Les auteurs tiennent à remercier l&#39;assistance technique de C. Zambataro pendant le tournage de ce protocole. Nous remercions chaleureusement le soutien de la Fondation Glenn pour la recherche médicale (SM), la fondation Hillblom, et le National Institutes of Health pour un prix de Nathan Centre Choc (P30AG025708) et PO1AG025901. JMF a été soutenue par T32AG000266 décerné à l&#39;Institut Buck pour la recherche sur le vieillissement.

<ol>
	<li>Wolska, B. M., Solaro, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+adult+mouse+cardiac+myocytes+for+studies+of+contraction+and+microfluorimetry.">Method for isolation of adult mouse cardiac myocytes for studies of contraction and microfluorimetry.</a> Am. J. Physiol. 271 (3 Pt 2), H1250-H1255 (1996).</li><li>Shioya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+isolating+healthy+heart+cells+from+mouse+models.">A simple technique for isolating healthy heart cells from mouse models.</a> J. Physiol. Sci. 57 (6), 327-335 (2007).</li><li>Guo, G., Huss, M., Tong, G. Q., Wang, C., Li Sun, L., Clarke, N. D., Robson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+cell+fate+decisions+revealed+by+single-cell+gene+expression+analysis+from+zygote+to+blastocyst.">Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst.</a> Dev. Cell. 18 (4), 675-685 (2010).</li><li>Narsinh, K. H., Sun, N., Sanchez-Freire, V., Lee, A. S., Almeida, P., Hu, S., Jan, T., Wilson, K. D., Leong, D., Rosenberg, J., Yao, M., Robbins, R. C., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+transcriptional+profiling+reveals+heterogeneity+of+human+induced+pluripotent+stem+cells.">Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells.</a> J. Clin. Invest. 121 (3), 1217-1221 (2011).</li><li>D'haene, B., Vandesompele, J., Hellemans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+and+objective+copy+number+profiling+using+real-time+quantitative+PCR.">Accurate and objective copy number profiling using real-time quantitative PCR.</a> Methods. 50 (4), 262-270 (2010).</li><li>Cook, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Anatomy+of+the+Laboratory+Mouse.">The Anatomy of the Laboratory Mouse.</a> Informatics. , (1965).</li><li>Gaillard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aorta.">Aorta.</a> Radiopaedia. , (2008).</li><li>St&#229;hlberg, A., Andersson, D., Aurelius, J., Faiz, M., Pekna, M., Kubista, M., Pekny, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+cell+populations+with+single-cell+gene+expression+profiling:+correlations+and+identification+of+astrocyte+subpopulations.">Defining cell populations with single-cell gene expression profiling: correlations and identification of astrocyte subpopulations.</a> Nucleic Acids Res. 39 (4), e24-e24 (2011).</li><li>Zhong, J. F., Chen, Y., Marcus, J. S., Scherer, A., Quake, S. R., Taylor, C. R., Weiner, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+processor+for+gene+expression+profiling+of+single+human+embryonic+stem+cells.">A microfluidic processor for gene expression profiling of single human embryonic stem cells.</a> Lab Chip. 8 (1), 68-74 (2008).</li><li>Bahar, R., Hartmann, C. H., Rodriguez, K. A., Denny, A. D., Busuttil, R. A., Dollé, M. E., Calder, R. B., Chisholm, G. B., Pollock, B. H., Klein, C. A., Vijg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+cell-to-cell+variation+in+gene+expression+in+ageing+mouse+heart.">Increased cell-to-cell variation in gene expression in ageing mouse heart.</a> Nature. 441 (7096), 1011-1014 (2006).</li><li>Raj, A., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature,+nurture,+or+chance:+stochastic+gene+expression+and+its+consequences.">Nature, nurture, or chance: stochastic gene expression and its consequences.</a> Cell. 135 (2), 216-226 (2008).</li><li>Elowitz, M., Levine, A., Siggia, E., Swain, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+gene+expression+in+a+single+cell.">Stochastic gene expression in a single cell.</a> Science. 297 (5584), 1183-1186 (2002).</li></ol>

<p class="jove_content"&gt; Accès libre et la production de cette de cet article est sponsorisé Sigma-Aldrich.</p

