多能干细胞衍生的心肌修复心肌细胞
Summary
我们提出三种新型和更有效的协议，用于人类诱导多能干细胞分化为心肌细胞，内皮细胞和平滑肌细胞，并且通过细胞注射补丁介导的细胞因子的递送组合提高移植细胞的植入的递送方法。
Abstract
人类诱导多能干细胞（人iPS细胞）必须充分分化为给药前的特定细胞类型，但常规协议分化人iPS细胞为心肌细胞（hiPSC-CMS），内皮细胞（hiPSC-ECS）和平滑肌细胞（SMC）常常由低收率，纯度和/或表型稳定性差的限制。这里，我们提出用于产生hiPSC-CMS，-ECs是基本上比传统方法更有效的新颖的协议，和-SMCs，以及用于细胞注射超过施用部位产生一个含有细胞因子的贴剂组合的方法。贴剂提高了注射细胞的同时保留，通过密封针轨道，以防止细胞从被挤出心肌，和细胞存活，通过在较长时间内释放胰岛素样生长因子（IGF）。在心肌缺血再灌注损伤的猪模型，植入率超过两倍大于当细胞与含细胞因子的贴剂比较的细胞而不补丁，和治疗与细胞和贴片都施用，但不能与单独的细胞，用心脏功能和梗塞大小显著改善有关。
Introduction
人类诱导多能干细胞（人iPS细胞）是用于再生细胞治疗的最有前途的试剂中，因为它们可以分化成不由病人的免疫系统拒绝的细胞的潜在无限范围和数量。然而，它们的自我复制和分化能力也能导致肿瘤的形成，因此，人iPS细胞需要充分分化成特定的细胞类型，如心肌细胞（CMS），内皮细胞（EC），和平滑肌细胞（SMC ），给药前。之一的细胞给药的最简单和最常用的方法是直接的心肌内注射，但是这是由天然心肌组织嫁接移植细胞的数量是非常低的。许多这种消耗可以归因于缺血组织的细胞毒性的环境;然而，当鼠胚胎干细胞（ESC）是直接注入未受损伤心中的心肌Ó交付被保留为3-5小时1，这表明施用的细胞的相当比例退出给药部位，这可能是因为它们是由过程中产生的高压通过针轨道挤出5百万细胞的唯一一句〜40％心肌收缩。
这里，我们提出新颖的和基本上更有效的方法，用于产生hiPSC衍生的心肌（hiPSC-CMS）2，内皮细胞（hiPSC-ECS）3，和平滑肌细胞（SMC）4。值得注意的是，此hiPSC-SMC协议是第一模仿广泛的通过引导细胞向一个主要合成的或收缩的SMC表型在体平滑肌5观察到的形态和功能特征。我们还提供细胞递送的方法，其通过创建一个含有细胞因子的血纤维蛋白p提高注射细胞的植入率ATCH在注射部位。补丁似乎改善两种细胞保留，通过密封针轨道，以防止细胞从离开心肌，和细胞存活，通过在一段至少三天的释放胰岛素样生长因子（IGF）。
Protocol
所有的实验过程符合阿拉巴马大学伯明翰大学的动物指导进行。 1.区分iPS细胞成hiPSC-CMS 外套预冷生长因子减少的凝胶状蛋白混合物6孔板于4℃过夜的孔中。吸使用前胶状蛋白质混合物。在5％CO 2和37℃下在mTeSR1介质补充有10μM的ROCK抑制剂播种人iPS细胞到预涂覆板，和培养的细胞（每孔1×10 5个细胞）。 每日刷新介质直到细胞达到90％汇合;然后，生…
Representative Results
差异化hiPSC-CMS，-ECs和-SMCs表征人iPS细胞的鉴别能力进行了评价2，3，4。流式细胞仪分析肌钙蛋白T（cTnT）的表达表明，最终hiPSC-CM人口的纯度超过90％（ 图1A，1B，面板B1）。几乎所有的细胞中的表达的慢肌球蛋白重链（ 图1B，</st…
Discussion
hiPSC-CMS的改善的产量/纯度
为了区分人类干细胞到cms传统的协议通常是由低产量和纯度的限制;例如，通过珀分离和心体形成得到的hESC的CM的只是35-66％表达慢肌球蛋白重链或肌钙蛋白6。分化hiPSC-CM群体的纯度可大幅增加通过选择已连接于启动子的报道基因的表达的CM-特异性蛋白7，8，9，但这…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
This work was supported by US Public Health Service grants NIH RO1s HL67828, HL95077, HL114120, and UO1 HL100407-project 4 (to JZ), an American Heart Association Scientist Development Grant (16SDG30410018) and a Research Voucher Award from University of Alabama at Birmingham Center for Clinical and Translational Science (to WZ).
