著者らは、このプロトコルの撮影の間にC. Zambataroの技術支援に感謝します。我々は感謝グレンネイサンショックセンター賞のための医学研究（SM）、Hillblom基礎、および国立衛生研究所財団（P30AG025708）とPO1AG025901のサポートを認める。 JMFは、老化研究のためにバック研究所に授与T32AG000266によってサポートされていました。

<ol>
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<p class="jove_content"この記事のこれの&gt;フリーアクセスと生産はSigma - Aldrich後援される。</p

このメソッドは、機能の数だけでなく、遺伝子発現研究のための心筋細胞を生成する可能性があります。単一細胞の検査は、遺伝子発現解析で急成長領域です。単一細胞のレベルでの転写レベルを調べることの利点は、全体の組織調製物から可能ではない純粋な細胞集団の検査を、できることです。さらに、単一細胞の分析は、以前に8,9均質であると考えられていた細胞集団を定義するために個々の細胞におけるmRNAレベルの確率的変動の検討が可能になります。それらの遺伝子発現10,11,12で潜在的に確率的な遺伝子を同定に加えて、この方法はまた、それらの遺伝子発現プロファイルで定義されている希少細胞集団の同定が可能になります。 このメソッドは、遺伝子発現解析でエキサイティングな潜在的な用途の数を提供する一方で、SOMがあります単一細胞の使用中の電子の注意点と考慮事項。単一細胞解析の主な制約は、例えば分散のために、関心のメトリックに関しては統計的有意性を達成するために実験に十分な細胞の集まりです。関心の組織から単離された細胞の数が制限されていない場合は、いずれかがこのような次世代シーケンシング、またはナノ流体アレイなどのアプローチを使用して、グループごとに細胞数百を調べることができます。しかし、いくつかのケースでは、関心の組織からの十分な細胞を得ることが困難があるかもしれません。これは、部分的に標的細胞のタイプの収集のための特異な時間集約的な手続きによって引き起こされる場合があります。しかし、単一のセルのコレクションを進めるために慎重な計画と事前の準備はまだ限られた技術的なエラーと厳密な単一細胞解析のためにできる可能性があります。結果を検査する際には、細胞を単離するために、この手順は、多数のstudieで使用されているにもかかわらずこと、を考慮する必要がありますsは（顕微鏡、電気生理学、等を含む）、分離自体は潜在的に遺伝子発現に影響ことができます。生物学的サンプルを操作する任意のメソッドと同様に、あなたの調査結果の結果は慎重に、単一細胞の発現を確実に検証しなければなりません組織自体ではなく、技術的なバイアスの代表です。このようなin situハイブリダイゼーションなどのメソッドは、無傷の組織でこれらの結果を検証するために有用であろう。最後に、生成されたデータは慎重に品質管理のために選択されていることを確認することが重要です。図8に示すデータは、qPCRアッセイはそのようなアッセイ＃13（A13）として、その特異性に非常に堅牢であることまたはそのようなアッセイ＃34（A34）のような技術的な分散につながることができる可変性の高いレベルを持っている可能性があることを示しています。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> NaClを</td><td>シグマアルドリッチ</td><td> 71378 </td><td></td></tr><tr><td>塩化カリウム</td><td>シグマアルドリッチ</td><td> 60128 </td><td></td></tr><tr><td>ピルビン酸</td><td>シグマアルドリッチ</td><td> P4562 </td><td></td></tr><tr><td> HEPES </td><td>シグマアルドリッチ</td><td> 54457 </td><td></td></tr><tr><td> MgCl 2の </td><td>シグマアルドリッチ</td><td> M1020 </td><td></td></tr><tr><td>のNaH 2 PO 4一塩基</td><td>シグマアルドリッチ</td><td> P9791 </td><td></td></tr><tr><td>デキストロース</td><td>シグマアルドリッチ</td><td> G6721 </td><td></td></tr><tr><td> NaOHで</td><td>シグマアルドリッチ</td><td> 72068 </td><td></td></tr><tr><td> EGTA </td><td>シグマアルドリッチ</td><td> O3777 </td><td></td></tr><tr><td> CaCl 2で </td><td>シグマアルドリッチ</td><td> 21115 </td><td></td></tr><tr><td>プロテアーゼ、タイプXIV </td><td>シグマアルドリッチ</td><td> P5147 </td><td></td></tr><tr><td>コー​​ラgenaseは、タイプI </td><td>ワージントン</td><td> C9891 </td><td></td></tr><tr><td> 1XのDNAのSuspension Buffer（10 mMトリス、pH 8.0、0.1mMのEDTA） </td><td> TEKnova、 </td><td> PN T0221 </td><td></td></tr><tr><td> BSA </td><td>シグマアルドリッチ</td><td> B8667 </td><td></td></tr><tr><td>ペントバルビタールナトリウム</td><td>ヘンリーシャインの獣医。供給* </td><td>発注するときはサプライヤーにお問い合わせください</td><td> *獣医師や実験動物専門家を通じて調達する必要があります。 </td></tr><tr><td> ITをExoSAP </td><td>アフィメトリクス/ USB </td><td> 78201 </td><td></td></tr><tr><td> 2倍RXNミックスをCellsDirect </td><td>インビトロジェン</td><td> 11737-030 </td><td></td></tr><tr><td>スーパースクリプトIII RTプラチナTaqポリメラーゼミックス</td><td>インビトロジェン</td><td> 10928-042 </td><td></td></tr><tr><td> RT - STAのプライマーミックス</td><td> IDT </td><td>カスタム</td><td></td></tr><tr><td>ヌクレアーゼフリー水</td><td>シグマアルドリッチ</td><td> W4502 </td><td></td></tr><tr><td> WTA2 </td><td>シグマアルドリッチ</td><td> WTA2 - 50rxn </td><td></td></tr><tr><td> QIAクイックPCR精製キット</td><td>キアゲン</td><td> 28104 </td><td></td></tr><tr><td> Qiacube </td><td>キアゲン</td><td> 9001292 </td><td></td></tr><tr><td>光度計分光光度計</td><td>光度計</td><td> 2000C </td><td></td></tr><tr><td>バイオアナライザDNA 7500キット</td><td>アジレント</td><td> 7500キット</td><td></td></tr><tr><td> Oneカラーラベリングキット</td><td>ロシュ、NimbleGen社</td><td> 5223555001 </td><td></td></tr><tr><td>ハツカネズミ12x135kの配列</td><td>ロシュ、NimbleGen社</td><td> 5543797001 </td><td></td></tr><tr><td> GenePix 4200Aスキャナ</td><td>分子デバイス</td><td> 4200A </td><td></td></tr><tr><td> TransPlex完全に全トランスクリプトーム増幅キット（WTA2キット） </td><td>シグマアルドリッチ</td><td> WTA2 - 50RXN </td><td></td></tr></tbody></table>

