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 authors declare no conflict of interest.

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>
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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...

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Analyzing the &amp;#945;-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. ...

This method can be used for the quantitative evaluation of sarcomere maturation in iPSC-derived cardiomyocytes. Using this super resolution-based approach, it is possible to detect even subtle alterations in sarcomere organization, which is not possible with conventional confocal imaging. At least three hours before use, switch on the microscope and bring the sample to room temperature.When the microscope is ready, add 300 microliters of PALM imaging buffer to a well of labeled cells and insert the chamber slide into the stage holder of the microscope. Select the 1.57 NA 100X oil objective and use the PALM mode in the imaging software and activate the TIRF settings. Set the number of frames to 5, 000 to 10, 000, the UV laser power to 0.1%and the 647 laser to 0.2%and the gain level to 50 to 100.When all of the acquisition parameters have been set, switch on the laser illumination and select a target cell. To acquire an image, increase the 647 laser power to 100%and reduce the gain to zero. Bleach the target cell for about five seconds, then increase the gain to 50 and initiate the PALM image acquisition.For reconstruction of the PALM data, at the end of the acquisition, open the data in ImageJ and open the Thunderstorm plugin. Select run analysis in the plugin and open the camera setup menu. Enter the pixel size and the electromagnetic gain.In the run analysis menu, set the B-spline order to three, the B-spline scale to two, the peak intensity threshold to stfwave. f1, the fitting radius to three, the initial sigma to 1.6, the magnification to five, the update frequency to 50, and the lateral shifts to two, and click OK.After reconstruction, select sigma in the plot histogram menu and use the rectangle tool to select a region of interest, excluding possible artifacts. Add the region of interest to the filter and add and uncertainty less than 25 to the region of interest values.In the remove duplicates tab, enter a distance threshold of 10 nanometers. In the merging tab, set the maximum distance to 20, the maximum frames per molecule to zero, and the maximum off frames to one. In the drift correction tab, select cross-correlation and set the number of bins and the magnification to five, then save the final PALM image and export the post-process data.To analyze the sarcomere length, import the reconstructed PALM image of interest into ImageJ and use the line tool to draw a line between the selected sarcomere structures perpendicular to the Z-disc to measure the shortest distance between the actin filaments. In the analysis menu, select plot profile and acquire the length between the two peaks. To analyze Z-disc thickness, convert the reconstructed PALM image into an eight-bit mode image and open the Ridge Detection plugin.Set the line width to 20, the high contrast to 230, the low contrast to 10, the sigma to 0.79, the lower threshold to 25.84, and the minimum line length to 20. Select estimate width, extend line, and display results, then click OK and use the mean line width from results table for further analysis. iPSC-derived cardiomyocyte and neonatal cells exhibit a similar alpha actinin pattern with irregular disarranged sarcomere structures.Quantitative assessment demonstrates that the length and thickness of the alpha actinin filaments are almost identical between the two groups of cells indicating a premature developmental state of iPSC-derived cardiomyocytes. In contrast, adult mature cardiomyocytes exhibit a regular sarcomere network with a slightly increased sarcomere length and reduced Z-disc thickness. A comparison of conventional confocal imaging and PALM reveals no significant difference in sarcomere length.However, a profound reduced Z-disc thickness is detected when iPSC cardiomyocytes are subjected to PALM imaging. A gain in resolution is observed when PALM is applied, which is supported by the corresponding intensity plots. Notably, sarcomere structures acquired using a low quality buffer appear to be thicker compared to structures captured under optimal imaging conditions.This lack of data accuracy is due to the reduced blinking properties of the fluorophore, resulting in fewer detected photons per localization event. Moreover, localization precision is decreased, lowering the overall resolution of the reconstructed PALM image. In addition, sample drift can affect the precise localization of the fluorescent molecules resulting in blurry images.To acquire proper PALM images, it is very important to allow thermal equilibration of the entire imaging system to avoid excessive sample drift during imaging. Following this procedure, other cellular structures such as mitochondria can be imaged by PALM to acquire additional parameters of cardiomyocyte maturation.

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JoVE Journal

Biology

6.6K visualizações.  Rostock University Medical Center. Este método pode ser utilizado para a avaliação quantitativa da maturação de sarcomere em cardiomiócitos derivados do iPSC. Usando essa abordagem baseada em super resolução, é possível detectar até mesmo alterações sutis na organização sarcomere, o que não é possível com imagens confocal convencionais. Pelo menos três horas antes do uso, ligue o microscópio e leve a amostra à temperatura ambiente.Quando o microscópio estiver pronto, adicione 300 microlitros de tam...