Cette méthode a la possibilité de générer des cardiomyocytes pour un certain nombre de fonctions ainsi que les études d&#39;expression génique. L&#39;examen des cellules individuelles est une région en plein essor dans l&#39;analyse de l&#39;expression génique. L&#39;avantage de l&#39;examen de niveau transcriptionnel au niveau de la cellule unique est qu&#39;il permet l&#39;examen d&#39;une population cellulaire pure, qui n&#39;est pas possible à partir des préparations de tissus ensemble. De plus, l&#39;analyse seule cellule permet à l&#39;examen de la variation stochastique des taux d&#39;ARNm dans des cellules individuelles pour définir les populations de cellules qui étaient auparavant considérés comme homogènes 8,9. En plus d&#39;identifier les gènes qui sont potentiellement stochastique dans leur expression des gènes 10,11,12, la méthode permet aussi l&#39;identification des populations de cellules rares définies par leurs profils d&#39;expression génique. Bien que cette méthode fournit un certain nombre de passionnantes utilisations potentielles dans l&#39;analyse de l&#39;expression des gènes, il ya des some mises en garde et considérations dans l&#39;utilisation des cellules individuelles. La principale limitation à l&#39;analyse unicellulaire, est la collecte de suffisamment de cellules dans l&#39;expérience pour atteindre une signification statistique en ce qui concerne la métrique d&#39;intérêt, de la variance par exemple. Dans le cas où le nombre de cellules isolées à partir du tissu d&#39;intérêt n&#39;est pas une limitation, on peut examiner des centaines de cellules par groupe, en utilisant des approches telles que séquençage de nouvelle génération, ou des tableaux nanofluidique. Toutefois, dans certains cas, il peut y avoir des difficultés à obtenir suffisamment de cellules du tissu d&#39;intérêt. Cela peut être en partie causée par le temps des procédures idiosyncratiques intensif pour la collection du type de cellules ciblées. Cependant, une planification minutieuse et la préparation avant de procéder à la collecte seule cellule peut encore permettre une analyse rigoureuse de cellule unique avec une erreur technique limitée. Lorsque l&#39;on examine les résultats, il doit être considéré que, même si cette procédure pour isoler les cellules a été utilisé dans de nombreux Studies (y compris la microscopie, l&#39;électrophysiologie, etc), l&#39;isolement se pourrait potentiellement effet l&#39;expression des gènes. Comme avec n&#39;importe quelle méthode qui manipule des échantillons biologiques, les résultats de vos résultats devraient être soigneusement validé pour garantir l&#39;expression cellule unique est représentatif du tissu lui-même et un biais non pas technique. Des méthodes telles que l&#39;hybridation in situ peut s&#39;avérer utile de vérifier ces résultats dans le tissu intact. Enfin, il est essentiel de s&#39;assurer que les données générées sont soigneusement vérifiées pour le contrôle qualité. Les données présentées dans la figure 8 montre que les dosages qPCR peut être très robustes dans leur spécificité, comme test n ° 13 (A13) ou ont un niveau élevé de variabilité qui peut conduire à la variance techniques telles que le dosage n ° 34 (A34).

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> NaCl </td><td> Sigma-Aldrich </td><td> 71378 </td><td></td></tr><tr><td> KCl </td><td> Sigma-Aldrich </td><td> 60128 </td><td></td></tr><tr><td> L&#39;acide pyruvique </td><td> Sigma-Aldrich </td><td> P4562 </td><td></td></tr><tr><td> Hepes </td><td> Sigma-Aldrich </td><td> 54457 </td><td></td></tr><tr><td> MgCl 2 </td><td> Sigma-Aldrich </td><td> M1020 </td><td></td></tr><tr><td> NaH 2 PO 4 monobasique </td><td> Sigma-Aldrich </td><td> P9791 </td><td></td></tr><tr><td> Dextrose </td><td> Sigma-Aldrich </td><td> G6721 </td><td></td></tr><tr><td> NaOH </td><td> Sigma-Aldrich </td><td> 72068 </td><td></td></tr><tr><td> EGTA </td><td> Sigma-Aldrich </td><td> O3777 </td><td></td></tr><tr><td> CaCl 2 </td><td> Sigma-Aldrich </td><td> 21115 </td><td></td></tr><tr><td> Protéase, type XIV </td><td> Sigma-Aldrich </td><td> P5147 </td><td></td></tr><tr><td> Collaoxygénase, Type I </td><td> Worthington </td><td> C9891 </td><td></td></tr><tr><td> Tampon 1x suspension l&#39;ADN (10 mM Tris, pH 8,0, EDTA 0,1 mM) </td><td> TEKnova, </td><td> PN T0221 </td><td></td></tr><tr><td> BSA </td><td> Sigma-Aldrich </td><td> B8667 </td><td></td></tr><tr><td> Pentobarbital sodique </td><td> Henry Schein Vet. * Alimentation </td><td> Contacter avec le fournisseur pour commander </td><td> * Doit être procuré par un vétérinaire ou un laboratoire animalier professionnel </td></tr><tr><td> IT ExoSAP </td><td> Affymetrix / USB </td><td> 78201 </td><td></td></tr><tr><td> CellsDirect 2x Mix Rxn </td><td> Invitrogen </td><td> 11737-030 </td><td></td></tr><tr><td> SuperScript III Platinum Taq Mix RT </td><td> Invitrogen </td><td> 10928-042 </td><td></td></tr><tr><td> Mélange d&#39;amorces de RT-STA </td><td> IDT </td><td> Personnalisé </td><td></td></tr><tr><td> Nucléase l&#39;eau libre </td><td> Sigma-Aldrich </td><td> W4502 </td><td></td></tr><tr><td> WTA2 </td><td> Sigma-Aldrich </td><td> WTA2-50rxn </td><td></td></tr><tr><td> Kit QIAquick PCR Purification </td><td> Qiagen </td><td> 28104 </td><td></td></tr><tr><td> QIAcube </td><td> Qiagen </td><td> 9001292 </td><td></td></tr><tr><td> Spectrophotomètre NanoDrop </td><td>NanoDrop </td><td> 2000c </td><td></td></tr><tr><td> BioAnalyzer DNA kit 7500 </td><td> Agilent </td><td> 7500 Kit </td><td></td></tr><tr><td> Un kit de marquage couleur </td><td> Roche-NimbleGen </td><td> 5223555001 </td><td></td></tr><tr><td> Mus musculus 12x135k array </td><td> Roche-NimbleGen </td><td> 5543797001 </td><td></td></tr><tr><td> GenePix 4200A Scanner </td><td> Molecular Devices </td><td> 4200A </td><td></td></tr><tr><td> Transplex Complete kit complet d&#39;amplification du transcriptome (WTA2 Kit) </td><td> Sigma-Aldrich </td><td> WTA2-50RXN </td><td></td></tr></tbody></table>