Materials
| Protocol 1 | |||
| mTeSR1 medium | Stem cell technologies | 5850 | |
| Growth-factor-reduced matrigel | Corning lifescience | 356231 | |
| Y-27632 | Stem cell technologies | 72304 | |
| B27 supplement, serum free | Fisher Scientific | 17504044 | |
| RPMI1640 | Fisher Scientific | 11875-119 | |
| Activin A | R&D | 338-AC-010 | |
| BMP-4 | R&D | 314-BP-010 | |
| bFGF | R&D | 232-FA-025 | |
| Collagenase IV | Fisher Scientific | NC0217889 | |
| Hanks Balanced Salt Solution (Dextrose, KCl, KH2PO4, NaHCO3, NaCl, Na2HPO4 anhydrous) | Fisher Scientific | 14175079 | |
| Fetal Bovine Serum | Fisher Scientific | 10438018 | |
| 6-well plate | Corning Lifescience | 356721 | |
| 10cm dish | Corning Lifescience | 354732 | |
| Cell incubator | Panasonic | MCO-18AC | |
| Materials | Company | Catalog Number | Comments |
| Protocol 2 | |||
| Versene | Fisher Scientific | 15040066 | |
| Fibrinogen | Sigma-Aldrich | F8630-5g | |
| Thrombin | Sigma-Aldrich | T7009-1KU | |
| EMB2 medium | Lonza | CC-3156 | |
| VEGF | ProSpec-Tany | CYT-241 | |
| EPO | Life Technologies | PHC9431 | |
| TGF-ß | Peprotech | 100-21C | |
| EGM2-MV medium | Lonza | CC-4147 | |
| SB-431542 | Selleckchem | S1067 | |
| CD31 | BD Bioscience | BDB555445 | |
| CD144 | BD Bioscience | 560411 | |
| 15 mL centrifuge tube | Fisher Scientific | 12565269 | |
| Eppendorff Centrifuge | Eppendorf | 5702R | |
| Materials | Company | Catalog Number | Comments |
| Protocol 3 | |||
| CHIR99021 | Stem cell technologies | 720542 | |
| PDGF-ß | Prospec | CYT-501-10ug | |
| Materials | Company | Catalog Number | Comments |
| Protocol 4 | |||
| Olive oil | Sigma-Aldrich | O1514 | |
| Gelatin | Sigma-Aldrich | G9391 | |
| Acetone | Sigma-Aldrich | 179124 | |
| Ethanol | Fisher Scientific | BP2818100 | |
| Glutaraldehyde | Sigma-Aldrich | G5882 | |
| Glycine | Sigma-Aldrich | G8898 | |
| IGF | R&D | 291-G1-01M | |
| Bovine serum albumin | Fisher Scientific | 15561020 | |
| Heating plate | Fisher Scientific | SP88850200 | |
| Water bath | Fisher Scientific | 15-462-10Q | |
| Materials | Company | Catalog Number | Comments |
| Protocol 5 | |||
| CaCl2 | Sigma-Aldrich | 223506 | |
 -aminocaproic acid |
Sigma-Aldrich | A0420000 | |
| MEM medium | Fisher Scientific | 12561-056 | |
| Syringe | Fisher Scientific | 1482748 | |
| Anesthesia ventilator | Datex-Ohmeda | 47810 | |
| Anesthesia ventilator | Ohio Medical | V5A | |
| Defibrillator | Physiol Control | LIFEPAK 15 | |
| 1.5T MRI | General Electric | Signa Horizon LX | |
| 7T MRI | Siemens | 10018532 | |
| Gadolinium Contrast Medium (Magnevist) | Berlex | 50419-188-02 | |
| 2-0 silk suture | Ethilon | 685H | |
| 3-0 silk suture | Ethilon | 622H | |
| 3-0 monofilament suture | Ethilon | 627H |
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Diesen Artikel zitieren
Zhu, W., Gao, L., Zhang, J. Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair. J. Vis. Exp. (120), e55142, doi:10.3791/55142 (2017).