マウス胚線維芽細胞の転写因子媒介リプログラミングに続いて心筋細胞のサブタイプを評価します
Summary
This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.
Abstract
Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs).
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.
Introduction
心臓は胚1、2に開発した最初の機能的器官です。循環器系に関連して、開発中に、酸素、栄養素、および廃棄物処理機構に供給する。三週間受精後、人間の心は初めて打つとその適切な規制は、心筋細胞（のCM）によって維持されています。これらの特殊化した細胞の不可逆的損失は、したがって、進行性の心不全の根底にある基本的な問題です。このようなゼブラフィッシュおよびアフリカツメガエルのようないくつかの生物は、心臓の再生の可能性を有しているが、大人の哺乳類の心臓は3、5、6、より限定されています。このように、心の重要な機能を考えると、心臓病は米国だけで60万人が死亡7を占め、世界における死亡の主な原因であることは驚くべきではありません。ザrefore、効率的に負傷した心筋を修理または交換するために、細胞ベースの治療は臨床上非常に興味深いものです。
山中ら8の精液の研究は、4つの転写因子の強制発現は、多能性幹細胞に完全に分化した線維芽細胞に変換するのに十分であることを示しました。しかし、すべての多能性幹細胞戦略の腫瘍形成能力は、治療目的のためのそれらの使用における重大な関心事となっています。これは、多能性段階を回避しながら細胞を分化転換するための代替方法を検索するための科学的なフィールドをやる気。最近、いくつかのグループは、転写の異所性発現は、GATA4、MEF2C、TBX5、および後に、Hand2（GMTとGHMT因子を用いて誘導心筋細胞様細胞（iCLMs）にマウス線維芽細胞の直接変換を表示することにより、この戦略の実現可能性を示していますそれぞれ）9,10。 Furthermore、同じ戦略は、 インビボおよびヒト由来の組織9、11、12で行うことができます。最近の研究では、追加の因子またはさらなる心臓再プログラミング効率13、14、15を改善するために調節することができるシグナル伝達経路を強調しています。まとめると、これらの研究は、再生治療のために向かう分化転換の可能性を示します。しかし、方法論的差異16にCMの再プログラミング、未知の分子機構、一貫性のない再現性の低効率、およびiCLMsの不均一な性質は、アドレス指定されないままです。
直接iCLM不均一性を評価するために、我々は、サルコメアの開発および心臓系統specificatioの同定のために離散的で堅牢な単一細胞アッセイを設計しましたn-2の機能的心筋細胞の必要な特性。それらの場所でユニークな電気的特性によって定義されたCMの少なくとも三つの主要なタイプは、心臓である：心房（AM）は、心室（VM）及びペースメーカー（PM）17、18、19、20。オーケストレーションの組み合わせで、それらは、血液の適切なポンピングを可能にします。心臓損傷の間、一つまたはすべてのサブタイプが影響を受ける可能性があり、および細胞治療のタイプは、ケースバイケースで対処する必要があります。少し作業は、サブタイプの仕様を調節する分子メカニズムを研究するために行われている間、現在、ほとんどの戦略は、心筋細胞の全体的な世代に焦点を当てています。
以下の研究は、適切によく組織サルコメアを定量化し、心筋細胞のサブタイプの多様なセットを識別する方法について詳しく説明します。ペースメーカー（PM）特異的レポーターマウスを用いて、Iを適用することができます誘導された心房のような筋細胞（IAM）、誘導された心室のような筋細胞（IVM）、および誘導されたPM-様筋細胞（IPMS）21を区別するためにmmunocytochemicalアプローチ。我々の観察に基づいて、サルコメア組織を示す細胞のみが自発的な拍動することが可能です。このユニークな再プログラミングプラットフォームは役割を評価することができます サルコメア組織、サブタイプの仕様、および単一セルの解像度でCMの再プログラミングの効率の特定のパラメータの。
Protocol
Representative Results
Discussion
本研究は、心臓転写のレトロウイルス媒介発現を介して、心臓のサブタイプの多様なセットにしたMEFの変換のためのダイレクトリプログラミング戦略を提供することはGATA4、MEF2C、TBX5、およびHand2（GHMT）要因。 PM固有レポーターマウスと組み合わせて多重免疫染色法を用いて、我々は、単一細胞の解像度でIAM、IVMS、およびIPMSを識別することができます。このようなアッセイは、サブタイプの…
Declarações
The authors have nothing to disclose.
Acknowledgements
A.F.-P. was supported by the National Science Foundation Graduate Research Fellowship under Grant No.2015165336. N.V.M was supported by grants from the NIH (HL094699), Burroughs Wellcome Fund (1009838), and the March of Dimes (#5-FY14-203). We acknowledge Young-Jae Nam, Christina Lubczyk, and Minoti Bhakta for their important contributions to protocol development and data analysis. We also thank John Shelton for valuable technical input and members of the Munshi lab for scientific discussion.
