechnology29,267-272,doi:10.1038/nbt.1788(2011).
12 Huang,S.X.et al.The in vitro generation of lung and airwayprogenitor cells from human plu ripotent stem cells.Nature protocols10,413-425,doi:10.1038/nprot.2015.023(2015).
13 Kumar,M.E.et al.Mesenchymal cells.Defining a mesenchymalprogenitor niche at single-cell resolution.Science 346,1258810,doi:10.1126/science.1258810(2014).
14 Hrycaj,S.M.et al.Hox5 Genes Regulate the Wnt2/2b-Bmp4-SignalingAxis during Lung Development.Cell reports 12,903-912,doi:10.1016/jcelrep.2015.07.020(2015).
15 Liu,L.et al.Hedgehog signaling in neonatal and adult lung.Americanjournal of respiratory cell and molecular blology 48,703-710,doi:10.1165/rcmb.2012-0347OC(2013).
16 Bellusci,S.et al.Involvement of Sonic hedgehog(Shh)in mouseembryonic lung growth and morphogenesis.Development 124,53-63(1997).
17 Pepicelli,C.V.,Lewis,P.M.&McMahon,A.P.Sonic hedgehog regulatesbranching morphogenesis in the mammalian lung.Current biology:CB 8,1083-1086(1998).
18 Que,J.et al.Multipledose-dependent roles for Sox2 in thepatterning and differentiation of ante rior foregut endoderm.Development 134,2521-2531,doi:10.1242/dev.003855(2007).
19 Alanis,D.M.,Chang,D.R.,Akiyama,H.,Krasnow,M.A.&Chen,J.Two nesteddevelopmental waves demarcate a compartment boundary in the mouse lung.Naturecommunications 5,3923,doi:10.1038/ncomms4923(2014).
20 Treutlein,B.et al.Reconstructing lineage hierarchies of the distallung epithelium using single-cell RNA-seq.Nature 509,371-375,doi:10.1038/nature13173(2014).
21 Sakurai,J.et al.Differential expression of the glycosylated formsof MUC1 during lung development.Eur J Histochem 51,95-102(2007).
22 Cutz,E.,Pan,J.,Yeger,H.,Domnik,N.J.&Fisher,J.T.Recent advances andcontraversies on the role of pulmonary neuroepithelial bodies as airwaysensors.Seminars in cell&developmental biology 24,40-50,doi:10.1016/j.semcdb.2012.09.003(2013).
23 Ban,N.et al.ABCA3 as a lipid transporter in pulmonary surfactantbiogenesis.The Journal of biological chemistry 282,9628-9634,doi:10.1074/jbc.M611767200(2007).
24 Ciancanelli,M.J.et al.Life-threatening influenza and impairedinterferon ampliffcation in human IRF7 deficiency.Science,doi:10.1126/science.aaa1578(2015).
25 Whitsett,J.A.,Wert,S.E.&Weaver,T.E.Diseases of pulmonarysurfactant homeostasis.Annual review of pathology 10,371-393,doi:10.1146/annurev-pathol-012513-104644(2015).
26 Jain,R.et al.Plasticity of Hopx(+)type I alveolar cells toregenerate type II cells in the lung.Nature communications 6,6727,doi:10.1038/ncomms7727(2015).
27 Liu,Y.&Hogan,B.L.Differential gene expression in the distal tipendoderm of the embryonic mouse lung.Gene expression patterns:GEP 2,229-233(2002).
28 Rockich,B.E.et al.Sox9 plays multiple roles in the lung epitheliumduring branching morphogenesis.Proc Natl Acad Sci U S A 110,E4456-4464,doi:10.1073/pnas.1311847110(2013).
29 Perl,A.K.,Kist,R.,Shan,Z.,Scherer,G.&Whitsett,J.A.Normal lungdevelopment and function after Sox9 inactivation in the respiratoryepithelium.Genesis 41,23-32,doi:10.1002/gene.20093(2005).
30 Roost,M.S.et al.KeyGenes,a Tool to Probe Tissue DifferentiationUsing a Human Fetal Transcriptional Atlas.Stem cell reports 4,1112-1124,doi:10.1016/jstemcr.2015.05.002(2015).
31 Florin,T.A.,Plint,A.C.&Zorc,J.J.Viral bronchiolitis.Lancet,doi:10.1016/S0140-6736(16)30951-5(2016).
32 Simoes,E.A.et al.Challenges and opportunities in developingrespiratory syncytial virus therapeutics.The Journal of infectious diseases211 Suppl 1,S1-S20,doi:10.1093/infdis/jiu828(2015).
33 Liesman,R.M.et al.RSV-encoded NS2 promotes epithelial cellshedding and distal airway obstruction.J Clin Invest 124,2219-2233,doi:10.1172/JCI72948(2014).
34 Huizing,M.,Helip-Wooley,A.,Westbroek,W.,Gunay-Aygun,M.&Gahl,W.A.Disorders of lysosome-related organelle biogenesis:clinical and moleculargenetics.Annu Rev Genomics Hum Genet 9.359-386,doi:10.1146/annurev.genom.9.081307.164303(2008).
35 Ryu,J.H.et al.Idiopathic pulmonary fibrosis:evolving concepts.MayoClinic proceedings 89,1130-1142,doi:10.1016/j.mayocp.2014.03.016(2014).
36 Young,L.R.et al.The alveolar epithelium determines susceptibilityto lung fibrosis in Hermansky-Pudlak syndrome.Am J Respir Crit Care Med 186,1014-1024,doi:10.1164/rccm.201207-1206OC(2012).
37 Armanios,M.Telomerase and idiopathic pulmonary fibrosis.Mutationresearch730,52-58,doi:i0.1016/j.mrfmmm.2011.10.013(2012).
38 Short,K.,Hodson,M.&Smyth,I.Spatial mapping and quantification ofdevelopmental branching morphogenesis.Development 140,471-478,doi:10.1242/dev.088500(2013).
39 Peng,T.et al.Coordination of heart and lung co-development by amultipotent cardiopulmonary progenitor.Nature 500,589-592,doi:10.1038/nature12358(2013).
参考文献列表2
1.Noble,P.W.,Barkauskas,C.E.&Jiang,D.Pulmonary fibrosis:pattems andPerpetrators.J Clin Invest 122,2756-2762(2012).
2.Ryu,J.H.et al.Idiopathic pulmonary fibrosis:evolving concepts.MayoClinic proceedings 89,1130-1142(2014).
3.McCurry,K.R.et al.Lung transplantation in the United States,1 998-2007.Am J Transplant 9,942-958(2009).
