髪の由来ケラチノサイトを使用した統合フリーヒト誘導多能性幹細胞の生成
Summary
This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.
Abstract
Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.
Introduction
ヒト誘導多能性幹細胞（hiPSCs）の発見は、患者特異的幹細胞1-3の生成のための実行可能な方法を提供し、再生医療の分野に革命をもたらしました。 hiPSCsは正常線維芽細胞4,5を含む種々の体細胞タイプ、造血細胞6,7、尿8およびケラチノサイト9,10からの腎上皮細胞から生成されています。これまで、皮膚線維芽細胞および造血細胞は、患者固有のiPS細胞を生成するための最も一般的に使用される細胞の供給源を表します。おそらく、これは、皮膚生検及び採血ルーチン医療処置および多くの国で確立された患者の血液または皮膚サンプルの大規模なバイオバンクであるという事実によるものです。
侵襲的な抽出方法を必要とする血液細胞および皮膚線維芽細胞とは対照的に、ケラチノサイトはhiPSCの生成のために容易にアクセスできる細胞型を表します。 KeratinocytESは、皮膚の外側表皮バリアを形成し、また、爪及び髪11に見出されるケラチン豊富な上皮細胞です。特に、ケラチノサイトは毛包、内毛根鞘（IRS）細胞（12、 図1）と一緒に毛幹をカバーし、外部の細胞膜の外毛根鞘（ORS）に記載されています。ヘアコレクションは医療関係者の支援を必要としない簡単な手順であるように、患者が収集し、大幅hiPSCの世代のための患者サンプルの収集を容易にするであろう実験室に自分の毛髪サンプルを送信するための機会を提供しています。表皮角化細胞はまた、hiPSCの生成9,13のための出発細胞としてのケラチノサイトを使用する利点に加えて、線維芽細胞と比較してより高い初期化の効率と高速再プログラミング速度を持っています。さらに、hiPSCsはまた毛包内の他の細胞集団を使用して生成することができ、毛包14,15の基部に位置毛乳頭細胞を含みます。
毛由来の細胞を用いてiPS細胞の発生以前の報告は、多くの場合、レトロウイルスまたはレンチウイルスベースの再プログラミング法9,14,15を利用します。しかし、これらのウイルスの方法は、再プログラミング中に外国の導入遺伝子の望ましくないゲノム組込みをご紹介します。対照的に、エピソームベクターの使用は、統合フリー性IPSC 4を生成するために実行可能な、非ウイルス性の再プログラミング方法を示しています。我々は以前に効率的にエピソームベクター13を使用してhiPSCsにケラチノサイトを再プログラムするための簡単な、費用対効果および非ウイルス法を開発しました。ここでは、hiPSCsを生成するためのケラチノサイトおよびその後の再プログラミングの撥髪、拡張やメンテナンスからケラチノサイトの導出を含むケラチノサイト由来hiPSCsの生成のための詳細なプロトコルを提供します。
Protocol
Representative Results
Discussion
患者特有のhiPSCsの生成は、in vitroでの病気の細胞型において病因を研究するためのユニークなアプローチを提供し、また、疾患の表現型を救出することができる新規分子を同定するための薬物スクリーニングのためのプラットフォームを提供します。 hiPSCsを使用して、この疾患のモデリングアプローチは、QT延長症候群、ハンチントン病、パーキンソン病および筋萎縮性側索硬化症…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors wish to thank Harene Ranjithakumaran and Stacey Jackson for technical support. This work was supported in part by grants from the National Health and Medical Research Council (R.C.B. Wong, A. Pébay), the University of Melbourne (R.C.B. Wong), Retina Australia (R.C.B. Wong, S.S.C. Hung, A. Pébay) and the Ophthalmic Research Institute of Australia (R.C.B. Wong, S.S.C. Hung, A. Pébay); Australian Research Council Future Fellowship (A. Pébay, FT140100047), Cranbourne Foundation Fellowship (R.C.B. Wong); intramural funding from the National Institutes for Health (R.C.B. Wong, S.S.C. Hung) and operational infrastructure support from the Victorian Government.
