En effektiv metode til rettet Hepatocyte-Lignende Cell Induktion fra Human Embryonale stamceller
Summary
Dette manuskript beskriver en detaljeret protokol for differentiering af humane embryonale stamceller (hESC’er) til funktionelle hepatocytlignende celler (HLC’ er) ved løbende at supplere Activin A og CHIR99021 under hESC-differentiering til endelig endoderm (DE).
Abstract
De potentielle funktioner i hepatocytlignende celler (HLC’er) fra menneskelige embryonale stamceller (HESC’er) rummer store løfter om sygdomsmodellering og lægemiddelscreeningsapplikationer. Forudsat her er en effektiv og reproducerbar metode til differentiering af HESCs i funktionelle HLCs. Etableringen af en endoderm afstamning er et vigtigt skridt i differentieringen til HLCs. Ved vores metode regulerer vi de vigtigste signalveje ved løbende at supplere Activin A og CHIR99021 under hESC-differentiering til endelig endoderm (DE), efterfulgt af generering af lever stamceller og endelig HLCs med typisk hepatocytmorfologi i en trinvis metode med helt definerede reagenser. De hESC-afledte NPC’er, der produceres ved denne metode, udtrykker scenespecifikke markører (herunder albumin, HNF4α nuclear receptor og natrium taurocholat cotransporterende polypeptid (NTCP)) og udviser særlige egenskaber i forbindelse med modne og funktionelle hepatocytter (herunder indokyaningrøn farvning, glykogenlagring, hæmatoboxyl-eosin farvning og CYP3-aktivitet) og kan udgøre en platform for udvikling af HLC-baserede anvendelser i undersøgelsen af leversygdomme.
Introduction
Leveren er et meget metabolisk organ, der spiller flere roller, herunder deoxidation, opbevaring af glykogen og sekretion og syntese af proteiner1. Forskellige patogener, stoffer og arvelighed kan forårsage patologiske ændringer i leveren og påvirke dens funktioner2,3. Hepatocytter, som den vigtigste funktionelle enhed i leveren, spiller en vigtig rolle i kunstig leverstøtte systemer og narkotika toksicitet elimination. Men, ressourcen af primære menneskelige hepatocytter er begrænset i celle-baseret behandling, samt i leversygdom forskning. Derfor er udvikling af nye kilder til funktionelle menneskelige hepatocytter en vigtig forskningsretning inden for regenerativ medicin. Siden 1998, hvor hESC’er er blevet etableret4, hESC’er er blevet bredt overvejet af forskere på grund af deres overlegne differentieringspotentiale (de kan differentiere sig til forskellige væv i et passende miljø) og høj grad af selvfornyelse og dermed give ideelle kildeceller til bioartificial lever, hepatocyttransplantation og endda levervævsteknik5.
I øjeblikket kan den hepatisk differentieringseffektivitet øges betydeligt ved at berige endoderm6. I differentieringsdifferentiering af stamceller til endoderm er niveauer af den transformerende vækstfaktor β (TGF-β) signalering og WNT-signalveje de vigtigste faktorer i lyden i endodermdannelsesfasen. Aktivering af et højt niveau af TGF-β og WNT signalering kan fremme udviklingen af endoderm7,8. Activin A er en cytokin, der tilhører TGF-β superfamilien. Derfor anvendes Activin A i vid udstrækning til endoderminduktion af menneskeskabte pluripotente stamceller (hiPSC’er) og HESC’er9,10. GSK3 er en serine-threonine protein kinase. Forskere har konstateret, at CHIR99021, en specifik hæmmer af GSK3β, kan stimulere typiske WNT-signaler og kan fremme stamcelledifferentiering under visse betingelser, hvilket tyder på, at CHIR99021 har potentiale til at inducere stamcelledifferentiering i endoderm 11,12,13.
Her rapporterer vi om en effektiv og reproducerbar metode til effektivt at reducere differentieringen af HESC’er til funktionelle HLC’er. Den sekventielle tilføjelse af Activin A og CHIR99021 producerede omkring 89,7±0,8% SOX17 (DE-markør)-positive celler. Efter at være blevet yderligere modnet in vitro, udtrykte disse celler leverspecifikke markører og udøvede hepatocytlignende morfologi (baseret på hematoxylin-eosin farvning (H &E)) og funktioner, såsom optagelse af indocyaningrøn (ICG), glykogenlagring og CYP3-aktivitet. Resultaterne viser, at HESC’er med succes kan differentieres til modne funktionelle HLC’er ved denne metode og kan danne grundlag for leversygdomsrelateret forskning og in vitro-lægemiddelscreening.
Protocol
Representative Results
Discussion
Her præsenterer vi en trinvis metode, der fremkalder HLC’er fra HESC’er i tre faser. I første fase blev Activin A og CHIR99021 brugt til at differentiere HESC’er til DE. I anden fase blev KO-DMEM og DMSO brugt til at differentiere DE til lever stamceller. I tredje fase blev HZM plus HGF, OSM og hydrocortison 21-hemisuccinat natriumsalt brugt til fortsat at differentiere lever stamceller til HLC’er.