4.Lawson,W.E.et al.Endoplasmic reticulum stress enhances fibroticremodeling in the lungs.Proc Natl Acad Sci U S A 108,10562-10567(2011).
5.Taniore,H.,Blackwell,T.S.&Lawson,W.E.Emerging evidence forendoplasmic reticulum stress in the pathogenesis of idiopathic pulmonaryfibrosis.American joumal of physiology.Lung cellular and molecular physiology302,L721-729(2012).
6.Araya,J.et al.Insufficient autophagy in idiopathic pulmonaryfibrosis.American joumal of physiology.Lung cellular and molecular physiology304,L56-69(2013).
7.Bueno,M.et al.PINK1 deficiency impairs mitochondrial homeostasisand promotes lung fibrcsis.J Clin Invest(2014).
8.Margaritopoulos,G.A.et al.Self-eating:friend or foe?The emergingrole of autophagy in idiopathic pulmonary fibrosis.BioMed researchinternational 2013,420497(2013).
9.Patel,A.S.et al.Autophagy in idiopathic pulmonary fibrosis.PLoS One7,e41394(2012).
10.Lawson,W.E.et al.Genetic mutations in surfactant protein C are arare cause of sporadic cases of IPF.Thorax 59,977-980(2004).
11.Nogee,L.M.et al.A mutation in the surfactant protein C geneassociated with familial interstitial lung disease.The New England journal ofmedicine 344,573-579(2001).
12.Thomas,A.Q.et al.Heterozygosity for a surfactant Protein C genemutation associated with usual interstitial pneumonitis and cellularnonspecific interstitial pneumonitis in one kindred.Am J Respir Crit Care Med165,1322-1328(2002).
13.Wang,Y.et al.Genetic defects in surfactant protein A2 areassociated with pulmonary fibrosis and lung cancer.American journal of humangenetics 84,52-59(2009).
14.Armanios,M.Telomerase and idiopathic pulmonary fibrosis.Mutationresearch 730,52-58(2012).
15.Loyd,J.E.Pulmonary fibrosis in families.American journal ofrespiratory cell and molecular biology 29,S47-50(2003).
16.Pierson,D.M.et al.Pulmonary fibrosis in hermansky-pudlaksyndrome.a case report and review.Respiration;international review ofthoracic diseases 73,382-395(2006).
17.Mahavadi,P.,Guenther,A.&Gochuico,B.R.Hermansky-Pudlak syndromeinterstitial pneumonia:it′s the epithelium,stupid!Am J Respir Crit Care Med186,939-940(2012).
18.Mahavadi,P.et al.Epithelial stress and apoptosis underlieHermansky-Pudlak syndrome-associated interstitial pneumonia.Am J Respir C ritCare Med 182,207-219(2010).
19.Young,L.R.et al.The alveolar epithelium determines susceptibilityto lung fibresis in Hermansky-Pudlak syndrome.Am J Respir Crit Care Med 186,1014-1024(2012).
20.Young,L.R.,Pasula,R.,Gulleman,P.M.,Deutsch,G.H.&McCormack,F.X.Susceptibility of Hermansky-Pudlak mice to bleomycin-induced type II cellapoptosis and fibrosis.American journal of respiratory cell and molecularbiology 37,67-74(2007).
21.Huizing,M.,Helip-Wooley,A.,Westbroek,W.,Gunay-Aygun,M.&Gahl,W.A.Disorders of lysosome-related organelle biogenesis:clinical and moleculargenetics.Annu Rev Genomics Hum Genet 9,359-386(2008).
22.Luzio,J.P.,Hackmann,Y.,Dieckmann,N.M.&Griffiths,G.M.The biogenesisof lysosomes and lysosome-related organelles.Cold Spring Harbor perspectivesin biology 6,a016840(2014).
23.Alder,J.K.et al.Telomere dysfunetion causes alveolar stem cellfailure.Proc Natl Acad Sci U S A 112,5099-5104(2015).
24.Barkauskas,C.E.et al.Type 2 alveolar cells are stem cells in adultlung.J Clin Invest(2013).
25.Desai,T.J.,Brownfield,D.G.&Krasnow,M.A.Alveolar progenitor andstem cells in lung development,renewal and cancer.Nature 507,190-194(2014).
26.Armanios,M.Y.et al.Telomerase mutations in families withidiopathic pulmonary fibrosis.The New England journal of medicine 356,1317-1326(2007).
27.Alder,J.K.et al.Short telomeres are a risk factor for idiopathicpulmonary fibrosis.Proc Natl Acad Sci U S A 105,13051-13056(2008).
28.Alder,J.K.et al.Ancestral mutation in telomerase causes defects inrepeat addition processivity and manifests as familial pulmonaryfibrosis.PLoS genetics 7,e1001352(2011).
29.Alder,J.K.et al.Exome sequencing identifies mutant TINF2 in afamily with pulmonary fibrosis.Chest(2014).
30.Armanios,M.Telomerase mutations and the pulmonary fibrosis-bonemarrow failure syndrome complex.The New England journal of medicine 367,384;author reply 384(2012).
31.Carmona-Rivera,C.etal.Clinical,molecular,and cellular features ofnon-Puerto Rican Hermansky-Pudlak syndrome patients of Hispanic descent.JInvest Dermatol 131,2394-2400(2011).
32.Wang,L.&Lyerla,T.Histochemical and cellular changes accompanyingthe appearance of lung fibrosis in an experimental mouse model for HermanskyPudlak syndrome.Histochemistry and cell biology 134,205-213(2010).
33.Smith,L.J.,Kaplan,N.B.&Brody,J.Response of normal and beige mousealveolar type 2 cells to lung injury.The American review of respiratorydisease 122,947-957(1980).
34.Chi,E.Y.,Lagunoff,D.&Koehler,J.K.Abnormally largelamellar bodiesin type II pneumocytes in Chediak-Higashi syndrome in beige mice.Laboratoryinyestigation;a journal of technical methods and pathology 34,166-173(1976).
35.Clarke,R.et al.Endoplasmic reticulum stress,the unfolded proteinresponse,autophagy,and the integrated regulation of breast cancer cellfate.Cancer research 72,1321-1331(2012).
36.Klionsky,D.J.et al.Guidelines forthe use and interpretation ofassays for monitoring autophagy.Autophagy 8,445-544(2012).
37.Ashrafi,G.&Schwarz,T.L.The pathways of mitophagy for qualitycontrol and clearance of mitochondria.Cell death and differentiation 20,31-42(2013).
38.Wolff,S.,Weissman,J.S.&Dillin,A.Differential scales of proteinquality control.Cell 157,52-64(2014).
