Culturas alternativas para Pluripotentes Stem Cell Production, mantenimiento y análisis genético
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 capacidad de hPSCs para diferenciar hacia los tejidos adultos multilinaje ha abierto nuevas vías para el tratamiento de pacientes que sufren de enfermedades graves que involucran cardiovasculares, hepáticas, pancreáticas, y los sistemas neurológicos 1-4. Varios tipos de células derivadas de hPSCs también proporcionarían plataformas móviles robustos para el modelado de la enfermedad, la ingeniería genética, la detección de drogas y 1,4 pruebas toxicológicas. La cuestión clave que garantiza sus futuras aplicaciones clínicas y farmacológicas es la generación de un gran número de hPSCs de grado clínico a través de cultivo celular in vitro. Sin embargo, los sistemas de cultivo actuales son insuficientes o intrínsecamente variables, diversos cultivos de alimentación y libres de alimentador de hPSCs como colonias 5,6.
Crecimiento de tipo colonia de acciones hPSCs muchas características estructurales de la masa celular interna (ICM) de embriones de mamíferos tempranos. El ICM es propenso a diferenciarse en las tres capas germinalesen un entorno multicelular debido a la existencia de gradientes de señalización heterogéneos. Por lo tanto, la adquisición de la heterogeneidad en el desarrollo embrionario temprano se considera como un proceso necesario para la diferenciación, pero una característica no deseada de la cultura HPSC. La heterogeneidad en la cultura HPSC es a menudo inducida por señales apoptóticas excesivas y diferenciación espontánea debido a las condiciones de crecimiento subóptimas. Por lo tanto, en el tipo de colonia de cultivo, las células heterogéneas se observan a menudo en la periferia de las colonias 7,8. También se ha demostrado que las células en células madre embrionarias (células madre) colonias de respuestas diferenciales de exposición a moléculas de señalización tales como BMP-9 4. Por otra parte, los métodos de cultivo de colonias producen rendimientos bajos de células, así como las tasas de recuperación muy bajo de células de la criopreservación, debido a las tasas de crecimiento incontrolable y señalización apoptótica pathways 6,9. En los últimos años, diversos cultivos en suspensión han sido desarrollados para hPSCs de cultivo, particulArly para la expansión de grandes cantidades de hPSCs en subordinado, y las condiciones de matriz libre 6,10-13. Obviamente, diferentes sistemas de cultivo tienen sus propias ventajas y desventajas. En general, la naturaleza heterogénea de hPSCs representa uno de los principales inconvenientes en los métodos de tipo colonia y cultura agregada, que son subóptimas para la entrega de materiales de ADN y de ARN en hPSCs para la ingeniería genética 6.
Claramente, existe una necesidad imperiosa de desarrollar nuevos sistemas que evitan algunas deficiencias de los métodos de cultivo actuales. Los descubrimientos de inhibidores de moléculas pequeñas (como el inhibidor de la ROCA Y-27632 y JAK inhibidor 1) que mejoran la supervivencia de una sola célula preparan el terreno para el cultivo disociado-HPSC 14,15. Con el uso de estas moléculas pequeñas, hemos desarrollado recientemente un método de cultivo basado en el tipo no-colonia (NCM) de crecimiento de-hPSCs disociadas 9. Este método de cultivo de la novela combina pases de una sola célula y de alta densidadchapado métodos, que nos permite producir grandes cantidades de hPSCs homogéneas bajo ciclos de crecimiento consistentes sin mayores anomalías cromosómicas 9. Alternativamente, la cultura NCM podría ser implementado con diferentes moléculas pequeñas y matrices definidas (tales como lamininas) con el fin de optimizar el método de cultivo para amplias aplicaciones. A continuación, presentamos varios protocolos de detalle sobre la base de la cultura NCM y delinear los procedimientos detallados para la ingeniería genética. Para demostrar la versatilidad de los protocolos de NCM, también a prueba la cultura NCM con inhibidores de ROCK diversos y con la única isoforma laminina 521 (es decir, LN-521).
Protocol
Representative Results
Discussion
Hay dos formas principales para hPSCs de cultivo in vitro: cultura convencional de tipo colonia (de las células en los comederos o matrices extracelulares) y de cultivo en suspensión de hPSCs como agregados sin alimentadores 6. Las limitaciones de los métodos de cultivo, tanto de tipo colonia y suspensión incluyen la heterogeneidad acumulada y los cambios epigenéticos heredables. Cultura NCM, basada tanto en los pases de una sola célula y de las planchas de células de alta densidad, r…
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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