Alternativa kulturer för Human pluripotenta stamceller Produktion, underhåll, samt genetisk analys
Summary
Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.
Abstract
Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.
Introduction
Kapaciteten i hPSCs att differentiera mot multilineage vuxna vävnader har öppnat nya möjligheter att behandla patienter som lider av svåra sjukdomar som involverar hjärt-, lever, bukspottkörtel, och neurologiska system 1-4. Olika celltyper härrör från hPSCs skulle också ge robusta cellulära plattformar för sjukdomsmodellering, genteknik, drog screening och toxikologiska tester 1,4. Den viktigaste frågan som garanterar deras framtida kliniska och farmakologiska tillämpningar är generering av ett stort antal kliniska kvalitet hPSCs genom cellodling in vitro. Men de nuvarande kultursystem är antingen otillräckliga eller inneboende variabel, olika feeder-och feeder-fria kulturer av hPSCs som kolonier 5,6.
Colony typ tillväxt av hPSCs delar många strukturella drag den inre cellmassan (ICM) i däggdjursembryon tidigt. ICM är benägen att differentiera till de tre bakterie-lageri en flercellig miljö på grund av förekomsten av heterogena signalering gradienter. Således är förvärv av heterogenitet i tidig embryonal utveckling betraktas som en erforderlig process för differentiering, men en oönskad egenskap hos HPSC kultur. Den heterogenitet i HPSC kulturen ofta induceras av alltför apoptotiska signaler och spontan differentiering på grund av suboptimala tillväxtbetingelser. Således, i koloni-typ kultur, de heterogena celler observeras ofta i periferin av kolonierna 7,8. Det har också visat att cellerna i mänskliga embryonala stamceller (hESC) kolonier uppvisar differential svar på signalmolekyler som BMP-4 9. Dessutom koloniodlingsmetoder producerar låga cellutbyten samt mycket låga cell återvinningen från frysförvaring på grund av okontrollerbara tillväxt och apoptotiska signalvägar 6,9. Under senare år har olika suspensionskulturer har utvecklats för odling hPSCs, particularly för expansion av stora mängder hPSCs i feeder-och matrisfria betingelser 6,10-13. Uppenbarligen olika kultursystem har sina egna fördelar och nackdelar. I allmänhet representerar den heterogena karaktären hos hPSCs en av de stora nackdelarna i koloni-typ och aggregerade odlingsmetoder som är suboptimal för att leverera DNA-och RNA-material till hPSCs för genteknik 6.
Uppenbarligen finns det ett tvingande behov av att utveckla nya system som kringgår vissa brister i nuvarande odlingsmetoder. Upptäckterna av småmolekylära hämmare (t.ex. ROCK hämmare Y-27632 och JAK-hämmare 1) som förbättrar encelliga överlevnad bana väg för dissocierade-HPSC kultur 14,15. Med hjälp av dessa små molekyler, har vi nyligen utvecklat en odlingsmetod som bygger på icke-koloni typ (NCM) tillväxt av dissocierade-hPSCs 9. Denna nya kultur-metoden kombinerar både encelliga aging och hög densitetplätering metoder, vilket gör att vi kan producera stora mängder av homogena hPSCs enligt konsekventa tillväxtcykler utan större kromosomavvikelser 9. Alternativt kan NCM kultur implementeras med olika små molekyler och definierade matriser (såsom lamininer) i syfte att optimera odlingsmetoden för breda tillämpningar. Här presenterar vi flera detaljerade protokoll som bygger på NCM kultur och avgränsa detaljerade förfaranden för genteknik. För att visa mångsidigheten hos NCM protokoll, testade vi också NCM företagskultur där mångfald ROCK-hämmare och med den enda laminin isoform 521 (dvs., LN-521).
Protocol
Representative Results
Discussion
Det finns två huvudsakliga sätt att kultur hPSCs in vitro: konventionell koloni-typ kultur (av celler på matare eller extracellulära matriser) och fjädring kultur av hPSCs som aggregat utan matare 6. Begränsningarna av både koloni-typ och hängodlingsmetoder inkluderar ackumulerade heterogenitet och ärftliga epigenetiska förändringar. NCM kultur, baserad på både encelliga aging och hög densitet cell plätering, representerar en ny kultur metod för HPSC tillväxt 6,18. Fastän…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Intramural Research Program of the National Institutes of Health (NIH) at the National Institute of Neurological Disorders and Stroke. We would like to thank Dr. Ronald D. McKay for his discussion and comments on this project.
Materials
| Countess automated cell counter | Invitrogen Inc. | C10227 | Automatic cell counting |
| Faxitron Cabinet X-ray System | Faxitron X-ray Corporation, Wheeling, IL | Model RX-650 | X-ray irradiation of MEFs |
| MULTIWELL six-well plates | Becton Dickinson Labware | 353046 | Polystyrene plates |
| DMEM | Invitrogen Inc. | 11965–092 | For MEF medium |
| mitomycin C | Roche | 107 409 | Mitotic inhibitor |
| Trypsin | Invitrogen Inc. | 25300-054 | For MEF dissociation |
| DMEM/F12 | Invitrogen Inc. | 11330–032 | For hPSC medium |
| Opti-MEM I Reduced Serum Medium | Invitrogen Inc. | 31985-062 | For hPSC transfection |
| Heat-inactivated FBS | Invitrogen Inc. | 16000–044 | Component of MEF medium |
| Knockout Serum Replacer | Invitrogen Inc. | 10828–028 | KSR, Component of hPSC medium |
| Dulbecco’s Phosphate-Buffered Saline | Invitrogen Inc. | 14190-144 | D-PBS, free of Ca2+/Mg2+ |
| Non-essential amino acids | Invitrogen | 11140–050 | NEAA, component of hPSC medium |
| L-Glutamine | Invitrogen | 25030–081 | Component of hPSC medium |
| mTeSR1 & Supplements | StemCell Technologies | 5850 | Animal protein-free |
| medium | |||
| TeSR2 & Supplements | StemCell Technologies | 5860 | Xeno-free medium |
| β-mercaptoethanol | Sigma | 7522 | Component of hPSC medium |
MEF (CF-1) ATCC |
American Type Culture Collection (ATCC) | SCRC-1040 | For feeder culture of hPSCs |
| hESC-qualified Matrigel | BD Bioscience | 354277 | For feeder-free culture of hPSCs |
| Laminin-521 | BioLamina | LN521-02 | Human recombinant protein |
| FGF-2 (recombinant FGF, basic) | R&D Systems, MN | 223-FB | Growth factor in hPSC medium |
| CryoStor CA10 | StemCell Technologies | 7930 | |
| Accutase | Innovative Cell Technologies | AT-104 | 1X mixed enzymatic solution |
| JAK inhibitor I | EMD4 Biosciences | 420099 | An inhibitor of Janus kinase |
| Y-27632 | EMD4 Biosciences | 688000 | ROCK inhibitor |
| Y-27632 | Stemgent | 04-0012 | ROCK inhibitor |
| Y-39983 | Stemgent | 04-0029 | ROCK I inhibitor |
| Phenylbenzodioxane | Stemgent | 04-0030 | ROCK II inhibitor |
| Thiazovivin | Stemgent | 04-0017 | A novel ROCK inhibitor |
| BD Falcon Cell Strainer | BD Bioscience | 352340 | 40-µm cell strainer |
| Nalgene 5100-0001 Cryo 1°C | Thermo Scientific | C6516F-1 | “Mr. Frosty” Freezing Container |
| Lipofectamine 2000 | Invitrogen Inc. | 11668-027 | Transfection reagents |
| DharmaFECT Duo | Thermo Scientific | T-2010-02 | Transfection reagent |
| Non-targeting miRIDIAN miRNA Transfection Control | Thermo Scientific | IP-004500-01-05 | Labeled with Dy547, to monitor the delivery of microRNAs |
| SMART-shRNA | Thermo Scientific | To be determined | Lentiviral vector |
| pmaxGFP | amaxa Inc (Lonza) | Included in every transfection kit | Expression plasmid for transfection control |
| 4-Oct | Santa Cruz Biotechnology | sc-5279 | Mouse IgG2b, pluripotent marker |
| SSEA-1 | Santa Cruz Biotechnology | sc-21702 | Mouse IgM, differentiation marker |
| SSEA-4 | Santa Cruz Biotechnology | sc-21704 | Mouse IgG3, pluripotent marker |
| Tra-1-60 | Santa Cruz Biotechnology | sc-21705 | Mouse IgM, pluripotent marker |
| Tra-1-81 | Santa Cruz Biotechnology | sc-21706 | Mouse IgM, pluripotent marker |
| CK8 (C51) | Santa Cruz Biotechnology | sc-8020 | Mouse IgG1, against cytokeratin 8 |
| α-fetoprotein | Santa Cruz Biotechnology | sc-8399 | AFP, mouse IgG2a |
| HNF-3β (P-19) | Santa Cruz Biotechnology | sc-9187 | FOXA2, goat polyclonal antibody |
| Troponin T (Av-1) | Thermo Scientific | MS-295-P0 | Mouse IgG1 |
| Desmin | Thermo Scientific | RB-9014-P1 | Rabbit IgG |
| Anti-NANOG | ReproCELL Inc, Japan | RCAB0004P-F | Polyclonal antibody |
| Rat anti-GFAP | Zymed | 13-0300 | Glial fibrillary acidic protein |
| Albumin (clone HSA1/25.1.3) | Cedarlane Laboratories Ltd. ( | CL2513A | Mouse IgG1, |
| Smooth muscle actin (clone 1A4) | DakoCytomation Inc | IR611/IS611 | Mouse IgG2a |
| Nestin | Chemicon International | MAB5326 | Rabbit polyclonal antibody |
| TUBB3 | Convance Inc | MMS-435P | Tuj1, mouse IgG2a |
| HNF4α (C11F12) | Cell Signaling Technologies | 3113 | Rabbit monoclonal antibody |
| Paraformaldehyde (solution) | Electron Microscopy Sciences | 15710 | PFA, fixative, diluted in D-PBS |
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