39.Rooney,S.A.Regulation of surfactant secretion.Comparativebiochemistry and physiology.Part A,Molecular&integrative physiology 129,233-243(2001).
40.Whitsett,J.A.,Wert,S.E.&Weaver,T.E.Alveolar surfactant homeostasisand the pathogenesis of pulmonary disease.Annual review of medicine 61,105-119(2010).
41.Huang,S.X.et al.Efficient generation of lung and airway epithelialcells from human pluripotent stem cells.Nature biotechnology 32,84-91(2014).
42.Green,M.D.et al.Generation of anterior foregut endoderm from humanembryonic and induced pluripotent stem cells.Nature biotechnology 29,267-272(2011).
43.Huang,S.X.et al.The in vitro generation of lung and airwayprogenitor cells from human pluripotent stem cells.Nature protocols 10,413-425(2015).
44.Oh,J.et al.Positional cloning of a gene for Hermansky-Pudlaksyndrome,a disorder of cytoplasmic organelles.Nat Genet 14,300-306(1996).
45.Inoue,H.,Nagata,N.,Kurokawa,H.&Yamanaka,S.iPS cells:a game changerfor future medicine.The EMBO journal 33,409-417(2014).
46.Rock,J.R.et al.Multiple stromalpopulations contribute to pulmonaryfibrosis without evidence for epithelial to mesenchymal transition.Proc Na tlAcad Sci U S A 108,E1475-1483(2011).
47.Hogan,B.L.et al.Repair and regeneration of the respiratory system:complexity,plasticity,and mechanisms of 1ung stem cell function.Cell StemCell 15,123-138(2014).
48.Rock,J.R.et al.Basal cells as stem cells of the mouse trachea andhuman airway epithelium.Proc Natl Acad Sci U S A 106,12771-12775(2009).
49.Rawlins,E.L.&Hogan,B.L.Ciliated epithelial cell lifespan in themouse trachea and lung.American journal of physiology.Lung cellular andmolecular physiology 295,L231-234(2008).
50.Rawlins,E.L.et al.The role of Scgb lal+Clara cells in the long-term maintenance and repair of lung airway,but not alveolar,epithelium.CellStem Cell 4,525-534(2009).
51.Kumar,P.A.et al.Distal airway stem cells yield alveoli in vitroand during lung regeneration following H1N1 influenza infection.Cell 147,525-538(2011).
52.Kim,C.F.et al.Identification of bronchioalveolar stem cells innormal lung and lung cancer.Cell 121,823-835(2005).
53.Zuo,W.et al.p63(+)Krt5(+)distal airway stem cells are essentialfor lung regeneration.Nature 517,616-620(2015).
54.Chapman,H.A.et al.Integrin alpha6beta4 identifies an adult distallung epithelial population with regenerative potential in mice.J Clin Invest121,2855-2862(2011).
55.Vaughan,A.E.et al.Lineage-negative progenitors mobilize tofegenerate lung epithelium after major injury.Nature 517,621-625(2015).
56.Mulugeta,S.,Nureki,S.&Beers,M.F.Lost after translatioin;insightsfrom pulmonary surfactant for understanding the role of alveolar epithelialdysfunction and cellular quality control in fibrotic lung disease.Americanjourmal of physiology.Lung cellular and molecular physiology 309,L507-525(2015).
57.Anikster,Y.et al.Mutation of a new gene causes a unique form ofHermansky-Pudlak syndrome in a genetic isolate of central Puerto Rico.NatGenet 28,376-380(2001).
58.Feng,G.H.,Bailin,T.,Oh,J.&Spritz,R.A.Mouse pale ear(ep)ishomologous to human Hermansky-Pudlak syndrome and contains a rare′AT-AC′intron.Human molecular genetics 6,793-797(1997).
59.Feng,L.et al.The Hermansky-Pudlak syndrome 1(HPS1)and HPS2 genesindependently contribute to the production and function of platelet densegranules,melanosomes,and lysosomes.Blood 99,1651-1658(2002).
60.Li,W.et al.Hermansky-Pudlak syndrome type 7(HPS-7)results frommutant dysbindin,a member of the biogenesis of lysosome-related organellescomplex 1(BLOC-1).Nat Genet 35,84-89(2003).
61.Suzu ki,T.et al.Hermansky-Pudlak syndrome is caused by mutationsin HPS4,the human homolog of the mouse light-ear gene.Nat Genet 30,321-324(2002).
62.Li,W.et al.Murine Hermansky-Pudlak syndrome genes:regulators oflysosome-related organelles.BioEssays:news and reviews in molecular,cellularand developmental biology 26,616-628(2004).
63.Atochina-Vasserman,E.N.et al.Early alveolar epithelial dysfunctionpromotes lung inflammation in a mouse model of Hermansky-Pudlak syndrome.Am JRespir Crit Care Med 184,449-458(2011).
64.Barbosa,M.D.et al.Identification of the homologous beige andChediak-Higashi syndrome genes.Nature 382,262-265(1996).
65.Cullinane,A.R.,Schaffer,A.A.&Huizing,M.The BEACH is hot:a LYST ofemerging roles for BEACH-domain containing proteins in human disease.Traffic14,749-766(2013).
66.Nakatani,Y.et al.Interstitial pneumonia in Hermansky-Pudlaksyndrome:significance of florid foamy swelling/degeneration(giant lamellarbody degeneration)of type-2pneumocytes.Virchows Arch 437,304-313(2000).
67.Whitsett,J.A.,Wert,S.E.&Weaver,T.E.Diseases of pulmonarysuffactant homeostasis.Annual review of pathology 10,371-393(2015).
68.Hawkins,A.et al.A non-BRICHOS SFTPC mutant(SP-CI73T)linked tointerstitial lung disease promotes a late block in macroautophagy disruptingcelluIar proteostasis and mitophagy.American journal of physiology.Lungcellular and molecular physiology 308,L33-47(2015).
69.Fernandez,I.E.&Eickelberg,O.The impact of TGF-beta on lungfibrosis:from targeting to biomarkers.Proc Am Thorac Soc 9,111-116(2012).
70.Chilosi,M.et al.Aberrant Wnt/beta-catenin pathway activation inidiopathic pulmonary fibrosis.The American journal of pathology 162,1495-1502(2003).
71.Bolanos,A.L.et al.Role of Sonic Hedgehog in idiopathic pulmonaryfibrosis.American journal of physiology.Lung cellular and molecularphysiology 303,L978-990(2012).
72.Crestani,B.et al.Hepatocyte growth factor and lung fibrosis.ProcAm Thorae Soc 9,158-163(2012).
