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
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;即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, 坎宁安等。(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 (Id1–4), 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 (IRX4、 MYL2) 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
Representative Results
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…
Disclosures
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) |
References
- Chong, J. J., et al. Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature. 510, 273-277 (2014).
- Kodo, K., et al. iPSC-derived cardiomyocytes reveal abnormal TGF-beta signalling in left ventricular non-compaction cardiomyopathy. Nature cell biology. 18, 1031-1042 (2016).
- Kim, C., et al. Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs. Nature. 494, 105-110 (2013).
- Colas, A. R., et al. Whole-genome microRNA screening identifies let-7 and mir-18 as regulators of germ layer formation during early embryogenesis. Genes & development. 26, 2567-2579 (2012).
- Cunningham, T. J., et al. Id genes are essential for early heart formation. Genes & development. , (2017).
- McKeithan, W. L., Colas, A. R., Bushway, P. J., Ray, S., Mercola, M. Serum-free generation of multipotent mesoderm (Kdr+) progenitor cells in mouse embryonic stem cells for functional genomics screening. Current protocols in stem cell biology. , 13 (2012).
- McKeithan, W. L., et al. An automated platform for assessment of congenital and drug-induced arrhythmia with hipsc-derived cardiomyocytes. Frontiers in physiology. 8, 766 (2017).
- Sharma, A., et al. High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells. Science translational medicine. 9, (2017).
- Burridge, P. W., et al. Chemically defined generation of human cardiomyocytes. Nature methods. 11, 855-860 (2014).
- Kattman, S. J., et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell stem cell. 8, 228-240 (2011).
- Laflamme, M. A., et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nature biotechnology. 25, 1015-1024 (2007).
- Lian, X., et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proceedings of the National Academy of Sciences of the United States of America. 109, 1848-1857 (2012).
- Willems, E., et al. Small molecule-mediated TGF-beta type II receptor degradation promotes cardiomyogenesis in embryonic stem cells. Cell stem cell. 11, 242-252 (2012).
- Yang, L., et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature. 453, 524-528 (2008).
- Palpant, N. J., et al. Generating high-purity cardiac and endothelial derivatives from patterned mesoderm using human pluripotent stem cells. Nature protocols. 12, 15-31 (2017).
- DeLaughter, D. M., et al. Single-cell resolution of temporal gene expression during heart development. Developmental cell. 39, 480-490 (2016).
- Liu, X., et al. The complex genetics of hypoplastic left heart syndrome. Nature genetics. 49, 1152-1159 (2017).
- Corrado, D., Link, M. S., Calkins, H. Arrhythmogenic right ventricular cardiomyopathy. New England journal of medicine. 376, 1489-1490 (2017).
- Kitamura, T., et al. Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics. Experimental hematology. 31, 1007-1014 (2003).