Materials
| Antibiotic Mix: | |||
| 250 ng/ml Antimycotic amphotericin B | Sigma | A2942-20ml | Antibiotic mix is made up in PBS. |
| 1X Penicillin/Streptomycin | Invitrogen | 15140-122 | |
| PBS (-) | Invitrogen | 14190-144 | |
| Knockout Serum Replacement (KSR) medium: | KSR medium is filtered using Stericup (Millipore, #SCGPU05RE) before use. bFGF is added fresh to the media before use. | ||
| 20% knockout serum replacement (KSR) | Invitrogen | 10828-028 | |
| DMEM/F12 with glutamax | Invitrogen | 10565-042 | |
| 1× MEM non-essential amino acid | Invitrogen | 11140-050 | |
| 0.5× Penicillin/Streptomycin | Invitrogen | 15140-122 | |
| 0.1 mM β-mercaptoethanol | Invitrogen | 21985 | |
| bFGF (10 ng/ml, added fresh) | Millipore | GF003 | |
| Keratinocyte medium: | |||
| EpiLife with 60 µM Calcium | Invitrogen | M-EPI-500-CA | |
| 1× Human keratinocyte growth supplement (HKGS) | Invitrogen | S-001-5 | |
| Fetal Bovine Serum (FBS) medium: | FBS medium is filtered using Stericup (Millipore, #SCGPU05RE) before use. | ||
| 10% fetal bovine serum (FBS) | Invitrogen | 26140079 | |
| DMEM | Invitrogen | 11995-073 | |
| 0.5× Penicillin/Streptomycin | Invitrogen | 15140-122 | |
| 2 mM L-glutamine | Invitrogen | 25030 | |
| 0.25% trypsin-EDTA | Invitrogen | 25200-056 | |
| Extracellular Matrix (ECM): | |||
| Matrigel | Corning | 354234 | Aliquot Matrigel stock and store in -80°C following manufacturer’s instructions. Stock concentration of Matrigel varies slightly from batch to batch (~9mg/ml). We recommend to use 200µl matrigel for coating a 12-well plate (~150µg/well). |
| Coating Matrix Kit | Invitrogen | R-011-K | |
| Plasmids: | Note that pCXLE-eGFP is only used for monitoring transfection efficiency and is not required for reprogramming. | ||
| - pCXLE-eGFP | Addgene | 27082 | |
| - pCXLE-hOct3/4-shP53F | Addgene | 27077 | |
| - pCXLE-hSK | Addgene | 27078 | |
| - pCXLE-hUL | Addgene | 27080 | |
| Transfection reagent Fugene HD | Promega | E231B | |
| Gelatin (from porcine skin) | Sigma | G1890 | Make up 0.1% gelatin in distilled water. Autoclave before use. |
| Reduced Serum medium: OPTI-MEM | Invitrogen | 31985062 | |
| Accutase | Sigma | A6964-100ml | |
| Mouse embryonic fibroblast (MEF) feeder | MEF can be inactivated by mitomycin C treatment or irradiation as described previously 16. | ||
| 26G needle | Terumo | NN2613R | |
| 6-well plate (tissue culture treated) | BD Biosciences | 353046 | |
| 12-well plate (tissue culture treated) | BD Biosciences | 353043 | |
| 10 cm dish (tissue culture treated) | BD Biosciences | 353003 | |
| Dispase | Invitrogen | 17105-041 | Use at 10mg/ml |
| Collagenase IV | Invitrogen | 17104-019 | Use at 1mg/ml |
| TRA-160 antibody | Millipore | MAB4360 | Use at 5µg/ml |
| OCT4 antibody | Santa Cruz | SC-5279 | Use at 5µg/ml |
| NANOG antibody | R&D Systems | AF1997 | Use at 10µg/ml |
| MycoAlert Detection kit | Lonza | LT07-418 |
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