73.Kramann,R.et al.Perivascular glil(+)progenitors are keycontributors to injury-indueed organ fibrosis.Cell Stem Cell 16,51-66(2015).
74.Zhong,Q.et al.Role of endoplasmic reticulum stress in epithelial-mesenchymal transition of alveolar epithelial cells:effects of misfoldedsurfactant protein.American joumal of respiratory cell and molecular biology45,498-509(2011).
75.Tanjore,H.et al.Alveolar epithelial cells undergo epithelial-to-mesenchymal transition in response to endoplasmic reticulum stress.TheJournal of biological chemistry 286,30972-30980(2011).
76.Kage,H.&Borok,Z.EMT and interstitial lung disease:a mysteriousrelationship.Current opinion in pulmonary medicine 18,517-523(2012).
77.Seibold,M.A.et al.A common MUC5B promoter polymorphism andpulmonary fibrosis.The New England journal of medicine 364,1503-1512(2011).
78.Peljto,A.L.et al.Association between the MUC5B promoterpolymorphism and survival in patients with idiopathic pulmonaryfibrosis.JAMA:the journal of the American Medical Association 309,2232-2239(2013).
79.Fingerlin,T.E.et al.Genome-wide association study identifiesmultiple susceptibility loci for pulmonary fibrosis.Nat Genet 45,613-620(2013).
80.Murry,C.E.&Keller,G.Differentiation of embryonic stem cells toclinically relevant populations:lessons from embryonic development.Cell 132,661-680(2008).
81.Hanna,J.H.,Saha,K.&Jaenisch,R.Pluripotency and cellularreprogramming:facts,hypotheses,unresolved issues.Cell 143,508-525(2010).
82.Takahashi,K.&Yamanaka,S.Induction of pluripotent stem cells frommouse embryonic and adult fibroblast cultures by defined factors.Cell 126,663-676(2006).
83.Yamanaka,S.A fresh look at iPS cells.Cell 1 37,13-17(2009).
84.Okita,K.&Yamanaka,S.Induced pluripotent stem cells:opportunitiesand challenges.Philos Trans R Soc Lond B Biol Sci 366,2198-2207(2011).
85.Park,I.H.et al.Reprogramming of human somatic cells to pluripotency with defined factors.Nature 451,l41-146(2008).
86.Yu,J.et al.Induced pluripotent stem cell lines derived from humansomatic cells.Science 318,1917-1920(2007).
87.Fox,I.J.et al.Stem cell therapy.Use of differentiated pluripotentstem cells as replacement therapy for treating disease.Science 345,1247391(2014).
88.Lancaster,M.A.&Knoblich,J.A.Organogenesis in a dish:modelingdevelopment and disease using organoid technologies.Science 345,1247125(2014).
89.Robinton,D.A.&Daley,G.Q.The promise of induced pluripotent stemcells in research and therapy.Nature 481,295-305(2012).
90.Mali,P.,Esvelt,K.M.&Chureh,G.M.Cas9 as a versatile tool forengineering biology.Nature methods 10,957-963(2013).
91.Sternberg,S.H.&Dondna,J.A.Expanding the Biologist′s Toolkit withCRISPR-Cas9.Molecular cell 58,568-574(2015).
92.Gonzalez,F.et al.An iCRISPR platform for rapid,multiplexable,andinducible genome editing in human pluripotent stem cells.Cell Stem Cell 15,215-226(2014).
93.Zhu,Z.,Gonzalez,F.&Huangfu,D.The iCRISPR platform for rapid genomeediting in human pluripotent stem cells.Methods in enzymology 546,215-250(2014).
94.Zhu,Z.&Huangfu,D.Human pluripotent stem cells:an emerging model indevelopmental biology.Development 140,705-717(2013).
95.Herriges,M.&Morrisey,E.E.Lung development:orchestrating thegeneration and regeneration of a complex organ.Development1 41,502-513(2014).
96.Morrisey,E.E.&Hogan,B.L.Preparing for the first breath:genetic andcellular mechanisms in lung development.Dev Cell 18,8-23(2010).
97.Warburton,D.et al.Lung organogenesis.Current topics indevelopmental biology 90,73-158(2010).
98.Herring,M.J.,Putney,L.F.,Wyatt,G.,Finkbeiner,W.E.&Hyde,D.M.Growthof alveoli during postnatal development in humans based on stereologicalestimation.American joumal of physiology.Lung cellular and molecularphysiology 307,L338-344(2014).
99.D′Amour,K.A.et al.Efficient differentiation of human embryonicstem cells to definitive endoderm.Nature biotechnology 23,1534-1541(2005).
100.Kubo,A.etal.Development of definitive endoderm from embryonicstem cells in culture.Development 131,1651-1662(2004).
101.Wells,J.M.&Melton,D.A.Early mouse endoderm is patterned bysoluble factors from a djacent germ layers.Development 127,1563-1572(2000).
102.Yasunaga,M.et al.Induction and monitoring of definitive andvisceral endoderm differentiation of mouse ES cells.Nature biotechnology 23,1542-1550(2005).
103.Zorn,A.M.&Wells,J.M.Vertebrate endoderm development and organformation.Annu Rev Cell Dev Biol 25,221-251(2009).
104.Nostro,M.C.&Keller,G.Generation of beta cells from humanpluripotent stem cells:Potential for regenerative mcdicine.Seminars in cell&developmental biology(2012).
105.Nostro,M.C.et al.Stage-specific signaling through TGFbeta familymembers and WNT regulates patterning and pancreatic specification of humanplu ripotent stem cells.Development 138,861-871(2011).
106.Goss,A.M.et al.Wnt2/2b and beta-catenin sigualing are necessaryand sufficient to specify lung progenitors in the foregut.Dev Cell 17,290-298(2009).
107.Bellusci,S.,Grindley,J.,Emoto,H.,Itoh,N.&Hogan,B.L.Fibroblastgrowth factor 10(FGF10)and branching morphogenesis in the embryonic mouselung.Development 124,4867-4878(1997).
108.Bellusci,S.,Henderson,R.,Winnier,G.,Oikawa,T.&Hogan,B.L.Evidencefrom normal expression and targeted misexpression that bone morphogeneticprotein(Bmp-4)plays a role in mouse embryonic lung morphogenesis.Development122,1693-1702(1996).
109.Domyan,E.T.et al.Signaling through BMP receptors promotesrespiratory identity in the foregut via repression of Sox2.Development 138,971-981(2011).
110.Li,Y.,Gordon,J.,Manley,N.R.,Litingtung,Y.&Chiang,C.Bmp4 isrequired for tracheal formation:a novelmouse model for trachealagenesis.Developmental biology 322,145-155(2008).
