Alternatieve Cultures for Human pluripotente stamcellen zijn Productie, Onderhoud, en genetische analyses
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
De capaciteit van hPSCs om onderscheid te maken in de richting van multilineage volwassen weefsels heeft nieuwe wegen geopend voor de behandeling van patiënten die lijden aan ernstige ziekten die hart-, lever-, pancreas-, en neurologische systemen 1-4 betrekken. Diverse soorten cellen afgeleid van hPSCs zou ook robuuste mobiele platforms voor ziektemodel, genetische manipulatie, drug discovery, en toxicologische testen 1,4. Het belangrijkste punt dat hun toekomstige klinische en farmacologische toepassingen zorgt is het genereren van grote aantallen klinische-grade hPSCs via in vitro celkweek. Echter, de huidige cultuur systemen zijn onvoldoende of inherent variabel, waarbij verschillende feeder en-feeder vrij culturen van hPSCs als kolonies 5,6.
Kolonie-achtige groei van hPSCs aandelen veel constructie-eigenschappen van de binnenste celmassa (ICM) van de vroege embryo's van zoogdieren. De ICM is gevoelig om te differentiëren tot drie kiem-lagenin een meercellige milieu vanwege het bestaan van heterogene signalering gradiënten. Aldus wordt de verwerving van heterogeniteit in de vroege embryonale ontwikkeling als een vereiste werkwijze voor differentiatie, maar een ongewenste eigenschap van HPSC cultuur. De heterogeniteit in HPSC cultuur wordt vaak veroorzaakt door overmatige apoptotische signalen en spontane differentiatie als gevolg van suboptimale groeiomstandigheden. Dus in kolonie-typen cultuur, de heterogene cellen worden vaak waargenomen in de periferie van de kolonies 7,8. Het is ook aangetoond dat de cellen in menselijke embryonale stamcellen (hESC) kolonies vertonen differentiële reacties op signaalstoffen zoals BMP-4 9. Bovendien kolonie cultuur methoden produceren een laag cel opbrengst evenals zeer lage cel herstel tarieven van cryopreservatie te wijten aan oncontroleerbare groei en apoptotische signaalwegen 6,9. De laatste jaren zijn er verschillende suspensiekweken ontwikkeld voor kweken hPSCs, particularly voor uitbreiding van grote hoeveelheden hPSCs in feeder-en matrix-vrije omstandigheden 6,10-13. Uiteraard, andere cultuur systemen hebben hun eigen voordelen en nadelen. In het algemeen, de heterogeniteit van hPSCs is een van de belangrijkste nadelen van de methoden kolonie-type en geaggregeerde cultuur, die optimaal voor het leveren van DNA en RNA materialen in hPSCs voor gentechnologie 6 zijn.
Het is duidelijk dat er een dwingende noodzaak om nieuwe systemen die een aantal tekortkomingen van de huidige kweekmethoden omzeilen ontwikkelen. De ontdekkingen van kleine molecule inhibitoren (zoals de ROTS remmer Y-27632 en JAK remmer 1) dat eencellige verbeteren overleven de weg vrijmaken voor gedissocieerde-HPSC cultuur 14,15. Met het gebruik van deze kleine moleculen, hebben we onlangs een kweekmethode ontwikkeld op basis van niet-kolonie soort (NCM) groei van gedissocieerd-hPSCs 9. Deze nieuwe kweekmethode combineert zowel eencellige passage en high-densityplating methoden, waardoor we grote hoeveelheden homogene hPSCs produceren onder consistente groei cycli zonder grote chromosomale afwijkingen 9. Alternatief kan NCM cultuur met verschillende kleine moleculen en gedefinieerde matrices (bijvoorbeeld laminins) worden uitgevoerd om de kweekmethode voor brede toepassingen te optimaliseren. Hier presenteren we een aantal gedetailleerde protocollen op basis van NCM cultuur en af te bakenen gedetailleerde procedures voor genetische manipulatie. Om de veelzijdigheid van NCM protocollen tonen we ook getest NCM cultuur met diverse ROCK inhibitoren en met de single laminine isovorm 521 (dwz LN-521).
Protocol
Representative Results
Discussion
Er zijn twee belangrijke manieren om cultuur hPSCs in vitro: conventionele kolonie type cultuur (van cellen op feeders of extracellulaire matrices) en ophanging cultuur van hPSCs als aggregaten zonder feeders 6. De beperkingen van kweekmethoden zowel kolonie-type en ophanging omvatten geaccumuleerde heterogeniteit en overerfbare epigenetische veranderingen. NCM cultuur, gebaseerd op zowel single-cell passage en high-density cel plating, vertegenwoordigt een nieuwe kweekmethode voor HPSC groei 6,…
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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