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.
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.
Kapaciteten af hPSCs at differentiere mod multilineage voksent væv har åbnet nye veje til behandling af patienter, der lider af alvorlige sygdomme, som medfører hjerte-kar-, lever-, pancreas, og neurologiske systemer 1-4. Forskellige celletyper afledt fra hPSCs ville også give robuste cellulære platforme for sygdom modellering, genteknologi, narkotika screening, og toksikologisk testning 1,4. Det centrale spørgsmål, der sikrer deres fremtidige kliniske og farmakologiske applikationer er dannelsen af et stort antal kliniske kvalitet hPSCs gennem in vitro cellekultur. De nuværende dyrkningssystemer er enten utilstrækkelige eller iboende variabel: forskellige feeder og feeder-fri kulturer af hPSCs som kolonier 5,6.
Vækst koloni-type hPSCs deler mange strukturelle træk ved den indre cellemasse (ICM) af tidlige pattedyr embryoner. ICM er tilbøjelig til at differentiere til de tre kim lagi en flercellede miljø på grund af eksistensen af heterogene signalsystemer gradienter. Således er købet af heterogenitet i den tidlige fosterudvikling betragtes som en nødvendig proces for differentiering, men et uønsket element i hPSC kultur. Den heterogenitet i hPSC kultur er ofte fremkaldt af overdreven apoptotiske signaler og spontan differentiering på grund af suboptimale vækstbetingelser. Således i koloni-typen kultur, heterogene celler ofte observeret i periferien af kolonierne 7,8. Det er også blevet påvist, at cellerne i humane embryonale stamceller (embryonale) kolonier udstille differentierede svar på signalmolekyler såsom BMP-4 9. Desuden koloni dyrkningsmetoder producere lave celleudbytter såvel som meget lav celle genanvendelsesprocenter fra kryopræservering grund af ukontrollable vækstrater og apoptotiske signalveje 6,9. I de seneste år er der blevet udviklet forskellige suspensionskulturer til dyrkning hPSCs, particulArly for udvidelse af store mængder hPSCs i feeder-og matrix-fri betingelser 6,10-13. Naturligvis forskellige dyrkningssystemer har deres egne fordele og ulemper. Generelt er den heterogene karakter hPSCs repræsenterer en af de store ulemper i koloni-type og aggregerede dyrkningsmetoder, som er suboptimale for at levere DNA og RNA materialer i hPSCs for genteknologi 6.
Der er tydeligvis et presserende behov for at udvikle nye systemer, der omgår nogle mangler ved de nuværende dyrkningsmetoder. Opdagelser småmolekyleinhibitorer (såsom ROCK inhibitor Y-27632 og JAK-hæmmer 1), som forbedrer encellede overlevelse bane vejen for dissocierede-hPSC kultur 14,15. Med brugen af disse små molekyler, har vi for nylig udviklet en kultur der er baseret på ikke-koloni type (NCM) vækst dissocierede-hPSCs 9. Denne hidtil ukendte kultur metode kombinerer både encellede passage og høj massefyldeplating metoder, der giver os mulighed for at producere store mængder af homogene hPSCs under konstant vækst cyklusser uden større kromosomafvigelser 9. Alternativt kan NMR kultur gennemføres med andre små molekyler og definerede matricer (f.eks lamininer) for at optimere dyrkningsmetode til brede anvendelser. Her præsenterer vi en række detaljerede protokoller baseret på NCM kultur og afgrænse detaljerede procedurer for genteknologi. For at demonstrere den alsidighed af Nordisk Ministerråds protokoller, vi også testet NCM kultur med diverse ROCK-hæmmere og med en enkelt laminin isoform 521 (dvs. LN-521).
Der er to vigtige måder til kultur hPSCs in vitro: konventionel koloni-typen kultur (af celler på foderautomater eller ekstracellulære matricer) og hjulophæng kultur hPSCs som aggregater uden foderautomater 6. Begrænsningerne i både koloni-type og affjedring kultur metoder omfatter akkumulerede heterogenitet og arvelige epigenetiske ændringer. NCM kultur, er baseret på både enkelt-celle-passage og high-density celleudpladning repræsenterer en ny kultur fremgangsmåde til hPSC vækst 6,…
The authors have nothing to disclose.
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.
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 |