111.Chen,F.et al.A retinoic acid-dependent network in the foregutcontrols formation of the mouse lung primordium.J Clin Invest 120,2040-2048(2010).
112.Nelson,W.J.&Nusse,R.Convergence of Wnt,beta-catenin,and cadherinpathways.Science 303,1483-1487(2004).
113.Willert,K.&Nusse,R.Wnt proteins.Cold Spring Harbor perspectivesin biology 4,a007864(2012).
114.Gonzales,L.W.,Guttentag,S.H.,Wade,K.C.,Postle,A.D.&Ballard,P.L.Differentiation of human pulmonary type II cells in vitro byglucocorticoid plus cAMP.American journal of physiology.Lung cellular andmolecular physiology 283,L940-951(2002).
115.Longmire,T.A.et al.Efficient derivation of purified lung andthyroid progenitors from embryonic stem cells.Cell Stem Cell 10,398-411(2012).
116.Kumar,M.E.et al.Mesenchymal cells.Defining a mesenchymalprogenitor niche at single-cell resolution.Science 346,1258810(2014).
117.Hrycaj,S.M.et al.Hox5 Genes Regulatethe Wnt2/2b-Bmp4-SignalingAxis during Lung Development.Cell reports 12,903-912(2015).
118.Liu,L.etal.Hedgehog signaling in neonatal and adultlung.Americanjoumal of respiratory cell and molecularbiology 48,703-710(2013).
119.Vokes,S.A.et al. Genomic characterization of Gli_activatortargets in sonic hedgehog-mediated neural patterning.Development 134,1977-1989(2007).
120.Bellusci,S.et al.Involvement of Sonic hedgehog(Shh)in mouseembryonic lung growth and morphogenesis.Development 124,53-63(1997).
121.Pepicelli,C.V.,Lewis,P.M.&McMahon,A.P.Sonic hedgehog regulatesbranching morphogenesis in the lnammalianlung.Current biology:CB 8,1083-1086(1998).
122.Que,J.et al.Multiple dose-dependent roles for Sox2 in thepatterning and differentiation of anterior foregut endoderm.Development 134,2521-2531(2007).
123.Treutlein,B.et al.Reconstructing lineage hierarchies of thedistal lung epithelium using single-cell RNA-seq.Nature 509,371-375(2014).
124.Sakurai,J.et al.Differential expression of the glycosylated formsof MUC1 during lung development.Eur J Histochem 51,95-102(2007).
125.Ban,N.et al.ABCA3 as a lipid transporter in pulmonary surfactantbiogenesis.The Journal of biological chemistry 282,9628-9634(2007).
126.Alanis,D.M.,Chang,D.R.,Akiyama,H.,Krasnow,M.A.&Chen,J.Two nesteddevelopmental waves demarcate a compartment boundary in the mouse lung.Naturecommunications 5,3923(2014).
127.Jain,R.et al.Plasticity of Hopx(+)type I alveolar cells toregenerate type II cells in the lung.Nature communications 6,6727(2015).
128.Labbadia,J.&Morimoto,R.I.The biology of proteostasis in aging anddisease.Annu Rev Biochem 84,435-464(2015).
129.Wells,R.G.Fibrogenesis.V.TGF-beta signaling pathways.Americanjournal of physiology.Gastrointestinal and liver physiology 279,G845-850(2000).
130.Lindquist,J.N.,Marzluff,W.F.&Stefanovic,B.Fibrogenesis.III.Posttranscriptional regulation of type I collagen.American journal ofphysiology.Gastrointestinal and liver physiology 279,G471-476(2000).
131.Ishida,Y.et al.Type I collagen in Hsp47-null cells is aggregatedin endoplasmic reticulum and deficient in N-propeptide processing andfibrillogenesis.Molecular biology of the cell 17,2346-2355(2006).
132.Hosokawa,N.,Hohenadl,C.,Satoh,M.,Kuhn,K.&Nagata,K.HSP47,acollagen-specific molecular chaperone,delaysthe secretion of type IIIprocollagen transfected in human embryonic kidney cell line 293:a possiblerole for HSP47 in collagen modification.Journal of biochemistry 124,654-662(1998).
133.Armanios,M.Syndromes of telomere shortening.Annu Rev Genomics HumGenet 10,45-61(2009).
134.Park,I.H.et al.Disease-specific induced pluripotent stemcells.Cell 134,877-886(2008).
135.Unternaehrer,J.J.&Daley,G.Q.Induced pluripotent stem cells formodelling human diseases.Philos Trans R Soc Lond B Biol Sci 366,2274-2285(2011).
136.De Los Angeles,A.,Loh,Y.H.,Tesar,P.J.&Daley,G.Q.Accessing naivehuman pluripotency.Current opinion in genetics&development 22,272-282(2012).
137.Ferrari,F.,Apostolou,E.,Park,P.J.&Hochedlinger,K.Rearranging thechromatin for pluripotency.Cell Cycle 13,167-168(2014).
138.Kim,K.et al.Epigenetic memory in induced pluripotent stemcells.Nature 467,285-290(2010).
139.Hiler,D.et al.Quantification of Retinogenesis in 3D CulturesReveals Epigenetic Memory and Higher Efficiency in iPSCs Derived from RodPhotoreceptors.Cell Stem Cell 17,101-115(2015).
140.Rouhani,F.et al.Genetic background drivestranscriptionalvariation in human induced pluripotent stem cells.PLoSgenetics 10,e 1004432(2014).
141.Nazarian,R.et al.An immunoblotting assay to facilitate themolecular diagnosis of Hermansky-Pudlak syndrome.Mol Genet Metab 93,134-144(2008).
142.Dell′Angelica,E.C.,Shotelersuk,V.,Aguilar,R.C.,Gahl,W.A.&Bonifacino,J.S.Altered trafficking of lysosomal proteins in Hermansky-Pudlaksyndrome due to mutations in the beta 3A subunit of the AP-3adaptor.Molecular cell 3,11-21(1999).
143.Coral,J.,Gonzalez-Conejero,R.,Pujol-Moix,N.,Domenech,P.&Vicente,V.Mutation analysis of HPS1,the gene mutated in Hermansky-Pudlak syndrome,inpatients with isolated platelet dense-granuledeficiency.Haematologica 89,325-329(2004).
144.Oh,J.et al.Mutation analysis of patients with Hermansky-Pudlaksyndrome:a frameshift hot spot in the HPS gene and apparent locusheterogeneity.American journal of human genetics 62,593-598(1998).
145.Huizing,M.et al.Nonsense mutations in ADTB3A cause completedeficiency of the beta3A subunit of adaptor complex-3 and severe Hermansky-Pudlak syndrome type 2.Pediatr Res 51,150-158(2002).
