大人の皮膚線維芽細胞からのインスリン分泌細胞を得るために安全かつ簡便な方法として、エピジェネティック変換
Summary
Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.
Abstract
Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a “highly permissive” state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated “epigenetic cell conversion”. It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.
Introduction
再生医療の基本的な目的は、修理または損傷置き換えるために使用することができる新しい機能細胞の生成は、組織を縮退します。別の細胞型からそれらを変換することによって、新しいものに容易に利用可能な成体細胞をリメイク、必要な細胞集団が豊富なまたはアクセスすることは困難ではない場合は特に、特に魅力的なアプローチです。しかし、成体細胞が著しく安定しています。彼らは成熟した端末専門に達すると、彼らは安定して1を保持し 、そのオプションの緩やかな制限を介して自分の分化状態を取得します。
体細胞（IPS）の多能性への再プログラミングを可能にするプロトコルの数が開発されている最後の年では、転写のセットは2,3因子の強制発現によって達成。あるいは、細胞の変換は、単一の4を導入し 、直接系列分化転換することによって得ることができます</supは>または転写の組み合わせは、5-7因子 。この戦略は、脱分化状態を経て移行を伴うが、特定の転写因子8の高発現を必要としません。
我々は最近、シチジン類似体5-アザシチジン（5-アザCR）、よく特徴付けられたDNAメチル化酵素阻害剤の脱メチル化特性に成体細胞の短時間の暴露に基づいて、変換プロトコルを開発しました。脱メチル化工程は、直ちに必要な端末の表現型を得ることを可能にする特異的な分化プロトコール9-11が続きます。この方法は、異なる系統の細胞への成熟、分化した細胞に変換することができ、ウイルスベクターを使用すると、任意の外因性の転写因子のトランスフェクションの両方を避けるために実質的な利点を有しています。セルの不安定性への安定した多能性状態の取得、及び関連する感受性の増加も避けられます。
<p class=「jove_content ">ここに提示され、完全に機能するインスリン分泌細胞への成人ヒト皮膚線維芽細胞の変換を可能にする詳細なプロトコール。しかし、種々の分化経路に向けてセルをアドレス指定するとき技術は、異なる細胞型に適用され、肯定的な結果を生成したことは注目に値します。さらに、エピジェネティックな変換が成功したアプローチの広い有効性と堅牢性を示唆している（原稿は提出）は、ヒトおよびブタの種9-13ならびに犬で使用されています。Protocol
Representative Results
Discussion
本原稿は、組織特異的な誘導プロトコールに続いて5-アザCR、過渡および短時間の曝露を介して、インスリン産生細胞へのヒト皮膚線維芽細胞の変換を可能にする方法を説明します。このアプローチは、転写因子またはマイクロRNAの強制発現や細胞がより不安定とミス14になりやすいなり安定した多能性状態の取得、なし、中胚葉から内胚葉関連細胞への切り替えを可能にします。
…Disclosures
The authors have nothing to disclose.
Acknowledgements
この作品は、Carraresi財団と欧州糖尿病財団（EFSD）によって賄われていました。 GPは、ミラノの大学のポスドクフェローシップによってサポートされています。著者は、COSTアクションFA1201 Epiconceptのメンバーである：エピジェネティクスとPericonception環境とコストのアクションBM1308共有は大型動物モデル（サラーム）に進みます。 TALBはCOSTアクションCM1406エピジェネティックなケミカルバイオロジー（EPICHEM）のメンバーです。
Materials
| Dulbecco's Phosphate Buffered Saline | Sigma | D5652 | PBS; for cell wash and solution preparation |
| Antibiotic Antimycotic Solution | Sigma | A5955 | Component of Fibroblast, HP and Pancreatic media |
| 100 mm petri dish | Sarstedt | 83.3902 | For Fibroblast isolation |
| Porcine Gelatin | Sigma | G1890 | For dish coating |
| Water | Sigma | W3500 | For solution preparation |
| 35 mm petri dishes | Sarstedt | 83.39 | For Fibroblast isolation |
| DMEM, high glucose, pyruvate | Life Technologies | 41966052 | For Fibroblast culture medium |
| Fetal Bovine Serum | Life Technologies | 10500064 | FBS; Component of Fibroblast and HP media |
| L-Glutamine solution | Sigma | G7513 | Component of Fibroblast, HP and Pancreatic media |
| Trypsin-EDTA solution | Sigma | T3924 | For Fibroblast dissociation |
| KOVA GLASSTIC SLIDE 10 WITH GRIDS | Hycor Biomedical | 87144 | Cell counting |
| 5-Azacytidine | Sigma | A2385 | 5-aza-CR, for increrase cell plasticity in fibroblasts |
| Ham's F-10 Nutrient Mix | Life Technologies | 31550031 | For HP medium |
| DMEM, low glucose, pyruvate | Life Technologies | 31885023 | For HP medium |
| KnockOut Serum Replacement | Life Technologies | 10828028 | Component of HP medium |
| MEM Non-Essential Amino Acids Solution | Life Technologies | 11140035 | Component of HP and Pancreatic Basal media |
| 2-Mercaptoethanol | Sigma | M7522 | Component of HP and Pancreatic Basal media |
| Guanosine | Sigma | G6264 | Nucleoside mix stock component of HP medium |
| Adenosine | Sigma | A4036 | Nucleoside mix stock component of HP medium |
| Cytidine | Sigma | C4654 | Nucleoside mix stock component of HP medium |
| Uridine | Sigma | U3003 | Nucleoside mix stock component of HP medium |
| Thymidine | Sigma | T1895 | Nucleoside mix stock component of HP medium |
| Millex-GS 0,22 µm | Millipore | SLGS033SB | For sterilizing of solution |
| FGF-Basic (AA 1-155) Recombinant Human Protein | Life Technologies | PHG0261 | bFGF; Component of HP and Pancreatic Basal medium |
| Bovine Serum Albumin | Sigma | A3311 | BSA; Component of Pancreatic Basal medium |
| DMEM/F-12 | Life Technologies | 11320074 | For Pancreatic Basal medium |
| B-27 Supplement Minus Vitamin A | Life Technologies | 12587010 | Component of Pancreatic medium |
| N-2 Supplement | Life Technologies | 17502048 | Component of Pancreatic Basal medium |
| Activin A Recombinant Human Protein | Life Technologies | PHG9014 | For Pancreatic medium |
| Retinoic Acid | Sigma | R2625 | For Pancreatic medium |
| Dimethyl sulfoxide | Sigma | D2650 | DMSO; for Retinoic Acid stock preparation |
| Insulin-Transferrin-Selenium | Life Technologies | 41400045 | ITS; for Pancreatic Final medium |
| Anti-Vimentin antibody | Abcam | ab8069 | For immunocytochemical analisys. Working dilution 1:100 |
| 4′,6-Diamidino-2-phenylindole dihydrochloride | Sigma | 32670 | DAPI. For immunocytochemical analisys. Working dilution 1µg/ml |
| 5-Methylcytidine | Eurogentec | MMS-900P-B | For immunocytochemical analisys. Working dilution 1:500 |
| Anti-C Peptide antibody | Abcam | ab14181 | For immunocytochemical analisys. Working dilution 1:100 |
| Anti-PDX1 antibody | Abcam | ab47267 | For immunocytochemical analisys. Working dilution 1:500 |
| Mercodia Insulin ELISA | Mercodia | 10-1113-10 | For insulin release detection |
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