人类心肌细胞的生成：从馈线人类诱导多能干细胞的分化协议
Summary
多能干细胞，胚胎或诱导多能干细胞（iPS），构成人类分化的细胞，包括心肌细胞的重要来源。在这里，我们将专注于心脏iPS细胞的诱导，展示如何使用它们来获得功能的人类心肌细胞通过胚状体为基础的协议。
Abstract
为了研究心脏发育的事件驱动和确定导致心肌人类疾病的分子机制，它是必不可少的，以产生人心肌功能的CM。使用这些细胞在药物发现和毒理学研究也将是非常有益的，允许人类来源的细胞要验证的预临床上用于治疗心脏疾病的新的药物分子。的CM的可能来源，诱导多能干细胞（iPS细胞）是最有前途的，因为它们可以容易获得的患者组织直接来源于具备的特性的主体1的所有类型的细胞产生的能力。已经提出了几种方法进入CMS分化的iPS细胞，包括从古典的胚状体（EBS）聚合方法已有化学定义的协议2,3。在这篇文章中，我们提出了一种基于EBS-协议，并展示如何METHOd可以高效地生成功能CM从馈线的iPS细胞样细胞。
Introduction
从历史上看，推动人类发展和疾病的遗传和分子机制的调查已基于上一代转基因动物模型。然而，众多的人类表型未能成功复制老鼠，主要是因为现有两个物种之间的生物差异。另一方面，获得人体组织可能会受到限制的，通常不允许足够的材料来获得深入的实验研究。心血管生物学领域遭受来自这两个限制：人体心脏的生理是显着的不同，鼠标和心脏组织是一个重大的数量只有验尸报告或在心脏手术期间访问。因此，找到一个最佳来源的分化功能的人类CMS成为心血管生物学中的中心话题，多已作出努力，来解决这个问题。已经提出了各种细胞类型，我ncluding骨骼肌成肌细胞，本地心脏干细胞，骨髓单核细胞，血管内皮前体细胞，间充质干细胞。然而，获得的数据，使用这些细胞并不一致4。
人类胚胎干细胞（ESC）的推导第5和Yamanaka和汤姆森6,7诱导多能干细胞（iPS）的开创性发现后似乎提供解决方案：这些细胞具有无限增长的能力和潜力给予上升到所有三个胚层的细胞衍生工具，包括CMS。 iPS细胞的使用提供了进一步的优点：来自自体成体细胞，它们携带的个人或患者衍生它们的相同的基因组。因此，他们允许在体外人类遗传性疾病的调查和机制的发展模式，推动发展的。凭借这一特性，iPS细胞也可以Øvercome问题相关的免疫排斥和伦理问题8。出于这些原因，尽管胚胎干细胞仍然是干细胞生物学领域的黄金标准，世界各地的研究人员现在正在向iPS细胞技术。
iPS细胞在体外人类疾病模型的各种条件，包括先天性心血管疾病9,10。近日，应用iPS细胞疾病模型已进行单基因心脏疾病（ 如长QT综合征，儿茶酚胺敏感性多形性室性心动过速）和病理学心脏缺陷是一个复杂的表型（ 即 Leopard和蒂莫西·综合征，扩张型心肌病）的一部分。这些报告证实， 在体内的疾病患者特异性的iPS细胞分化成CMS显示类似的表型和功能特性。
然而，有仍有许多挑战，提高疗效诱导的iPS细胞转化为心脏谱系。通过聚集形成从人类胚胎干细胞自发产生的CMS称为胚状体（EB）已被证明成功的17。由于这一发现，许多其他的方法已经被提出。许多研究小组已经大大提高了工作效率的分化协议，并已走向化学定义和动物产品的培养试剂（见马默里，C. 等，流通研究 （2012年）进行全面检讨现有的所有方法3 ）。
然而，传统的方法基于EB聚集的仍然是最常用的执行功能研究和调查疾病机制。我们建议的协议是基于iPS细胞的聚集成胚体培养在血清和抗坏血酸的存在下，这已被证明，以提高心脏的分化过程，与positively影响这些细胞的成熟18,19。在这篇文章中，我们将通过这种方法，在细节和将展示如何使用无饲养层的iPS细胞系产生特定病人的iPS衍生CMS。
Protocol
Representative Results
Discussion
多能干细胞有潜力分化成CMS自发，虽然与线之间的低效率和高变异。因此，发展新型感应方法集中在提高效率的过程和走向更明确的协议。然而，大多数这些新的分化方法是相当复杂的，需要精心设计的培养条件下，定时的精细控制，试剂浓度，与昂贵的细胞因子和生长因子的治疗。此外，各种iPS细胞株中的内源性的细胞因子信号电平的差异使其难以标准化，这往往必须调整到适当的水平，每种?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
BS支持大学，米兰比可卡暑期学生科研训练计划，研究卫生意大利外交部和意大利教育部，大学及研究基金会马尼塔斯的GC资金支持，我们感谢迈克尔的VG拉特罗尼科为批判地阅读手稿。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Media and Reagents | |||
| Human ESC-qualified Matrix | BD Biosciences | 354277 | Thaw the 5 ml bottle at 4 °C on ice O/N, aliquot in pre-chilled cryo-tubes on ice, according to the data sheet dilution instructions and store at -20 °C . |
| Fibronectin, from human plasma, 0.1% solution | Sigma | F0896-2MG | Working concentration: 5 μg/cm2 |
| Laminin | Sigma | L2020-1MG | Working concentration: 5 μg/cm2 |
| PBS (no Mg and Ca), 10X | Lonza | BE17-515F | |
| PBS (with Mg and Ca), 10X | Bio Sera | XC-S2067/500 | |