146.Morgan,N.V.et al.A germline mutation in BLOC1S3/reducedpigmentation causes a novelvariant of Hermansky-Pudlak syndrome(HPS8).American journal of human genetics 78,160-166(2006).
147.Cullinane,A.R.et al.A BLOC-1 mutation screen reveals a novelBLOC1S3 mutation in Hermansky-Pudlak Syndrome type 8.Pigment Cell MelanomaRes 25,584-591(2012).
148.Nagle,D.L.et al. Identification and mutation analysis of thecomplete gene for Chediak-Higashi syndrome.Nat Genet 14,307-311(1996).
149.Fukai,K.et al.Homozygosity mapping of the gene for Chediak-Higashi syndrome to chromosome 1q42-q44 in a segment of conserved syntenythatincludes the mouse beige locus(bg).American journal of human genetics 59,620-624(1996).
150.Karim,M.A.et al.Apparent genotype-phenotype correlation inchildhood,adolescent,and adult Chediak-Higashi syndrome.Am J Med Genet 108,16-22(2002).
151.Winkler,T.et al.Defective telomere elongation and hematopoiesisfrom telomerase-mutant aplastic anemia iPSCs.J Clin Invest 123,1952-1963(2013).
152.Batista,L.F.et al.Telomere shortening and loss of self-renewal indyskeratosis congenita induced pluripotent stem cells.Nature 474,399-402(2011).
153.Gonzalez,R.F.,Allen,L.,Gonzales,L.,Ballard,P.L.&Dobbs,L.G.HTII-280,a biomarker specific to the apical plasma membrane of human lung alveolartype II cells.The joumal of histochemistry and cytochemistry:official journalof the Histochemistry Society 58,891-901(2010).
154.Chilosi,M.et al.Abnormal re-epithelialization and lung remodelingin idiopathic pulmonary fibrosis:the role of deltaN-p63.Laboratoryinvestigation;a journal of technical methods and pathology 82,1335-1345(2002).
155.Luchsinger,L.L.,de Almeida,M.J.,Corrigan,D.J.,Mumau,M.&Snoeck,H.W.Mitofusin 2 maintains haematopoietic stem cells with extensive lymphoidpotential.Nature(2016).
156.Barth,S.,Glick,D.&Macleod,K.F.Autophagy:assays and artifacts.TheJournal of pathology 221,117-124(2010).
157.Zhu,J.,Dagda,R.K.&Chu,C.T.Monitoring mitophagy in neuronal cellcultures.Methods Mol Biol793,325-339(2011).
158.Narendra,D.,Tanaka,A.,Suen,D.F.&Youle,R.J.Parkin is recruitedselectively to impaired mitochondria and promotes their autophagy.The Journalof cell biology 183,795-803(2008).
159.Pickrell,A.M.&Youle,R.J.The roles of PINK1,parkin,andmitochondrial fidelity in Parkinson′s disease.Neuron 85,257-273(2015).
160.Youle,R.J.&van der Bliek,A.M.Mitochondrial fission,fusion,andstress.Science 337,1062-1065(2012).
161.Lamb,C.A.,Yoshimori,T.&Tooze,S.A.The autophagosome:originsunknown,biogenesis complex.Nature reviews.Molecular cell biology 14,759-774(2013).
162.Santaguida,S.,Vasile,E.,White,E.&Amon,A.Aneuploidy-inducedcellular stresses limit autophagic degradation.Genes Dev 29,2010-2021(2015).
163.Korfei,M.et al.Epithelial endoplasmic reticulum stress andapoptosis in sporadic idiopathic pulmonary fibrosis.Am J Respir Crit Care Med178,838-846(2008).
164.Han,J.&Kaufman,R.J.Measurement of the unfolded protein responseto investigate its role in adipogenesis and obesity.Methods in enzymology538,135-150(2014).
165.Shang,J.Quantitative measurement of events in the mammalianunfolded protein response.Methods in enzymology 491,293-308(2011).
166.Carroll,T.P.,Greene,C.M.&McElvaney,N.G.Measurement of theunfolded protein response(UPR)in monocytes.Methods in enzymology 489,83-95(2011).
167.Moeller,A.,Ask,K.,Warburton,D.,Gauldie,J.&Kolb,M.The bleomycinanimal moel:a useful tool to investigate treatment options for idiopathicpulmonary fibrosis?The international journal of biochemistry&cell biology 40,362-382(2008).
168.Moore,B.B.&Hogaboam,C.M.Murine models of pulmonaryfibrosis.American journal of physiology.Lung cellular and molecularphysiology 294,L152-160(2008).
169.Matute-Bello,G.,Frevert,C.W.&Martin,T.R.Animal models of acutelung injury.American joumal of physiology.Lung cellular and molecularphysiology 295,L379-399(2008).
170.Morimoto,R.I.&Cuervo,A.M.Proteostasis and the aging proteome inhealth and disease.The journals of gerontology.Series A,Biological sciencesand medical sciences 69Suppl 1,S33-38(2014).
171.Raghu,G.et al.An official ATS/ERS/JRS/ALAT statement:idiopathicpulmonary fibrosis:evidence-based guidelines for diagnosis and management.Am.J.Respir.Crit.Care Med.183,788-824(2011).
172.Loh,Y.H.et al.Reprogramming of T cells from human peripheralblood.Cell Stem Cell 7,15-19(2010).
173.Staerk,J.et al.Reprogramming of human peripheral blood cells toinduced pluripotent stem cells.Cell Stem Cell 7,20-24(2010).
174.Brown,M.E.et al.Derivation of induced pluripotent stem cells fromhuman peripheral blood T lymphocytes.PLoS One 5,e11373(2010).
175.Seki,T.,Yuasa,S.&Fukuda,K.Generation of induced pluripotent stemcells from a small amount of human peripheral blood using a combination ofactivated T cells and Sendai virus.Nature protocols 7,718-728(2012).
