从多能干细胞使用BMP拮抗剂Grem2心房心肌细胞分化
Summary
Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.
Abstract
用于从多能干细胞的心肌细胞的种群的协议已被开发，但是这些通常会产生混合的表型的细胞。热衷于追求涉及特定肌细胞亚型研究的研究人员需要更定向分化的方法。通过处理小鼠胚胎干细胞（ES）与Grem2，分泌的BMP拮抗剂，它是必要的体内心房腔室的形成，可以产生具有一个心房的表型的大量心脏细胞的。改造的MYH6-DsRed的-NUC多能干细胞系的使用允许识别，选择和心肌细胞的纯化。在这个协议中胚状体是从使用悬滴法MYH6-DsRed的-NUC细胞产生的，并保持在悬浮液中，直到分化第4天（D4）。在D4细胞用Grem2处理并铺板于明胶包被的平板。 D8之间-D10大承包领域的文化观察，继续扩大和M通过D14 ATURE。分子，组织学和electrophysiogical分析表明细胞Grem2处理的细胞获得心房状特性提供一种在体外模型来研究心房心肌细胞的生物学和它们对各种药剂的响应。
Introduction
多能干细胞用于产生和从困难宿主研究细胞到访问组织的基础研究和临床前研究，特别是在人类中1-5的有力工具。发育信号通路的合适的调制可以直接多能干细胞分化为所需表型的命运。许多协议已被开发，以产生从多能干细胞6-14心肌（CMS）。这些协议通常包括TFGβ超家族（活化素，BMP和TGF-β）和Wnt通过定时加入外源性生长因子和/或小分子10,12-15途径调制。这些协议是在增加了成为的CM细胞的百分比通常是有效的，但缺乏特异性，产生代表心房，心室和节点/传导系统谱系细胞的混合群。为了研究心肌特异性亚型更定向分化Appro公司ACH是必需的。
Gremlin2（Grem2，也被称为蛋白相关丹和地狱犬或PRDC的简称）是一种分泌BMP拮抗剂，在斑马鱼16心脏发育过程中是必要进行适当的心脏分化和心房腔的形成。治疗分化胚胎干细胞与Grem2在分化第4天，只是中胚层标记物的T的Brachyury和Cerberus的样1的峰表达后，增加的CM的产率，并产生主要心房谱系14的细胞池。
重组Grem2用于治疗的分化细胞，并且可以使用标准蛋白质的生产技术17制成，或者可以商购买到。它是在水溶液中高度可溶，并且可以在期望的时间点外源添加到培养物。
分化可以利用RT-qPCR的量化标记代表的表达来跟踪心血管祖细胞，心脏祖细胞，并致力于CMS。免疫也可用于识别和可视化心脏细胞类型的空间分布。
使用报告系统，以鉴定和分离的CM时，需要纯群体应用更容易进行。为此，我们推出了αMHC-DsRedNuc建设成为鼠标CGR8胚胎干细胞系14。 CGR8细胞生长和保持多能性无饲养细胞，促进扩增和分化测定18。在ES细胞系含有与心脏特异性的α-肌球蛋白重链（αMHC或MYH6）基因启动子下一个核定位信号的DsRed荧光蛋白编码序列。利用这些细胞，CM的可以容易地鉴定和分离为定量，细胞分选，电生理学，药物筛选，并研究心房分化的机制。
Protocol
Representative Results
Discussion
该协议通常产生培养物与那些心房谱系特征的CM的高百分比。正如任何分化方案之前，分化mESCs的质量应给予特别的注意。 mESCs应定期监测适当的形态（ 图1A）。该形成的EB的前发生的任何自发分化将严重限制心脏发生的效率，应传代（ 图1B）之前被移除。 EB大小也影响心脏发生。 200和1000元之间的EB启动细胞的数量已经过测试，每500 EB常规的细胞产生的CM的最高数字。被前…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
“：在调查培训计划在心血管机制”（JB）这项工作是由美国国立卫生研究院资助HL083958和HL100398（AKH）和2T32HL007411-33支持。
Materials
| GMEM | Life Technologies | 11710 | |
| FBS | Life Technologies | 10082 | |
| Glutamax | Life Technologies | 35050 | |
| LIF | EMD Millipore | ESG1107 | |
| ß-Mercaptoethanol | Sigma | M3148 | |
| IMDM | Sigma | I3390 | |
| Non-Essential Amino Acids | Sigma | M7145 | |
| 10 cm Tissue Culture Plates | Sarstedt | 83.3902 | |
| 10 cm Bacterial Petri Dishes | VWR | 25384-342 | |
| 6 cm Bacterial Petri Dishes | VWR | 25384-092 | |
| 6-well tissue culture plates | Sarstedt | 83.3920 | |
| Gremlin 2 recombinant protein | R&D Systems | 2069-PR-050 | |
| Sterile filter units | Thermo Fisher | 09-741-02 | |
| Gelatin (from porcine skin) | Sigma | G1890 | |
| 10X PBS, Sterile | Sigma | P5493 | |
| BSA | Sigma | 5470 | |
| 0.05% Trypsin-EDTA solution | Life Technologies | 25300054 | |
| DPBS, no Calcium, no Magnesium | Life Technologies | 14200 |
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