Alternative Kulturer til human pluripotent Stem Cell produksjon, vedlikehold, og Genetic Analysis
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
Kapasiteten på hPSCs å skille mot multilineage voksen vev har åpnet nye veier for å behandle pasienter som lider av alvorlige sykdommer som involverer hjerte, lever, bukspyttkjertel, og nevrologiske systemer 1-4. Ulike celletyper som stammer fra hPSCs ville også gi robuste mobilplattformer for sykdom modellering, genteknologi, narkotika screening, og toksikologiske tester 1,4. Det sentrale spørsmålet som sikrer deres fremtidige kliniske og farmakologiske applikasjoner er generering av et stort antall klinisk-grade hPSCs gjennom in vitro cellekultur. Men dagens kultur-systemer er enten utilstrekkelig eller iboende variable, som involverer ulike mater og mater frie kulturer av hPSCs som kolonier 5,6.
Koloni-type vekst av hPSCs aksjer mange strukturelle trekk ved den indre cellemasse (ICM) av tidlige pattedyr embryoer. ICM er tilbøyelige til å skille ut de tre bakterie-lagi et flercellet miljøet på grunn av eksistensen av heterogene signaloverganger. Dermed er oppkjøpet av heterogenitet i tidlig embryoutvikling anses som en nødvendig prosess for differensiering, men en uønsket funksjon i hPSC kultur. Heterogenitet i hPSC kultur er ofte forårsaket av overdreven apoptotiske signaler og spontan differensiering på grunn av suboptimale vekstforhold. Således, i koloni-type kultur, de heterogene celler er ofte observert i periferien av koloniene 7,8. Det er også vist at cellene i humane embryonale stamceller (hESC) kolonier oppviser differensielle responser til signalmolekyler som BMP-4 9. Videre koloni kultur metoder produsere lave celleutbytte samt svært lav celle utvinning fra nedfrysing skyldes ukontrollerbare vekstrater og apoptotisk signalveier 6,9. I de senere årene har ulike suspensjonskulturer er utviklet for dyrking hPSCs, spesielarly for utvidelse av store mengder hPSCs i mater-og matrisefrie forhold 6,10-13. Tydeligvis, ulike kultur-systemer har sine egne fordeler og ulemper. Generelt er den heterogene karakter av hPSCs representerer en av de store ulemper i koloni-type og aggregert kultur metoder, som er suboptimal for å levere DNA-og RNA-materiale inn hPSCs for genteknologi 6..
Klart, det er en viktig grunn til å utvikle nye systemer som omgår noen svakheter i dagens kultur metoder. Funn av små molekyl-hemmere (for eksempel ROCK inhibitor Y-27632 og JAK inhibitor 1) som forbedrer encellede overlevelse bane vei for skilt-hPSC kultur 14,15. Med bruk av disse små molekylene, har vi nylig utviklet en dyrkningsmetode basert på ikke-kolonitype (NCM) vekst av dissosierte-hPSCs 9. Denne romanen kultur metoden kombinerer både encellede aging og høy tetthetplating metoder, slik at vi kan produsere store mengder homogene hPSCs etter jevn vekst sykluser uten store kromosomavvik ni. Alternativt kan NMR kultur gjennomføres med forskjellige små molekyler og definerte matriser (f.eks laminins) for å optimalisere den dyrkningsmetode for store applikasjoner. Her presenterer vi flere detaljerte protokoller basert på NCM kultur og avgrense detaljerte prosedyrer for genteknologi. For å demonstrere allsidigheten NCM protokoller, vi også testet NCM kultur med diverse ROCK hemmere og med singelen laminin isoformen 521 (dvs., LN-521).
Protocol
Representative Results
Discussion
Det er to viktige måter til kultur hPSCs in vitro: konvensjonell koloni-type kultur (av celler på brett eller ekstracellulære matriser) og suspensjon kultur av hPSCs som tilslag uten matere seks. Begrensningene av både koloni-type og fjæring kultur metoder inkluderer akkumulert heterogenitet og arvbare epigenetiske endringer. NCM kultur, basert på både encellede aging og høy tetthet celle plating, representerer en ny kultur metode for hPSC vekst 6,18. Selv om forskjellige encellede…
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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