Claims (25)
1.用于在3D基质中由哺乳动物多能干细胞制备支化肺芽类器官(BLBO)的方法,其包括
a)从前部前肠内胚层细胞选择具有折叠结构的d20-d25肺芽类器官(LBO),其中所述前部前肠内胚层细胞已由所述多能干细胞产生,和
b)将所述LBO嵌入3D基质且在诱导支化的培养基存在下以及在允许由所述LBO形成支化LBO(BLBO)的条件下培养所述LBO持续一段时间,其中支化培养基包含BMP4、视黄酸、FGF10、FGF7和CHIR99021,以及
其中步骤b)中的嵌入包括(i)在容器中固化第一量的3D基质以形成固化的3D基质的底部,(ii)使具有折叠结构的类器官与100%3D基质的混合物在底部部分的顶部固化以形成固化的中心部分;和(iii)在固化的中心部分上使另一量的3D基质固化以形成顶部。
2.根据权利要求1所述的方法,其中所述多能干细胞为RUES2细胞系或诱导性多能干细胞(iPSC)。
3.根据权利要求2所述的方法,其中所述ESC或iPSC已突变成具有肺病相关突变。
4.根据权利要求3所述的方法,其中所述ESC或iPSC已突变成具有以下各者的完全缺失或以下各者的突变:HPS1、HPS2、HPS4、hTERT、hTERC、角化不良蛋白、CFTR、DKC1、SFPTB、SFTPC、SFTPA1、SFTPA2、MUC5B、SHH、PTCH、SMO基因,或ABCA3的多态性,或前述任一者的组合。
5.根据权利要求1所述的方法,其中所述3D基质为基质胶。
6.根据权利要求1所述的方法,其中所述具有折叠结构的LBO通过以下制备:i)在形成类胚体的条件下培养所述多能干细胞;ii)在形成LBO的条件下于前部化培养基中培养所述类胚体,以及iii)在形成具有折叠结构的LBO的条件下于腹侧化培养基中培养所述LBO。
7.根据权利要求1所述的方法,其中进行步骤b)中的培养持续20-270天。
8.根据权利要求1所述的方法,其中进行步骤b)中的培养持续至少100天。
9.根据权利要求1所述的方法,其中步骤b)中的所述条件包括在常氧孵育箱中培养。
10.一种哺乳动物分离的BLBO,其通过根据权利要求1所述的方法制得。
11.根据权利要求1所述的方法,其中所述多能干细胞携带HPS1、HPS2、HPS4、HPS5、HPS8、LYST hTERT、hTERC、角化不良蛋白、CFTR、DKC1、SFPTB、SFTPC、SFTPA1、SFTPA2、MUC5B、SHH、PTCH、SMO基因突变,或ABCA3基因的多态性,或前述任一者的组合。
12.根据权利要求11所述的方法,其中所述LBO或BLBO出现一种或多种选自由以下组成的组的纤维化特性:与非突变的LBO或BLBO相比,EPCAM阴性间充质细胞的存在增加、间充质细胞标记物的表达增加,和/或细胞外基质沉积增强。
13.一种哺乳动物分离的BLBO,其通过根据权利要求11所述的方法制得。
14.一种用于鉴别治疗肺病的药剂的基于细胞的测定方法,其包括:提供一种或多种根据权利要求11所述的突变的LBO或BLBO的对照和测试样品;使所述测试样品与测试剂接触;测定所述对照和所述测试样品中以下各者的水平:(a)肺和气道上皮细胞标记物的表达,(b)细胞外基质沉积,(c)代谢,(d)内体转运,(e)细胞应激响应,(f)未折叠蛋白响应,(g)线粒体自噬,(h)自体吞噬,(i)表面活性物质分泌或(j)循环,或它们的组合;以及如果所述测试样品中有任何测定水平与所述对照显著不同,则选择所述测试剂。
15.根据权利要求14所述的方法,其还包括突变的LBO或BLBO的第二对照样品,其中所述LBO或BLBO携带LYST、HPS3、HPS5或HPS8突变。
16.根据权利要求1所述的方法,其中所述多能干细胞携带与表面活性物质分泌缺陷相关的突变。
17.根据权利要求16所述的方法,其中与表面活性物质分泌疾病相关的突变为SFTPA、SFTPC、SFTPB或ABCA3突变。
18.一种哺乳动物LBO或BLBO,其根据权利要求16或17所述的方法制得。
19.根据权利要求1所述的方法,其还包括用RSV或引起呼吸道疾病的其他病毒物种感染所述BLBO。
20.一种哺乳动物BLBO,其根据权利要求19制得。
21.一种用于鉴别治疗肺病的药剂的基于细胞的测定方法,其包括:(a)提供根据权利要求11所述的突变LBO或BLBO的对照和测试样品,其具有一种或多种纤维化特征,所述特征包括:间充质细胞的存在增加;2)细胞外基质的沉积增加;3)上皮应激增加;或4)异常的类器官形态;b)使所述测试样品与所述测试剂接触;c)确定与所述测试剂的接触是否导致以下中的一种或多种:间充质细胞的存在减少;细胞外基质的沉积减少;上皮应激的迹象减少;或类器官形态完全或部分正常化。
22.根据权利要求2所述的方法,其中所述iPSC细胞来自具有肺病基因突变的受试者且其中所述iPSC细胞已被遗传改变以修正所述基因突变。
23.根据权利要求22所述的方法,其中所述iPSC经由Crispr/cas系统进行遗传改变。
24.一种哺乳动物LBO,其根据权利要求22制得。
25.根据权利要求21所述的测定方法,其中所述间充质细胞的存在增加包括EPCAM阴性间充质细胞的存在增加。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201762476335P | 2017-03-24 | 2017-03-24 | |
US62/476,335 | 2017-03-24 | ||
PCT/US2018/024383 WO2018176044A1 (en) | 2017-03-24 | 2018-03-26 | Generation of lung bud organoids with branching structures and uses thereof for lung disease modeling |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110494555A CN110494555A (zh) | 2019-11-22 |
CN110494555B true CN110494555B (zh) | 2024-04-02 |
Family
ID=63585794
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201880020727.2A Active CN110494555B (zh) | 2017-03-24 | 2018-03-26 | 具有分支结构的肺芽类器官的生成和其用于肺疾病建模的用途 |
Country Status (7)
Country | Link |
---|---|
US (1) | US11918702B2 (zh) |
EP (1) | EP3601529A4 (zh) |
JP (1) | JP7183175B2 (zh) |
CN (1) | CN110494555B (zh) |
AU (1) | AU2018239660A1 (zh) |
IL (1) | IL269058A (zh) |
WO (1) | WO2018176044A1 (zh) |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11857697B2 (en) * | 2017-05-31 | 2024-01-02 | The Regents Of The University Of Michigan | Compositions and methods for obtaining 3-dimensional lung-like epithelium and related uses thereof |
WO2019217429A1 (en) * | 2018-05-07 | 2019-11-14 | The Trustees Of Columbia University In The City Of New York | Lung and airway progenitors generated from human pluripotent stem cells and related treatments |
CN109576211A (zh) * | 2018-11-01 | 2019-04-05 | 上海淮泊生物科技有限公司 | 一种人多能干细胞定向肺类器官分化流程 |
KR102221230B1 (ko) * | 2018-12-20 | 2021-03-02 | 한국화학연구원 | 인간 줄기세포 유래 폐포 대식세포를 포함하는 3차원 폐 오가노이드의 제조방법 |
KR102461222B1 (ko) * | 2019-10-30 | 2022-10-31 | 가톨릭대학교 산학협력단 | 사람 폐 조직으로부터 2형 폐포 세포 분리 및 계대배양 방법, 및 이를 이용한 폐 오가노이드 제작 방법 |
JPWO2021241493A1 (zh) * | 2020-05-25 | 2021-12-02 | ||
TW202200786A (zh) * | 2020-05-27 | 2022-01-01 | 日商大塚製藥股份有限公司 | 自肺上皮細胞或肺癌細胞製造類器官之方法 |
CN112410282B (zh) * | 2020-11-26 | 2023-03-24 | 安徽大学 | 一种体外高效诱导高级分支状肺类器官的方法及实验模型和化合物组合 |
CN113151149B (zh) * | 2021-03-10 | 2023-07-25 | 安徽大学 | 一种诱导肺类器官的方法及实验模型的建立 |
WO2023240053A2 (en) * | 2022-06-06 | 2023-12-14 | The Trustees Of Columbia University In The City Of New York | Generation of lung organoids from pluripotent stem cells and use thereof |