single cell transcriptional profiling of adult mouse cardiomyocytes

多くの研究は、心臓組織のホモジネートからの遺伝子発現の変化を検討してきたが、これは組織内の細胞間の固有の確率的変動を研究する防ぎます。マウスの心臓のコラゲナーゼ灌流を通して純粋な心筋細胞集団の分離は、全トランスクリプトーム遺伝子発現、またはナノ流体アレイを使用して特定のターゲットの定量PCRのための単一細胞マイクロアレイの生成を促進する。ここでは、心臓から単離された心筋細胞の単一細胞の遺伝子発現プロファイルを調べるために手順を説明します。このパラダイムでは、遺伝子発現の評価（図1）ため、従来の全組織のワークフローを使用する場合は不可能である（組織内の細胞の間に例の分散のための）意味に依存していない関心のメトリックの評価することができます。我々は、iのマイクロアレイの利用を促進する二本鎖cDNAの単セルのトランスクリプトーム収量マイクログラムの堅牢な増幅を実現していますndividual細胞。手順では、使用すると、そのデザインをカスタマイズする機能の容易さのために選択されたNimbleGen社配列を使​​用する方法を説明します。また、逆転写酵素 - 特定のターゲットの増幅（RT - STA）反応は、ナノ流体PCRによるターゲットの数百の定量PCRが可能になります。これらのアプローチのいずれかを使用して、それは細胞間の発現の変動だけでなく、組織内から珍しい種類の細胞の発現プロファイルを調べることを検討することが可能です。全体的に、単一細胞の遺伝子発現のアプローチは、潜在的に全体の組織から生成される典型的なホモジネートから細胞の数百万の発現を調べるときに典型的に平均化されている特異な発現プロファイルを識別できるデータを生成することができます。

Watch this Scientific Journal Video about Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes at JoVE.com

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

単一細胞の発現プロファイリングは、個々の細胞の詳細な遺伝子発現解析が可能になります。我々は心筋細胞の単離のための方法を説明し、特定のターゲットの全トランスクリプトームのマイクロアレイや定量PCRのどちらかの結果のライセートを調製した。

成体マウスの心筋細胞の単一細胞転写プロファイリング

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

single-cell-transcriptional-profiling-of-adult-mouse-cardiomyocytes

Research

JoVE Journal

Biology

17.3K ビュー.  Buck Institute for Research on Aging. 単一細胞の発現プロファイリングは、個々の細胞の詳細な遺伝子発現解析が可能になります。我々は心筋細胞の単離のための方法を説明し、特定のターゲットの全トランスクリプトームのマイクロアレイや定量PCRのどちらかの結果のライセートを調製した。

ビデオ: Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

新生仔マウス心筋細胞の単離および培養

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

生物学

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

マウス心筋細胞の単離と生理解析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

細胞シグナリングのための成体マウスの心筋細胞の単離と文化<em&gt; in vitroで</em&gt;心肥大

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

個々の細胞タイプの転写プロファイリングのためのツールと​​してのレーザ支援顕微解剖（LAM）

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

成体マウスの心筋細胞の単離、培養および形質導入

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

トランス内部細胞のキメリズム率が高いため、胚性幹細胞の大量注入

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

This protocol describes the general processes and quality control checks necessary for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations. Sequencing parameters, read alignment, and downstream single-cell bioinformatic analysis are also provided.

哺乳類の成体の液滴バーコード ベースの単一細胞トランスクリプトーム

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

マウス肝臓における細胞型特異的遺伝子発現プロファイリング

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

成人マウスおよびラット心筋細胞の分離、トランスフェクション、長期培養

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

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.

Analyzing the &#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.

一分子局在顕微鏡を用いたヒトiPSC由来心筋細胞におけるαアクチニンネットワークの解析

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