Le colture per pluripotenti umane alternative staminali produzione di celle, manutenzione e analisi genetica
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
La capacità di hPSCs di differenziare verso tessuti adulti multilineage ha aperto nuove strade alla cura dei pazienti che soffrono di malattie gravi che coinvolgono cardiovascolare, epatico, pancreatico e sistemi neurologici 1-4. Vari tipi di cellule derivate da hPSCs sarebbe anche fornire piattaforme cellulari robuste per la modellazione della malattia, l'ingegneria genetica, screening di farmaci e test tossicologici 1,4. La questione chiave che assicura loro applicazioni cliniche e farmacologiche futuri è la generazione di un gran numero di hPSCs clinico-grade in coltura cellulare in vitro. Tuttavia, i sistemi di coltura attuali sono insufficienti o intrinsecamente variabile, che coinvolge vari alimentazione e senza alimentatore culture del hPSCs come colonie 5,6.
Colony-tipo crescita delle quote hPSCs molte caratteristiche strutturali della massa cellulare interna (ICM) di embrioni di mammiferi. L'ICM è incline a differenziarsi in tre strati germinaliin un ambiente multicellulare causa dell'esistenza di gradienti di segnalazione eterogenei. Pertanto, l'acquisizione di eterogeneità in sviluppo embrionale precoce è considerata come un processo necessario per la differenziazione, ma una caratteristica indesiderata della cultura HPSC. L'eterogeneità nella cultura HPSC è spesso indotto da eccessivi segnali apoptotici e differenziazione spontanea a causa di condizioni di crescita ottimali. Così, in colonia-tipo coltura, le cellule eterogenee si osservano spesso nella periferia delle colonie 7,8. È stato anche dimostrato che le cellule in cellule staminali embrionali umane (hESC) Colonie risposte differenziali Esporre a molecole di segnalazione come BMP-4 9. Inoltre, metodi di coltura colonia producono produzioni di cellule basse così come i tassi di recupero molto bassi cellulare da crioconservazione a causa di tassi di crescita incontrollabili e segnale apoptotico Percorsi di 6,9. Negli ultimi anni, diverse culture sospensioni sono state sviluppate per hPSCs coltura, soprattarly per l'espansione di grandi quantità di hPSCs in-e alimentatore condizioni prive di matrice 6,10-13. Ovviamente, diversi sistemi di coltura hanno i loro vantaggi e svantaggi. In generale, l'eterogeneità hPSCs rappresenta uno dei principali inconvenienti di tipo colonia e cultura aggregato metodi, che sono ottimali per la consegna di DNA e RNA materiali in hPSCs per l'ingegneria genetica 6.
Chiaramente, vi è la necessità imperativa di sviluppare nuovi sistemi che aggirano alcune carenze delle attuali metodi di coltura. Le scoperte di piccoli inibitori della molecola (come l'inibitore ROCK Y-27632 e JAK inibitore 1) che migliorano la sopravvivenza a cella singola spianare la strada per dissociato-HPSC cultura 14,15. Con l'uso di queste piccole molecole, abbiamo recentemente sviluppato un metodo di coltura in base al tipo non-colonia (NCM) crescita di dissociate-hPSCs 9. Questo metodo di coltura romanzo combina passaging cella singola e ad alta densitàplaccatura metodi, che ci permette di produrre grandi quantità di hPSCs omogenee sotto cicli di crescita costante senza grandi anomalie cromosomiche 9. In alternativa, cultura NCM potrebbe essere implementato con diverse piccole molecole e matrici definite (quale laminine) al fine di ottimizzare il metodo di coltura per le applicazioni di larghezza. Qui vi presentiamo diversi protocolli dettagliati basato sulla cultura NCM e delineare le procedure dettagliate per l'ingegneria genetica. Per dimostrare la versatilità dei protocolli di NCM, abbiamo provato anche la cultura NCM con inibitori ROCK diversi e con il singolo laminina isoforma 521 (vale a dire, LN-521).
Protocol
Representative Results
Discussion
Ci sono due modi principali per hPSCs coltura in vitro: coltura convenzionale colonia-tipo (di celle alimentatori o matrici extracellulari) e cultura sospensione delle hPSCs gli aggregati senza alimentatori 6. Le limitazioni di entrambi colonia-tipo e sospensione metodi di coltura comprendono eterogeneità accumulati e cambiamenti epigenetici ereditabili. Cultura NCM, sulla base sia passaging singola cellula e cellula placcatura ad alta densità, rappresenta un nuovo metodo di coltura per la crescita…
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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