KR20240029430A (ko) * | 2022-08-26 | 2024-03-05 | 가톨릭대학교 산학협력단 | 폐포세포 유래 유도만능줄기세포를 이용한 폐포 오가노이드 제작 방법 |
CN117286108B (zh) * | 2023-11-24 | 2024-03-01 | 领因生物科技(上海)有限公司 | 一种乳腺癌类器官专用培养基及培养方法 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011139628A1 (en) | 2010-04-25 | 2011-11-10 | Mount Sinai School Of Medicine | Generation of anterior foregut endoderm from pluripotent cells |
US9988606B2 (en) | 2012-07-24 | 2018-06-05 | The Trustees Of Columbia University In The City Of New York | Generation of airway and lung progenitors and epithelial cells and three-dimensional anterior foregut spheres |
WO2014066649A1 (en) | 2012-10-26 | 2014-05-01 | The Regents Of The University Of California | A strategy for engineering various 3d tissues, organoids and vasculature |
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 |
GB201317869D0 (en) | 2013-10-09 | 2013-11-20 | Cambridge Entpr Ltd | In vitro production of foregut stem cells |
EP3556858A3 (en) | 2014-04-09 | 2020-01-22 | Editas Medicine, Inc. | Crispr/cas-related methods and compositions for treating cystic fibrosis |
-
2018
- 2018-03-26 EP EP18771973.7A patent/EP3601529A4/en active Pending
- 2018-03-26 AU AU2018239660A patent/AU2018239660A1/en active Pending
- 2018-03-26 WO PCT/US2018/024383 patent/WO2018176044A1/en active Application Filing
- 2018-03-26 CN CN201880020727.2A patent/CN110494555B/zh active Active
- 2018-03-26 US US16/495,557 patent/US11918702B2/en active Active
- 2018-03-26 JP JP2019552163A patent/JP7183175B2/ja active Active
-
2019
- 2019-09-02 IL IL26905819A patent/IL269058A/en unknown
Non-Patent Citations (3)
Title |
---|
A bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids;DYE,B.R. 等;《eLIFE》;20160928;正文第1-18页 * |
efficient generation of lung and airway epithelial cells from human pluripotent stem cells;HUANG, S. et al;《nature biotechnology》;20131201;84-94 * |
Modeling Human Branching Morphogenesis Using Human Embryonic Stem Cells in vivo, ISSCR;CHEN et al,;《ISSCR 2015》;20150625;摘要 * |
Also Published As
Publication number | Publication date |
---|---|
JP7183175B2 (ja) | 2022-12-05 |
AU2018239660A1 (en) | 2019-10-03 |
IL269058A (en) | 2019-11-28 |
US11918702B2 (en) | 2024-03-05 |
EP3601529A1 (en) | 2020-02-05 |
CN110494555A (zh) | 2019-11-22 |
US20200093959A1 (en) | 2020-03-26 |
EP3601529A4 (en) | 2021-03-17 |
WO2018176044A1 (en) | 2018-09-27 |
JP2020511968A (ja) | 2020-04-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110494555B (zh) | 具有分支结构的肺芽类器官的生成和其用于肺疾病建模的用途 | |
Chen et al. | A three-dimensional model of human lung development and disease from pluripotent stem cells | |
KR102142364B1 (ko) | 유도 연장 만능 줄기 세포, 이의 제조방법 및 사용 방법 | |
EP2450707B1 (en) | Lung tissue model | |
Le et al. | Enhanced telomere rejuvenation in pluripotent cells reprogrammed via nuclear transfer relative to induced pluripotent stem cells | |
US20220112492A1 (en) | Compositions and methods for induced tissue regeneration in mammalian species | |
US20230314413A1 (en) | Heart tissue model | |
CN110573609A (zh) | 双分化或多分化类器官 | |
JP7232201B2 (ja) | 胆管細胞の増殖方法 | |
AU2010244121A1 (en) | Lung tissue model | |
JP2022511757A (ja) | 肝細胞増殖方法 | |
WO2013131000A1 (en) | Emt-inducing transcription factors cooperate with sox9 | |
JP2019503703A (ja) | 幹細胞由来外胚葉系統前駆体を分化する方法 | |
JP7274683B2 (ja) | 多能性幹細胞から樹状分岐した集合管を伴う腎臓構造を作製する方法 | |
Schruf | Recapitulating aspects of alveolar epithelial dysfunction related to idiopathic pulmonary fibrosis utilizing an iPSC-derived air-liquid interface model system | |
WO2024092181A2 (en) | Compositions and methods for obtaining human alveolar cells and related uses thereof | |
EP1961810A1 (en) | Method for obtaining intestinal stem-precursor cell | |
Foltz | Patient-Derived Induced Pluripotent Stem Cells for Modeling Splicing Factor Retinitis Pigmentosa in Retinal Pigmented Epithelial Cells and Retinal Organoids | |
WO2001023531A9 (en) | Recognition of differences in cell cycle structure between stem and differentiated cells | |
JP2019050771A (ja) | 高機能分化誘導細胞の濃縮方法及び高機能分化誘導細胞集団 | |
Rosenfeld | GATA4 regulates distinct developmental programs during formation of the embryonic heart | |
Dvořák et al. | BALANCING ENDOGENOUS AND EXOGENOUS FGF2 ACTION IN HUMAN PLURIPOTENT STEM CELLS |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |