利用单分子定位显微镜分析人类 iPSC 衍生肌细胞中的α-Actinin 网络
Summary
形成适当的沙康网络对于 iPSC 衍生心肌细胞的成熟非常重要。我们提出了一种基于超分辨率的方法，允许对干细胞衍生心肌细胞的结构成熟进行定量评估，以改善促进心脏发育的培养条件。
Abstract
iPSC衍生心肌细胞的成熟是它们在再生治疗、药物测试和疾病建模中应用的一个关键问题。尽管开发了多种分化方案，但类似成人表型的 iPSC 心肌细胞的生成仍然具有挑战性。心肌细胞成熟的一个主要方面是形成组织良好的沙科动物网络，以确保高收缩能力。在这里，我们提出了一种基于超分辨率的方法，用于对心肌细胞中α-actinin网络进行半定量分析。利用光激活定位显微镜，对从新生儿组织分离的 iPSC 衍生心肌细胞和心脏细胞的沙康长度和 z-disc 厚度进行了比较。同时，我们证明了适当的成像条件对于获得可靠数据的重要性。结果表明，该方法适合定量监测具有高空间分辨率的心脏细胞的结构成熟度，能够检测到沙康组织甚至细微的变化。
Introduction
心血管疾病（CVD）（如心肌梗塞或心肌病）仍然是西方世界的主要死因。由于人类心脏只有不良的再生能力，因此需要制定战略来促进心血管疾病的恢复。这包括细胞替代疗法，以补充丢失的心肌细胞（CM），以及开发新的抗心律失常药物，以高效和安全的药物干预。诱导多能干细胞（iPSC）已被证明是一个有希望的细胞来源，无限代人类CM在体外，适合再生疗法，疾病建模，并用于药物筛选测定,2，3，43的发展。2
虽然存在许多不同的心脏分化协议，iPSC衍生的CM仍然缺乏某些表型和功能方面，阻碍体外和体内应用5,5，6。除了电生理、代谢和分子变化，心脏成熟过程涉及沙状物的结构组织，这是力生成和细胞收缩所需的基本单位。虽然成人CMs表现出组织良好的收缩装置，iPSC衍生的CMs通常表现出不一和的沙康丝，与收缩能力降低和改变收缩动力学8,8，9。与显示单轴收缩模式的成熟CM相比，未成熟CM中的迷失方向结构导致整个细胞的径向收缩或促进收缩焦点9、10,的出现。
为了改善心脏成熟，已经应用了多种方法，包括3D细胞培养方法，电和机械刺激，以及使用细胞外基质模仿体内条件11，12，13。11,12,13为了评估这些不同培养条件的成功和效率，需要技术来监测和估计 iPSC CM 的结构成熟程度，例如，通过微观技术。与传统共体成像相比，光激活定位显微镜 （PALM） 的分辨率高出约 10 倍。这种技术反过来允许更准确的分析，检测甚至微妙的变化的细胞结构14。考虑到PALM成像的高分辨率，该方法的总体目标是通过精确测定z-Disc厚度和沙康长度，对psC衍生的CMs中沙科勒成熟度进行微观评估。在以前的研究中，这些结构特征已被证明是评估心脏成熟度15的适当参数。例如，与野生细胞16相比，缺乏全长肌营养不良素的病症 iPSC-CM 表现出减少的沙康长度和 z 波段宽度。同样，测量单个沙形病的长度，以调查地形图线索对心脏发育的影响16。因此，我们运用这种方法对 iPSC-CM 中沙康器网络的结构成熟度进行了定量测量，从而测量了沙康卡长度和 z-disc 厚度。
Protocol
本议定书中涉及新生儿和成年小鼠的所有步骤都按照罗斯托克大学医学中心动物护理的道德准则执行。 1. iPSC衍生心肌细胞的培养和分离 使用 2D 单层方法区分 hiPSC-VM 25 天，如前面所述。 预热分离介质至37°C，支持中室温度。 用 PBS 清洗细胞两次。 将预预热分离介质添加到细胞中，在37°C和5%CO2下孵育12分钟。 向?…
Representative Results
为了估计CM、新生儿、完全成熟的成人和 iPSC CM 的结构成熟程度，最初用 α-actinin 抗体标记 CM 以可视化沙康网络。在PALM采集后，对图像进行了重建，并使用基于插件的图像处理软件测量了z-disc厚度，以自动检测单个细丝的宽度。通过测量两个相邻强度峰之间的距离（对应于相邻的长丝）计算了 Sarcomere 长度。 图 1 显示了 iPSC 派生 CM 中对 sarcomere 组织的评估。 <p class="jove_c…
Discussion
体外功能 iPSC 衍生 CM 的生成对于再生疗法、疾病建模和药物筛选平台的发展非常重要。然而，这些CM的成熟度不足是心血管研究20的主要障碍。在这方面，需要高分辨率成像技术，以便监测 iPSC 衍生 CM 的结构成熟状态。同时，超分辨率显微镜可以是一个有价值的工具，可以精确分析特定蛋白质的功能，需要适当的沙康，最近证明的滴定和肌蛋白10，21，22。,<…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究得到了欧盟结构基金（ESF/14-BM-A55-0024/18）的支持。此外，H.L.还得到罗斯托克大学医学中心（889001和889003）和约瑟夫和克林茨基金会（T319/29737/2017）的 FORUN 方案的支持。C.I.L. 得到罗斯托克大学医学中心临床科学家计划的支持。R.D 由 DFG （DA1296/6-1）、DAMP 基金会、德国心脏基金会 （F/01/12） 和 BMBF （VIP+ 00240） 支持。
我们感谢玛德琳·巴奇在 iPSC 细胞培养和心脏分化方面给予的技术支持。
Materials
| human iPSC cell line | Takara | Y00325 | |
| µ-Slide 8 Well Glass Bottom | ibidi | 80827 | |
| 0.5ml eppendorf tube | Eppendorf | 30121023 | |
| Bovine serum albumin | Sigma Aldrich | A906 | |
| Cardiomyocyte Dissociation Kit | Stem Cell Technologies | 05025 | |
| Catalase | Sigma Aldrich | C40-1G | |
| Cyclooctatetraene | Sigma Aldrich | 138924-1G | |
| Cysteamine | Sigma Aldrich | 30070-10g | |
| Dulbecco's phosphate-buffered saline without Ca2+ and Mg2+ | Thermo Fisher | 14190169 | |
| F(ab')2-Goat anti-Mouse IgG Alexa Fluor 647 | Thermo Fisher | A-21237 | |
| Fiji image processing software (Image J) | |||
| Glucose | Carl Roth | X997.2 | |
| Hydrochloric acid | Sigma Aldrich | H1758 | |
| LSM 780 ELYRA PS.1 system | Zeiss | ||
| Paraformaldehyde | Merck | 8187150100 | |
| Pyranose oxidase | Sigma Aldrich | P4234-250UN | |
| sarcomeric α-actinin antibody [EA-53] | Abcam | ab9465 | |
| Sodium chloride | Sigma Aldrich | S7653 | |
| sterile water | Carl Roth | 3255.1 | |
| Triton X-100 | Sigma Aldrich | X100 | |
| Trizma base | Sigma Aldrich | T1503 | |
| β-Mercaptoethanol | Sigma Aldrich | 63689 |
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Citazione di questo articolo
Johann, L., Chabanovska, O., Lang, C. I., David, R., Lemcke, H. Analyzing the α-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy. J. Vis. Exp. (165), e61605, doi:10.3791/61605 (2020).