Følgende kritiske trin skal tages i betragtning, når protokollen bruges. Den oprindelige dif…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af National Natural Science Foundation of China (Nr. 81870432 og 81570567 til X.L.Z.), (Nr. 81571994 til P.N.S.); Natural Science Foundation of Guangdong-provinsen, Kina (nr. 2020A1515010054 til P.N.S.), Li Ka Shing Shantou University Foundation (nr. L1111 2008 til P.N.S.). Vi vil gerne takke professor Stanley Lin fra Shantou University Medical College for nyttige råd.
Materials
| 2-Mercaptoethanol | Sigma | M7522 | For hepatic progenitor differentiation |
| 488 labeled goat against mouse IgG | ZSGB-BIO | ZF-0512 | For IF,second antibody |
| 488 labeled goat against Rabbit IgG | ZSGB-BIO | ZF-0516 | For IF,second antibody |
| Accutase | Stem Cell Technologies | 7920 | For cell passage |
| Activin A | peproTech | 120-14E | For definitive endoderm formation |
| Anti – Albumin (ALB) | Sigma-Aldrich | A6684 | For IF and WB, primary antibody |
| Anti – Human Oct4 | Abcam | Ab19587 | For IF, primary antibody |
| Anti – α-Fetoprotein (AFP) | Sigma-Aldrich | A8452 | For IF and WB, primary antibody |
| Anti -SOX17 | Abcam | ab224637 | For IF, primary antibody |
| Anti-Hepatocyte Nuclear Factor 4 alpha (HNF4α) | Sigma-Aldrich | SAB1412164 | For IF, primary antibody |
| B-27 Supplement | Gibco | 17504-044 | For definitive endoderm formation |
| BSA | Beyotime | ST023-200g | For cell blocking |
| CHIR99021 | Sigma-Aldrich | SML1046 | For definitive endoderm formation |
| DAPI | Beyotime | C1006 | For nuclear staining |
| DEPC-water | Beyotime | R0021 | For RNA dissolution |
| DM3189 | MCE | HY-12071 | For definitive endoderm formation |
| DMEM/F12 | Gibco | 11320-033 | For cell culture |
| DMSO | Sigma-Aldrich | D5879 | For hepatic progenitor differentiation |
| DPBS | Gibco | 14190-144 | For cell culture |
| GlutaMAX | Gibco | 35050-061 | For hepatic progenitor differentiation |
| H&E staining kit | Beyotime | C0105S | For H&E staining |
| Hepatocyte growth factor (HGF) | peproTech | 100-39 | For hepatocyte differentiation |
| HepatoZYME-SFM (HZM) | Gibco | 17705-021 | For hepatocyte differentiation |
| Hydrocortisone-21-hemisuccinate | Sigma-Aldrich | H4881 | For hepatocyte differentiation |
| Indocyanine | Sangon Biotech | A606326 | For Indocyanine staining |
| Knock Out DMEM | Gibco | 10829-018 | For hepatic progenitor differentiation |
| Knock Out SR Multi-Species | Gibco | A31815-02 | For hepatic progenitor differentiation |
| Matrigel hESC-qualified | Corning | 354277 | For cell culture |
| MEM NeAA | Gibco | 11140-050 | For hepatic progenitor differentiation |
| mTesR 5X Supplement | Stem Cell Technologies | 85852 | For cell culture |
| mTesR Basal Medium | Stem Cell Technologies | 85851 | For cell culture |
| Oncostatin (OSM) | peproTech | 300-10 | For hepatocyte differentiation |
| P450 – CYP3A4 (Luciferin – PFBE) | Promega | V8901 | For CYP450 activity |
| PAS staining kit | Solarbio | G1281 | For PAS staining |
| Pen/Strep | Gibco | 15140-122 | For cell differentiation |
| Peroxidase-Conjugated Goat anti-Mouse IgG | ZSGB-BIO | ZB-2305 | For WB,second antibody |
| Primary Antibody Dilution Buffer for Western Blot | Beyotime | P0256 | For primary antibody dilution |
| ReverTraAce qPCR RT Kit | TOYOBO | FSQ-101 | For cDNA Synthesis |
| RNAiso Plus | TaKaRa | 9109 | For RNA Isolation |
| RPMI 1640 | Gibco | 11875093 | For definitive endoderm formation |
| Skim milk | Sangon Biotech | A600669 | For second antibody preparation |
| SYBR Green Master Mix | Thermo Fisher Scientific | A25742 | For RT-PCR Analysis |
| Torin2 | MCE | HY-13002 | For definitive endoderm formation |
| Tween | Sigma-Aldrich | WXBB7485V | For washing buffer preparation |
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