评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程
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
心脏是在胚胎1，2，开发的第一个功能器官。与循环系统相结合，其提供氧，营养物，和开发过程中的废物处理机制。受精后三个星期，人的心脏跳动，第一次和适当的调整则是由心肌细胞（CMS）维护。因此，这些特殊细胞的不可逆损失是潜在的进行性心脏衰竭的根本问题。而一些生物如斑马鱼和爪蟾具有用于心脏再生的潜力，成年哺乳动物心脏较为有限3，5，6。因此，由于心脏的重要功能，是不令人吃惊的是心脏疾病是全世界死亡的首要原因，占仅7美国60万人死亡。该refore，基于细胞的疗法有效地修理或更换损伤心肌是伟大的具有临床意义。
山中和同事8的开创性的研究表明，四种转录因子的强制表达足以完全分化的成纤维细胞转化为多能干细胞。不过，所有的多能干细胞的策略的致瘤能力已在其用于治疗目的使用的一个关键问题。这促使科学领域寻找替代方法转分化的细胞，同时避免多能的阶段。最近，一些团体已经通过与转录异位表达显示小鼠成纤维细胞直接转化为诱导的心肌样细胞（iCLMs）显示了这一战略的可行性因素GATA4，MEF2C，Tbx5中，后来，HAND2（GMT和GHMT分别）9，10。 Furthermore，同样的策略可以在体内和人衍生的组织9，11，12来进行。最近的研究已强调了额外的因素或可被调制以进一步改善心脏重编程效率13，14，15的信号通路。总之，这些研究表明定向转分化为再生疗法的潜力。然而，CM重新编程，未知的分子机制，重复性不一致由于方法上的差异16低效率，iCLMs的异质性仍未得到解决。
为了直接评价iCLM异质性，我们设计了一个离散的和强大的单细胞测定为肌节发展和心脏谱系specificatio的识别正两个功能的心肌细胞的必要特征。存在由它们的位置和独特的电特性定义在心脏的至少三个主要类型的CM：心房（AM），心室（VM）和起搏器（PM）17，18，19，20。在一个精心策划的组合，他们让血液正常抽水。在心脏损伤，一个或所有亚型可能会受到影响，细胞治疗的类型，需要根据具体情况逐案予以解决。目前，大多数战略，注重整体代心肌细胞，而少量的工作正在做研究，调节亚型规范的分子机制。
下面详细研究如何正确量化井井有条肌节，并确定一组不同的亚型心肌的。使用心脏起搏器（PM）专用鼠标的记者，我们可以申请一个我mmunocytochemical方法来区分诱发房状细胞（IAM），诱发室状细胞（IVM），并诱导般的PM肌细胞（IPMS）21。根据我们的观察，只表现出肌组织细胞能够自发跳动。这种独特的重新编程平台可以评估的作用 在肌组织，子规范，CM重编程效率的某些参数在单细胞分辨率。
Protocol
Representative Results
Discussion
本研究通过心脏转录的逆转录病毒介导的表达提供的MEF转换的直接重新编程战略转化为一组不同的亚型心脏因素GATA4，MEF2C，Tbx5中，和HAND2（GHMT）。在使用具有特定的PM记者鼠标组合多重染色的方法，我们能够确定（IAM），网络视频监控软件，并在IPMS单细胞分辨率。这种试验可以在隔离能力个别转录因子对亚型多样性和肌节发展的贡献的体外系统的实验研究。与此同时，这可能会带?…
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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