Materials
| DMEM | Sigma | D5796 | Component of iCLM media, Plat-E media, fibroblast, and Transfection media |
| Medium 199 | Thermo Fisher Scientific | 11150059 | Component of iCLM media |
| Fetal bovine serrum (FBS) | Sigma | F2442 | Component of iCLM media, Plat-E media, fibroblast, and Transfection media |
| Insulin-Transferrin-Selenium G | Thermo Fisher Scientific | 41400-045 | Component of iCLM media |
| MEM vitamin solution | Thermo Fisher Scientific | 11120-052 | Component of iCLM media |
| MEM amino acids | Thermo Fisher Scientific | 1601149 | Component of iCLM media |
| Non-Essential amino acids | Thermo Fisher Scientific | 11140-050 | Component of iCLM media |
| Antibiotic-Antimycotics | Thermo Fisher Scientific | 15240062 | Component of iCLM media |
| B-27 supplement | Thermo Fisher Scientific | 17504044 | Component of iCLM media |
| Heat-Inactivated Horse Serum | Thermo Fisher Scientific | 26050-088 | Component of iCLM media |
| NaPyruvate | Thermo Fisher Scientific | 11360-70 | Component of iCLM media |
| Penicillin/Streptomycin | Thermo Fisher Scientific | 1514022 | Component of Plat-E media and fibroblast media |
| Puromycin | Thermo Fisher Scientific | A11139-03 | Component of Plat-E media |
| Blasticidin | Gemini Bio-Products | 400-128P | Component of Plat-E media |
| Glutamax | Thermo Fisher Scientific | 35050-061 | Component of Fibroblast media |
| Confocal laser scanning LSM700 | Zeiss | For confocal analysis | |
| FuGENE 6 transfection Reagent | Promega | E2692 | Transfection reagent |
| Opti-MEM Reduced Serum Medium | Thermo Fisher Scientific | 31985-070 | Transfection reagent |
| Polybrene | Millipore | TR-1003-G | Induction reagent. Use at a final concentration of 8um/mL |
| Platinium-E (PE) Retroviral Packagin Cell Line, Ecotropic | CellBiolabs | RV-101 | Retroviral pacaking cell line |
| Trypsin 0.25% EDTA | Thermo Fisher Scientific | For MEFs and Plat-E dissociation | |
| Mouse anti α-Actinin (Clone EA-53) | Sigma | A7811 | Antibody for confocal analysis. Use at 1:200 |
| Chicken anti-GFP IgY | Thermo Fisher Scientific | A10262 | Antibody for confocal analysis. Use at 1:200 |
| Rabbit Pab anti-NPPA | Abgent | AP8534A | Antibody for confocal analysis. Use at 1:400 |
| Rabbit Pab anti Myl2 IgG | ProteinTech | 10906-1-AP | Antibody for confocal analysis. Use at 1:200 |
| Vectashield solution with DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Vector Labs | H-1500 | Dye for confocal analysis |
| Superfrost Plus Microscope slides | Thermo Fisher Scientific | 12-550-15 | 25 x 75 x 1.0 mm |
| BioCoat Fibronectin 12mm coverslips | NeuVitro Corp | GG-12-1.5 | Coverslips for confocal analysis |
| 100um cell strainer | Thermo Fisher Scientific | 08-771-19 | |
| 0.45um Syringes filters SFCA 25MM | Thermo Fisher Scientific | 09-740-106 | For virus filtration |
| 6ml Syringes | Covidien | 8881516937 | For virus filtration |
| Goat anti-Chicken IgY (H&L) A488 | Abcam | AB150169 | Secondary antibody for confocal analysis. Use at 1:400 |
| Donkey anti-rabbit A647 IgG(H+L) | Thermo Fisher Scientific | A31573 | Secondary antibody for confocal analysis. Use at 1:400 |
| Goat anti-mouse IgG(H+L) A555 | Thermo Fisher Scientific | A21422 | Secondary antibody for confocal analysis. Use at 1:400 |
| Triton X-100 | Sigma | 93443-100ml | For cell permeabilization |
| Dulbecco's PBS without CaCl2 and MgCl2 (D-PBS) | Sigma | D8537 | |
| Power Block 10X Universal Blocking reagent | Thermo Fisher Scientific | NC9495720 | Dilute to 1X in H20 |
| 16% Paraformaldehyde aqueous solution (PFA) | Electro Microscopy Sciences | 15710 | Use at 4% diluted in dH20 |
| 6 cm plates | Olympus | 25-260 | |
| 6-well plates | Genesee Scientific | 25-105 | |
| 24-well plates | Genesee Scientific | 25-107 | |
| 10 cm Tissue culture dishes | Corning | 4239 | |
| 15 cm Tissue culture dishes | Thermo Fisher Scientific | 5442 | |
| 15 ml Conical tubes | Corning | 4308 | |
| 50 ml Conical tubes | Corning | 4249 | |
| 0.4% Trypan blue solution | Sigma | T8154 | For viability |
| Ethyl Alcohol 200 proof | Thermo Fisher Scientific | 7005 | |
| Bleach | Thermo Fisher Scientific | 6009 |
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