Summary

人多潜能干细胞第一心场样心脏祖细胞和心室样细胞的生成

Published: June 19, 2018
doi:

Summary

在这里, 我们描述了一种可伸缩的方法, 使用激活 a 和慢病毒北疆介导的 Id1-overexpression 的简单组合, 从人类多潜能干细胞中生成第一个心场样的心脏祖细胞和心室样心肌细胞。

Abstract

大量的功能性人类多潜能干细胞的产生是基于细胞的心脏治疗和疾病建模的先决条件。我们最近表明, Id 基因是必要的, 足以指定第一个心脏领域祖细胞在脊椎动物的发展。这种分化协议利用这些发现, 并使用 Id1 过度表达结合激活 A 作为有力的指定线索, 以产生第一个心脏场状 (FHF) 祖细胞。重要的是, 由此产生的祖细胞有效地区分 (~ 70–90%) 成心室状心肌。这里我们描述了一个详细的方法到 1) 生成 Id1-overexpressing hPSCs 和 2) 区分可伸缩数量的 cryopreservable FHF 祖细胞和心室样心肌。

Introduction

大规模生产人多潜能干细胞 (hPSCs) 源性心脏祖细胞和心肌细胞是干细胞治疗的先决条件1, 疾病建模2,3和快速表征新的路径调节心脏分化4,5,6和生理学7,8。虽然一些研究9,10,11,12,13,14,15以前描述高效心脏分化协议从 hPSCs, 没有解决导致心肌细胞的心脏领域起源, 尽管辨认重要分子区别左 (第一心脏领域) 和权利 (第二心脏领域)心室心肌细胞16与心场特异性先天性心脏病的存在;发育不良左心综合征17或致心律失常右心室发育不良18。因此, hPSCs 的心源和心肌细胞的产生是提高其作为治疗和疾病建模工具相关性的必要条件。

这个协议依赖于 Id1 的本构表达, 最近确定的5第一心脏领域-指定提示, 结合激活 a, 是必要的, 足以启动 cardiogenesis 在 hPSCs。值得注意的是, 坎宁安。(2017)5显示 Id1-induced 祖细胞特别表达第一心脏领域 (HCN4, TBX5), 但不是第二心脏领域标记 (SIX2, ISL1), 当他们接受心脏分化。此外, 作者还表明, 转基因小鼠胚胎缺乏完整的基因家族 (Id14), 发展不形成第一心脏领域的心脏祖细胞, 而更多的内侧和后心脏祖细胞 (第二心脏领域) 仍然可以形成, 从而表明, Id 蛋白是必不可少的启动第一心脏领域 cardiogenesis在体内。方便地, Id1-induced 祖细胞可冷冻保存, 自发分化为心肌样特征, 包括心室特异性标记 (IRX4MYL2) 表达和心室状动作电位。

在这里, 我们描述了一个简单的和可伸缩的方法, 以产生第一个心脏场样 (FHF) 心脏祖细胞和心室样心肌 Id1 overexpressing hPSCs。该协议的一个重要特点是, 通过一个方便的超低温保存步骤, 拆的心肌祖细胞产生的可能性。总之, 本协议详细说明了必要步骤 (1) 生成 Id1-overexpressing hPSCs, (2) 从 hPSCs 生成 FHF 心祖细胞, (3) cryopreserve 产生祖细胞, (4) 恢复 FHF-l 心祖分化, 生成高度丰富的 (> 70–90%) 跳动的心室状心肌细胞。

Protocol

1. Id1 病毒的制备和感染 由转染 pCMVDR8.74、pMD2、pCDH-EF1-Id1-PGK-PuroR (Addgene: 质粒 #107735) 生成 Id1 overexpressing 慢病毒北疆 HEK293T 细胞。收集病毒粒子, 滤液, 从上清纯化, 并存储在-80 摄氏度, 如在次郎等。(2003)19. 或者, 通过向慢病毒北疆生产的供应商发送 pCDH-EF1-Id1-PGK-PuroR, 在商业上生产 Id1 慢病毒北疆。注意: 请遵守慢病毒载体安全法规和标准操作协议。注意: 重要?…

Representative Results

hPSCs 的产生Id1线条hPSCs 感染慢病毒北疆调解 Id1 过度表达 (图 1a)。一旦 hPSCId1生成, 转基因的表达是量化的 qRT PCR (图 1B)。只有 hPSCId1线表达Id1 mRNA 的水平大于0.005 倍的GAPDH应用于分化。 <p class="jove_content" fo:keep-together.within-…

Discussion

为成功鉴别, 请务必密切遵循上面列出的指示。此外, 我们在这里强调的关键参数, 强烈影响分化的结果。在开始分化之前, 应观察以下三种形态学参数: hPSCsId1的茎形态、高细胞压实和0天的高汇流 (> 90%)。在这方面, 最佳分化条件最好是以电镀分离 hPSCsId1为单细胞, 并允许它们形成紧密的聚类膜, 在2到3天的过程中增长到90% 汇合。反之, 如果 hPSCId1文化表现出较差的细胞和菌落…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

我们感谢可乐实验室的成员对稿件进行有益的讨论和评论。这项研究得到了 NIH/NIEHS R44ES023521-02 和 CIRM DISC2-10110 对可乐博士的资助。

Materials

ACTC1 antibody Sigma A7811
Activin A Stem Cell Technologies Hu Recom Activin A
Antibiotic Antimycotic (Anti-Anti) Thermo Fisher Scientific 15240062
B27 supplement Thermo Fisher Scientific 17504044
B27 supplement w/o – insulin Thermo Fisher Scientific A1895601
B27 supplement w/o – vitamin A Thermo Fisher Scientific 12587001
CDH5 antibody R&D Systems AF938
CryoStor CS10 Stem Cell Technologies 7930 Cryopreservation reagent
DMEM high Glucose Mediatech 10-013-CV 
DPBS w/ Ca & Mg Corning 21-030-CV
EDTA Thermo Fisher Scientific 15575-038
FBS VWR 89510-186
FluoVolt membrane potential kit   Thermo Fisher Scientific F10488 For optical action potential acquisition, please refer to McKeithan et al. 2017
KnockOut Serum Replacement Gibco 10828010
Matrigel, Growth Factor Reduced Corning 356231 Coating reagent
mTeSR1 media kit Stem Cell Technologies 5850
PBS w/o Ca & Mg Corning 21-040-CV
Penicillin-Streptomycin Gibco
Puromycin  Acros 227422500
ReLeSR Stem Cell Technologies 5872 Enzyme-free dissociation reagent
RPMI 1640 Thermo Fisher Scientific 11875-093
TAGLN antibody Abcam ab14106
Thiazovivin Stem Cell Technologies 72254 RHO/ROCK pathway inhibitor
TrypLE Express Thermo Fisher Scientific 12605 -010 1X enzyme-containing dissociation reagent
Tyrodes solution mix packets   Sigma T2145-10X1L (For optical action potential acquisition, please refer to McKeithan et al. 2017)

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Yu, M. S., Spiering, S., Colas, A. R. Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells. J. Vis. Exp. (136), e57688, doi:10.3791/57688 (2018).

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