心外膜生长培养测定和<em>离体</em>心外膜源性细胞迁移的评估
Summary
Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.
Abstract
心外膜细胞系单层的心脏，提供刺激心肌细胞增殖旁分泌因子和发育和疾病过程中直接促进心血管祖细胞。而许多因素已在外膜的细胞（EPDC）动员牵连，管理其后续的迁移和分化的机制知之甚少。这里，我们目前在体外和体外的战略研究EPDC运动和分化。首先，我们描述了从胚胎小鼠心脏由生长的文化获得初级心外膜细胞的方法。我们还介绍了一个详细的协议，以评估在器官培养系统标记EPDC的三维迁移。我们提供了使用这些技术，在心外膜心肌素相关的转录因子基因缺失削弱EPDC迁移的证据。这种方法作为一个平台来评估EPDC候选修饰符生物学和可用于开发遗传或化学筛选，以确定EPDC动员的新颖调节可能是对心脏修复有用。
Introduction
心外膜是间皮细胞单层的所有行心脏和影响心脏发育，成熟和修理。通过旁分泌信号的高度协调交流，心肌和心外膜之间的亲密对话，是心脏的生长和非心源性心肌细胞谱系1的形成是必不可少的。心外膜衍生细胞（EPDC）从心外膜细胞的子集出现通过上皮至间质转变（EMT）2，侵入外膜下空间和潜在的心肌，并分化主要成冠状血管壁细胞和心脏成纤维细胞，并以一其次，内皮细胞和心肌细胞3-9。
调节外膜EMT和EPDC入侵机制已被归因于各种分子，包括分泌配体10-13，细胞表面受体和粘附分子14,15，单组的协调动作心尖基极性16,17，小GTP酶18,19，和转录因子lators 2,20,21。虽然许多EPDC迁移的分子效应已经确定，更好地理解激发调动EPDC在胚胎可能会加速发展战略的生理信号来操纵在成年改善心脏修复这个过程。
旨在查明EPDC动员新的监管机构研究依赖于这种细胞群的净化或追踪个人心外膜细胞的迁移。一个或标记基因，包括Wt1的 ，TCF21，Tbx18，和/或ALDH1A2的组合，通常用于确定胎儿心外膜1。然而，使用这些标记来跟踪迁移EPDC不是最佳的，因为外膜标志物的表达减弱在心脏发育的过程中，常常失去时心外膜细胞进行transdifferentiation到间质细胞。
在的Cre / loxP系统的应用，在与谱系追踪记者一起已经在永久性标记心脏发育过程中的心外膜和其后代，并在成人7,22,23以下缺血性损伤是有用的。几个心外膜限制性酶Cre线已经产生，并且广泛用于标记EPDC和有条件基因缺失策略1。这些研究已经导致各种心外膜谱系的表征，和EPDC运动和分化的关键介体的鉴定。然而，越来越多的证据也表明心外膜是祖细胞4,24,25的异质群体。因此，仅心外膜细胞的一个子集将使用一个给定的Cre司机进行定位。
为了标签的整个外膜的无论酶Cre特异性，许多实验室已经利用生长尽头TURE系统或体外器官培养的方法来隔离忠实或标签心外膜细胞而不取决于遗传谱系的标志物表达26-28。对于先体外后体内的迁移研究，胚胎的心前心外膜EMT并在补充有绿色荧光蛋白（GFP）-expressing腺病毒（广告/ GFP）9,18培养基中培养分离的。这种方法允许对整个心外膜的有效的标记，而不是利用Cre介导的重组的细胞的子集。心脏培养物暴露于EMT的已知诱导剂，刺激EPDC 28,29的动员。 体外 和体内分析通过体外心外膜外植体培养，这是为探索驱动EPDC迁移的详细机制的特别有用的方法是补充。
在这里，我们描述了用于分离主心外膜细胞在体外 epicardi研究方法人EMT，以及用于离体的器官培养系统中分析EPDC蠕动。我们最近通过遗传调制信号轴操纵EPDC 9的基于肌动蛋白的迁移的心肌素相关转录因子（MRTF）/血清应答因子（SRF）证明了该方法的实用性和鲁棒性。虽然我们的结果强调必要调节EPDC迁移一个信令通路，这些方法适用于解开，它们共同协调EPDC迁移和分化的机制。此外，外植体和体外培养系统能够在功能屏幕来实现，以确定EPDC动员新的监管机构在心脏再生治疗应用。
Protocol
注意：用小白鼠试验全部由大学委员会动物资源在罗切斯特大学的认可。 1.心外膜生长培养试验（图1A） 准备工作通过用5％胎牛血清（FBS）和1％青霉素/链霉素（青霉素/链霉素）补充培养基199（M199）准备5毫升原心外膜细胞的分离介质中的。 预暖培养基A和100毫升的Hanks'平衡盐溶液（HBSS）在37℃水浴中。 允许胶原包被的玻片温热至室温30分钟。 将100?…
Representative Results
心外膜可以通过采取其外观的位置优势，剥削发育可塑性和EPDC内在的迁移行为采用生长培养法进行有效隔离。小鼠胚胎的心都在E11.5心外膜EMT和培养的背面朝下放在胶原涂层玻片前隔离26（ 图1A，B）。植心将继续萎缩;然而，心外膜将坚持胶原基质和从心肌培养24小时（ 图1C）内迁移关闭。可以用鹅卵石样的形态，表明身份间皮（ 图1…
Discussion
在这里，我们列出了详细的方法，通过产物分离主心外膜细胞和跟踪体外心脏文化EPDC迁移。对生长培养物，在适当的时间以除去外膜贫心脏实验之间略有不同。过夜温育后的外植体的定期监测建议来衡量心外膜增生的程度和成纤维细胞出现之前提取心脏。以确保纯度，心应explanting防止非心外膜谱系的积累的24小时内被除去。修剪掉流出道和之前的心脏转移到玻片还可以减少污染的细胞类型…
Declarações
The authors have nothing to disclose.
