Name: analyzing the &alpha;-actinin network in human ipsc-derived cardiomyocytes using single molecule localization microscopy
Uploaded: 2020-11-03T13:00:00
Description: Watch this Scientific Journal Video about Analyzing the ÃŽÂ±-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy at JoVE.com

This study was supported by the EU structural Fund (ESF/14-BM-A55-0024/18). In addition, H.L. is supported by the FORUN Program of Rostock University Medical Centre (889001 and 889003) and the Josef and K&#228;the Klinz Foundation (T319/29737/2017). C.I.L. is supported by the Clinician Scientist Program of the Rostock University Medical Center. R.D is supported by the DFG (DA1296/6-1), the DAMP foundation, the German Heart Foundation (F/01/12) and the BMBF (VIP+ 00240).
We thank Madeleine Bartsch for her technical support in iPSC cell culture and cardiac differentiation.

All steps in this protocol involving neonatal and adult mice were performed according to the ethical guidelines for animal care of the Rostock University Medical Centre.
1. Cultivation and dissociation of iPSC-derived cardiomyocytes
<ol>
	<li>Differentiate hiPSC-CMs for 25 days using a 2D monolayer method as described previously17.</li>
	<li>Prewarm dissociation medium to 37 &#176;C and support medium to room temperature.</li>
	<li>Wash cells twice with PBS.</li>
	<li...

<ol>
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The generation of functional iPSC-derived CM in vitro is important for regenerative therapies, disease modeling and the development of drug-screening platforms. However, insufficient maturity of these CM is a major obstacle in cardiovascular research20. In this regard, high resolution imaging techniques are needed that enable monitoring of the structural maturation state of iPSC-derived CM. At the same time, super resolution microscopy can be a valuable tool to precisely analyze the function of sp...

Cardiovascular diseases (CVD) such as myocardial infarction or cardiomyopathy remain the major cause of death in the western world1. As the human heart possesses only poor regenerative capacity, there is a need for strategies to promote the recovery from CVDs. This includes cell replacement therapies to replenish lost cardiomyocytes (CM), as well as the development of new anti-arrhythmic drugs for efficient and safe pharmaceutical intervention. Induced pluripotent stem cell (iPSC) have been shown to be a promising cell source for the unlimited generation of human CM in vitro, suitable for regenerative therapies, disease modeling, and for the development of drug screening assays2,3,4.
Although many different cardiac differentiation protocols exist, iPSC-derived CM still lack certain phenotypical and functional aspects that impede the in vitro and in vivo application5,6. Beside electrophysiologic, metabolic, and molecular changes, the cardiac maturation process involves the structural organization of sarcomeres, which are the fundamental units required for force generation and cell contraction7. While adult CMs exhibit a well-organized contractile apparatus, iPSC-derived CMs commonly demonstrate disarranged sarcomere filaments, associated with a reduced contraction ability and altered contraction dynamics8,9. In contrast to mature CM that show uniaxial contraction pattern, the disoriented structures in immature CM results in a radial contraction of the whole cell or promote the appearance of contraction focal points9,10.
For improving cardiac maturation, multiple approaches have been applied, including 3D cell culture methods, electrical and mechanical stimulation, as well as the use of extracellular matrices mimicking in vivo conditions11,12,13. To evaluate the success and efficiency of these different culture conditions, techniques are needed to monitor and estimate the degree of the structural maturation of iPSC CM, e.g., by microscopic techniques. In contrast to conventional confocal imaging, the resolution in case of photoactivated localization microscopy (PALM) is approximately 10x higher. This technique in turn allows for a more accurate analysis, detecting even subtle alterations of cellular structures14. Considering the high resolution of PALM-based imaging, the overall goal of this method was the microscopic evaluation of sarcomere maturity in iPSC-derived CMs by precise determination of z-Disc thickness and sarcomere length. In previous studies, these structural features have been shown to be appropriate parameters to assess cardiac maturity15. For example, diseased iPSC-CM lacking full length dystrophin exhibit reduced sarcomere length and z-band width when compared to wild type cells16. Likewise, the length of individual sarcomeres was measured to investigate the impact of topographic cues on cardiac development16. Hence, we applied this approach to evaluate the structural maturation of the sarcomere network in iPSC-CM by quantitatively measuring the sarcomere length and z-disc thickness.

<table>
	<tbody>
		<tr>
			<td>human iPSC cell line</td>
			<td>Takara</td>
			<td>Y00325</td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;-Slide 8 Well Glass Bottom</td>
			<td>ibidi</td>
			<td>80827</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5ml eppendorf tube</td>
			<td>Eppendorf</td>
			<td>30121023</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>A906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiomyocyte Dissociation Kit</td>
			<td>Stem Cell Technologies</td>
			<td>05025</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma Aldrich</td>
			<td>C40-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclooctatetraene</td>
			<td>Sigma Aldrich</td>
			<td>138924-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cysteamine</td>
			<td>Sigma Aldrich</td>
			<td>30070-10g</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline without Ca2+ and Mg2+</td>
			<td>Thermo Fisher</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>F(ab&#39;)2-Goat anti-Mouse IgG Alexa Fluor 647</td>
			<td>Thermo Fisher</td>
			<td>A-21237</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji image processing software (Image J)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Carl Roth</td>
			<td>X997.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 780 ELYRA PS.1 system</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck</td>
			<td>8187150100</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyranose oxidase</td>
			<td>Sigma Aldrich</td>
			<td>P4234-250UN</td>
			<td></td>
		</tr>
		<tr>
			<td>sarcomeric &#945;-actinin antibody [EA-53]</td>
			<td>Abcam</td>
			<td>ab9465</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile water</td>
			<td>Carl Roth</td>
			<td>3255.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>63689</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing the &alpha;-actinin network in human ipsc-derived cardiomyocytes using single molecule localization microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

For estimating the degree of structural maturation of CM, neonatal, fully mature adult, and iPSC CM were initially labeled the CM with &#945;-actinin antibody to visualize the sarcomere network. Following PALM acquisition, images were reconstructed, and z-disc thickness was measured using plugin-based image processing software for the automatic detection of the width of individual filaments. Sarcomere length was calculated by measuring the distance between two adjacent intensity peaks, corresponding to neighboring filame...

Watch this Scientific Journal Video about Analyzing the ÃŽÂ±-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy at JoVE.com

Analyzing the ÃƒÅ½Ã‚Â±-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...

Research

JoVE Journal

Biology

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Single-Molecule Imaging of Nuclear Transport

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Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

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Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

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Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

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We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...