| Dispase II, neutral protease, grade II | Roche | 4942078001 | Stock: 10 mg/ml in DMEM-F12 at -20 °C, working solution: 1 mg/ml in PBS (no Mg/Ca) |
| Trypsin/EDTA, 0.5% | Sigma | T3924 | |
| Collagenase type 2 | Worthington | 4176 | Dilute to 480 U/ml (e.g. 1.6 mg/ml) in PBS with Mg and Ca |
| DMEM-F12 | Life Technologies | 21331-020 | |
| Knockout DMEM | Life Technologies | 10829-018 | |
| Knockout Serum Replacement (KSR) | Life Technologies | 10828-028 | Thaw at 4 °C, aliquot and store at -20 °C |
| Foetal Bovine Serum (origin South America) | Invitrogen | 10270-106 | Batches should be tested for their cardiogenic potential. Thaw at 4 °C, heat-inactivate at 56 °C for 30 min, aliquot and store at -20 °C |
| PenStrep, penicillin-Streptomycin, 100X | Life Technologies | 15140-122 | |
| GlutaMAX, 100X | Life Technologies | 35050-038 | |
| MEM NEAA, 100X | Invitrogen | 11140-135 | |
| N2 supplement, 100X | Life Technologies | 17502-048 | |
| B27 supplement, 50X | Life Technologies | 12587-010 | |
| 2-mercaptoethanol | Invitrogen | 31350-010 | |
| Gelatin, from porcine skin | Sigma | G1890-100G | Make stock at 1% in water, store at -20 °C. Dilute to 0.1% in PBS and keep at 4 °C |
| mTeSR1 | Stem Cell Technologies | 5850 | Combine Supplement 5X with the basic medium, aliquot and store at -20 °C. Do not keep complete medium at 4 °C for more than 1 week |
| Nutristem hESC XF | Stemgent from Biological Industries | 05-100-1A | Thaw at 4 °C O/N, aliquot and store at -20 °C until use. Aliquots may be kept at 4 °C for no more than a week. |
| mFreSR | Stem Cell Technologies | 5853 | |
| Ascorbic acid | Sigma | A4544-25G | Prepare stocks at 10 mg/ml in water. Store aliquots at -20 °C in the dark. Add to EBM at the final concentration (500X) |
| Plasticware | |||
| 35 mm culture dishes | Becton Dickinson | 353001 | |
| Cell scraper | Corning Incorporated | 3008 | |
| 12-well plates | Becton Dickinson | 353043 | |
| Permanox 2-well chamber slides | Thermo Fisher Scientific | 177437 | |
| Glass 2-well chamber slides | Thermo Fisher Scientific | 177380 | |
| 6-well plates, ultra-low-attachment surface | Corning Incorporated | 3471 | |
| 18G needle | Becton Dickinson | 304622 | |
| Glass pasteur pipettes, 230 mm | VWR International | 612-1702 | |
| 2.5 ml syringe | Becton Dickinson | 300188 | |
| Equipments | |||
| IVF Workstation | KSystem, by Nikon | ||
| Nikon SMZ1500 Stereomicroscope | Nikon |
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