Acknowledgements
EMS是由来自卫生研究院（NIH）的国家机构的资金支持[授权号R01HL120919]。美国心脏协会[授权号10SDG4350046]。来自美国国立卫生研究院[授权号UL1 TR000042]罗切斯特CTSA奖的大学;从AAB CVRI启动资金。 MAT是由授予医学和牙科学院罗彻斯特来自霍华德休斯医学研究所医学成 – 格拉德倡议大学的部分资助;从NIH [授权号GM068411]一个机构露丝L. Kirschstein国家研究服务奖。
Materials
| Dulbecco's Modified Eagle Medium | Hyclone | SH3002201 | DMEM |
| Hanks' Balanced Salt Solution | Hyclone | SH3003102 | HBSS |
| Medium 199 | Hyclone | SH3025301 | M199 |
| Dulbecco’s Phosphate-Buffered Saline | Hyclone | SH3002802 | DPBS |
| Fetal Bovine Serum | Gemini | 100-106 | FBS |
| Penicillin/Streptomycin Solution | Hyclone | SV30010 | |
| Corning Biocoat Collagen I, 4-Well Culture Slides | Corning | 354557 | |
| Multiwell 24-well plates | Falcon | 08-772-1 | |
| Dissecting microscope | Leica | M50 | Equipped with Leica KL300 LED might source |
| Confocal miscroscope | Olympus | 1X81 | Equipped with muli-argon laser 405/488/515, FV10-MCPSU laser 405/440/635, 559 laser, and mercury lamp |
| Bioruptor | Diagenode | UCD-200 | |
| Transforming growth factor beta 1 | R&D Systems | 100-B-001 | TGF-β1 |
| Platelet-derived growth factor BB | R&D Systems | 220-BB-010 | PDGF-BB |
| Trizol Reagent | Applied Biosystems | 15596-026 | Caution, hazardous material |
| TURBO DNA-free Kit | Life Technologies | AM1907 | DNaseI |
| iScript cDNA Synthesis Kit | BioRad | 1708891 | |
| UltraPure Glycogen | Life Technologies | 10814-010 | |
| SYBR Green | BioRad | 170-8880 | |
| Protease inhibtor cocktail tablets | Roche | 11836170001 | |
| Alexa Goat-anti-rabbit 488 | Life Technologies | A11008 | Secondary antibody |
| Alexa Goat anti-rabbit 594 | Life Technologies | A-11012 | Secondary antibody |
| Alexa Goat anti-mouse 594 | Life Technologies | A-11005 | Secondary antibody |
| Mouse anti-smooth muscle alpha actin, Cy3-conjugated | Sigma-Aldrich | C6198 | monoclonal antibody, clone 1A4 |
| Mouse anti-Wilms tumor 1 (WT1) | Life Technologies | MA1-46028 | monoclonal antibody, clone 6F-H2 |
| Rabbit anti-ZO1 | Life Technologies | 40-2200 | polyclonal antibody |
| Rabbit anti-Collagen Type 4 | Millipore | AB756P | polyclonal antibody |
| 4',6-Diamidino-2-Phenylindole, Dihydrochloride | Life Technologies | D1306 | DAPI |
| Fluorescent mounting medium | DAKO | S3023 | |
| 16% Paraformaldehyde solution | Electron Microscopy Sciences | 15710 | PFA diluted to 4% in DPBS. Caution, hazardous material |
| Sucrose | Sigma-Aldrich | S0389 | |
| Tissue Freezing Medium | Triangle Biomedical Sciences | TFM-B | |
| Pap pen | DAKO | S2002 | |
| Disposable mictrotome blades | Sakura Finetek | 4689 | |
| Microscope slides | Globe Scientific | 1358W | positively charged, 25 x 75 x 1mm |
| Cryostat | Leica | CM1950 | |
| Forceps | Fine Science Tools | 11295-00 | |
| Surgical Scissors | Fine Science Tools | 91460-11 | |
| Disposable base molds | Fisher Scientific | 22-363-553 | |
| Ketamine | Hospira | NDC 0409-2051-05 | |
| Xylazine | Akorn | NADA 139-236 |
Referências
- von Gise, A., Pu, W. T. Endocardial and epicardial epithelial to mesenchymal transitions in heart development and disease. Circ Res. 110, 1628-1645 (2012).
