ヒト多能性のための代替培養はセル生産、メンテナンス、および遺伝子解析幹
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
多系列成体組織に向かって差別化するhPSCsの容量は、心血管、肝臓、膵臓、および神経システム1-4を伴う重篤な疾患に苦しむ患者の治療に新たな道を開いた。 hPSCsから派生した様々な細胞型はまた、疾患のモデリング、遺伝子工学、薬物スクリーニング、および毒性試験1,4のための堅牢な携帯プラットフォームを提供するであろう。将来の臨床的および薬理学的用途を保証する重要な問題は、 インビトロでの細胞培養を介して臨床グレードhPSCs多数の生成である。しかし、現在の培養系ではコロニー5,6としてhPSCsの様々なフィーダーフィーダーフリーの培養液を含む、不十分であったり、本質的に可変のどちらかである。
hPSCs株哺乳動物の初期胚の内部細胞塊（ICM）の多くの構造的特徴のコロニー型の増殖。 ICMは3生殖層に分化する傾向がある多細胞の環境であるため、異種シグナル勾配の存在。このように、初期胚発生における不均一性の獲得は、分化に必要なプロセスであると考えますが、HPSC文化の不要な機能です。 HPSC培養における不均一性は、多くの場合、過度のアポトーシスシグナルおよび準最適な成長条件による自発的分化によって誘導される。従って、コロニータイプの培養で、不均一な細胞は、しばしば、コロニー7,8の周囲に観察される。また、ヒト胚性幹細胞（hESCの）中の細胞が、例えばBMP-4 9とシグナル伝達分子に展示差動応答をコロニーことが示されている。また、コロニー培養法は、制御不能の成長率およびアポトーシスシグナル伝達経路6,9に低い細胞収量だけでなく、凍結保存と非常に低い細胞回収率を生成する。近年、種々の懸濁培養は、培養のためにhPSCs particulが開発されているフィーダーマトリックスを含まない条件下6,10-13中hPSCs、大量の拡張のためアルリー。明らかに、別の培養システムは、独自の長所と短所を有する。一般に、hPSCsの不均一な性質を遺伝子工学6 hPSCsにDNAとRNAの物質を送達するための準最適であるのコロニー型および凝集培養法における主な欠点のうちの1つを表す。
明らかに、現在の培養方法のいくつかの欠点を回避する新しいシステムを開発する必要性が不可欠である。単一細胞の生存を改善する（このようなROCK阻害剤Y-27632およびJAK阻害剤1のような）低分子阻害剤の発見は解離し、HPSC文化14,15のための道を開く。これらの小分子を使用することで、我々は最近、非コロニー型（NCM）を解離·hPSCs 9の成長に基づいた培養法を開発した。この新規培養法は、単一細胞継代と高密度の両方を兼ね備え、メッキ法、私たちは主要な染色体異常9なしで一貫性のある成長サイクルの下で均質hPSCsを大量に生成することができます。あるいは、NCM培養は広い用途のための培養方法を最適化するために、異なる小分子と（例えば、ラミニンなど）が定義行列を用いて実施され得る。ここでは、NCMのカルチャに基づいて、いくつかの詳細なプロトコルを提示し、遺伝子工学のための詳細な手順を描く。 NCMプロトコルの汎用性を実証するために、我々はまた、多様なROCK阻害剤で、単一のラミニンアイソフォーム521（ すなわち 、LN-521）でNCM文化をテストしました。
Protocol
Representative Results
Discussion
従来の（フィーダまたは細胞外マトリックス上の細胞の）コロニー型の文化やフィーダー6のない集合体としてhPSCsの懸濁培養：in vitroでの培養hPSCsには、2つの主要な方法があります。コロニー型とサスペンションの両方培養法の制限は累積異質と継承エピジェネティックな変化があります。 NCM培養物は、単一細胞継代及び高密度細胞播種の両方に基づいて、HPSC成長6,18?…
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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