- Martinez-Estrada, O. M., et al. Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin. Nat Genet. 42, 89-93 (2010).
- Gittenberger-de Groot, A. C., Vrancken Peeters, M. P., Mentink, M. M., Gourdie, R. G., Poelmann, R. E. Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions. Circ Res. 82, 1043-1052 (1998).
- Katz, T. C., et al. Distinct compartments of the proepicardial organ give rise to coronary vascular endothelial cells. Dev Cell. 22, 639-650 (2012).
- Mikawa, T., Gourdie, R. G. Pericardial mesoderm generates a population of coronary smooth muscle cells migrating into the heart along with ingrowth of the epicardial organ. Dev Biol. 174, 221-232 (1996).
- Wilm, B., Ipenberg, A., Hastie, N. D., Burch, J. B., Bader, D. M. The serosal mesothelium is a major source of smooth muscle cells of the gut vasculature. Development. 132, 5317-5328 (2005).
- Zhou, B., et al. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature. 454, 109-113 (2008).
- Dettman, R. W., Denetclaw, W., Ordahl, C. P., Bristow, J. Common epicardial origin of coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts in the avian heart. Dev Biol. 193, 169-181 (1998).
- Trembley, M. A., Velasquez, L. S., de Mesy Bentley, K. L., Small, E. M. Myocardin-related transcription factors control the motility of epicardium-derived cells and the maturation of coronary vessels. Development. 142, 21-30 (2015).
- Mellgren, A. M., et al. Platelet-derived growth factor receptor beta signaling is required for efficient epicardial cell migration and development of two distinct coronary vascular smooth muscle cell populations. Circ Res. 103, 1393-1401 (2008).
- von Gise, A., et al. WT1 regulates epicardial epithelial to mesenchymal transition through beta-catenin and retinoic acid signaling pathways. Dev Biol. 356, 421-431 (2011).
- Lavine, K. J., et al. Endocardial and Epicardial Derived FGF Signals Regulate Myocardial Proliferation and Differentiation In Vivo. Dev Cell. 8, 85-95 (2005).
- Sanchez, N. S., Barnett, J. V. TGFbeta and BMP-2 regulate epicardial cell invasion via TGFbetaR3 activation of the Par6/Smurf1/RhoA pathway. Cell Signal. 24, 539-548 (2012).
- Dettman, R. W., Pae, S. H., Morabito, C., Bristow, J. Inhibition of 4-integrin stimulates epicardial-mesenchymal transformation and alters migration and cell fate of epicardially derived mesenchyme. Dev Biol. 257, 315-328 (2003).
- Rhee, D. Y., et al. Connexin 43 regulates epicardial cell polarity and migration in coronary vascular development. Development. 136, 3185-3193 (2009).
- Wu, M., et al. Epicardial spindle orientation controls cell entry into the myocardium. Dev Cell. 19, 114-125 (2010).
- Hirose, T., et al. PAR3 is essential for cyst-mediated epicardial development by establishing apical cortical domains. Development. 133, 1389-1398 (2006).
- Baek, S. T., Tallquist, M. D. Nf1 limits epicardial derivative expansion by regulating epithelial to mesenchymal transition and proliferation. Development. 139, 2040-2049 (2012).
- Lu, J., et al. Coronary smooth muscle differentiation from proepicardial cells requires rhoA-mediated actin reorganization and p160 rho-kinase activity. Dev Biol. 240, 404-418 (2001).
- Combs, M. D., Braitsch, C. M., Lange, A. W., James, J. F., Yutzey, K. E. NFATC1 promotes epicardium-derived cell invasion into myocardium. Development. 138, 1747-1757 (2011).
- Acharya, A., et al. The bHLH transcription factor Tcf21 is required for lineage-specific EMT of cardiac fibroblast progenitors. Development. 139, 2139-2149 (2012).
- Zhou, B., von Gise, A., Ma, Q., Hu, Y. W., Pu, W. T. Genetic fate mapping demonstrates contribution of epicardium-derived cells to the annulus fibrosis of the mammalian heart. Dev Biol. 338, 251-261 (2010).
- Zhou, B., et al. Adult mouse epicardium modulates myocardial injury by secreting paracrine factors. J Clin Invest. 121, 1894-1904 (2011).
- Singh, M., Epstein, J. Epicardial Lineages and Cardiac Repair. Journal of Developmental Biology. 1, 141-158 (2013).
- Braitsch, C. M., Combs, M. D., Quaggin, S. E., Yutzey, K. E. Pod1/Tcf21 is regulated by retinoic acid signaling and inhibits differentiation of epicardium-derived cells into smooth muscle in the developing heart. Dev Biol. 368, 345-357 (2012).
- Austin, A. F., Compton, L. A., Love, J. D., Brown, C. B., Barnett, J. V. Primary and immortalized mouse epicardial cells undergo differentiation in response to TGFbeta. Developmental dynamics : an official publication of the American Association of Anatomists. 237, 366-376 (2008).
- Kim, J., Rubin, N., Huang, Y., Tuan, T. L., Lien, C. L. In vitro culture of epicardial cells from adult zebrafish heart on a fibrin matrix. Nat Protoc. 7, 247-255 (2012).
- Compton, L. A., Potash, D. A., Mundell, N. A., Barnett, J. V. Transforming growth factor-beta induces loss of epithelial character and smooth muscle cell differentiation in epicardial cells. Developmental dynamics : an official publication of the American Association of Anatomists. 235, 82-93 (2006).
- Smith, C. L., Baek, S. T., Sung, C. Y., Tallquist, M. D. Epicardial-derived cell epithelial-to-mesenchymal transition and fate specification require PDGF receptor signaling. Circ Res. 108, 15-26 (2011).
- Shea, K., Geijsen, N. Dissection of 6.5 dpc Mouse Embryos. JOVE. 2, (2007).
- Witty, A. D., et al. Generation of the epicardial lineage from human pluripotent stem cells. Nature biotechnology. 32, 1026-1035 (2014).
- Iyer, D., et al. Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells. Development. 142, 1528-1541 (2015).
- Takeichi, M., Nimura, K., Mori, M., Nakagami, H., Kaneda, Y. The transcription factors Tbx18 and Wt1 control the epicardial epithelial-mesenchymal transition through bi-directional regulation of Slug in murine primary epicardial cells. PloS one. 8, e57829 (2013).
- Wong, M. L., Medrano, J. F. Real-time PCR for mRNA quantitation. BioTechniques. 39, 75-85 (2005).
- Schmittgen, T. D., Livak, K. J. Analyzing real-time PCR data by the comparative CT method. Nature Protocols. 3, 1101-1108 (2008).
- Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry. 72, 248-254 (1976).
- Moore, A. W., McInnes, L., Kreidberg, J., Hastie, N. D., Schedl, A. YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis. Development. 126, 1845-1857 (1999).
- Kim, J., et al. PDGF signaling is required for epicardial function and blood vessel formation in regenerating zebrafish hearts. Proc Natl Acad Sci U S A. 107, 17206-17210 (2010).
- Kalluri, R., Weinberg, R. A. The basics of epithelial-mesenchymal transition. J Clin Invest. 119, 1420-1428 (2009).
- Dong, X. R., Maguire, C. T., Wu, S. P., Majesky, M. W. Chapter 9 Development of Coronary Vessels. Methods in Enzymology. 445, 209-228 (2008).
- Ruiz-Villalba, A., Ziogas, A., Ehrbar, M., Perez-Pomares, J. M. Characterization of epicardial-derived cardiac interstitial cells: differentiation and mobilization of heart fibroblast progenitors. PLoS One. 8, e53694 (2013).
- Garriock, R. J., Mikawa, T., Yamaguchi, T. P. Isolation and culture of mouse proepicardium using serum-free conditions. Methods. 66, 365-369 (2014).
- Smart, N., Riley, P. Derivation of epicardium-derived progenitor cells (EPDCs) from adult epicardium. Curr Protoc Stem Cell Biol. , (2009).
- Zhou, B., Pu, W. T. Isolation and characterization of embryonic and adult epicardium and epicardium-derived cells. Methods Mol Biol. 843, 155-168 (2012).
- Morabito, C. J., Dettman, R. W., Kattan, J., Collier, J. M., Bristow, J. Positive and negative regulation of epicardial-mesenchymal transformation during avian heart development. Dev Biol. 234, 204-215 (2001).
- Grieskamp, T., Rudat, C., Ludtke, T. H., Norden, J., Kispert, A. Notch signaling regulates smooth muscle differentiation of epicardium-derived cells. Circ Res. 108, 813-823 (2011).
Citar este artigo
Trembley, M. A., Velasquez, L. S., Small, E. M. Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration. J. Vis. Exp. (109), e53750, doi:10.3791/53750 (2016).