single cell transcriptional profiling of adult mouse cardiomyocytes

Alors que de nombreuses études ont examiné les changements d&#39;expression génique à partir des homogénats de tissu cardiaque, ce qui empêche l&#39;étude de la variation stochastique inhérente entre les cellules dans un tissu. L&#39;isolement des populations pures cardiomyocyte à travers une perfusion de collagénase des cœurs de souris facilite la génération de puces à cellule unique pour l&#39;expression génique toute transcriptome, ou qPCR d&#39;objectifs spécifiques en utilisant des tableaux nanofluidique. Nous décrivons ici une procédure pour examiner les profils de simples cellules d&#39;expression génétique des cardiomyocytes isolés du coeur. Ce paradigme permet l&#39;évaluation des mesures d&#39;intérêt qui ne sont pas dépendants de la moyenne (par exemple pour la variance entre les cellules dans un tissu) qui n&#39;est pas possible lors de l&#39;utilisation traditionnelle des workflows tissu entier pour l&#39;évaluation de l&#39;expression génique (figure 1). Nous avons réalisé l&#39;amplification robuste du transcriptome des cellules microgrammes seule cession d&#39;ADNc double brin qui facilite l&#39;utilisation de puces sur les iertaines cellules. Dans la procédure que nous décrivons l&#39;utilisation de tableaux de NimbleGen, qui ont été sélectionnés pour leur facilité d&#39;utilisation et de possibilité de personnaliser leur conception. Alternativement, une transcriptase inverse - amplification de la cible spécifiques (RT-STA) de réaction, permet de qPCR de centaines de cibles en nanofluidique PCR. En utilisant une de ces approches, il est possible d&#39;examiner la variabilité d&#39;expression entre les cellules, ainsi que l&#39;examen des profils d&#39;expression de types de cellules rares à partir d&#39;un tissu. Globalement, l&#39;approche cellule unique permet l&#39;expression des gènes à la génération de données qui peuvent permettre de définir des profils d&#39;expression idiosyncrasique qui sont généralement en moyenne à l&#39;examen d&#39;expression de millions de cellules à partir d&#39;homogénats typiques générés à partir des tissus ensemble.

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

Simple cellule de profilage de l&#39;expression génique permet l&#39;analyse détaillée d&#39;expression des cellules individuelles. Nous décrivons des méthodes pour l&#39;isolement des cardiomyocytes, et la préparation des lysats résultant soit pour tout ou biopuces transcriptome qPCR d&#39;objectifs spécifiques.

Profilage Single Cell transcriptionnelle des cardiomyocytes de souris adultes

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

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17.3K vues.  Buck Institute for Research on Aging. Simple cellule de profilage de l'expression génique permet l'analyse détaillée d'expression des cellules individuelles. Nous décrivons des méthodes pour l'isolement des cardiomyocytes, et la préparation des lysats résultant soit pour tout ou biopuces transcriptome qPCR d'objectifs spécifiques.

Vidéo: 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.

Isolement et culture de cardiomyocytes de souris néonatales

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

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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.

L&#39;isolement et l&#39;analyse physiologique de cardiomyocytes de souris

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.

Isolement et culture de cardiomyocytes adultes de souris pour Signalisation cellulaire et<em&gt; In vitro</em&gt; Hypertrophie cardiaque

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.

assistée par laser microdissection (LAM) comme outil de profilage transcriptionnel des types cellulaires individuels

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.

L&#39;isolement, la culture et la transduction des adultes cardiomyocytes souris

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.

TRANS-intérieur cellulaire massive Injection de cellules souches embryonnaires conduit à des taux plus élevés de chimérisme

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.

Transcriptomique cellule unique axée sur le codage à barres de gouttelettes de tissus de mammifères adultes

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.

Profilage de l'expression génique spécifique au type cellulaire dans le foie de souris

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.

Isolation, transfection et culture à long terme des cardiomyocytes adultes de souris et de rats

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.

Analyse du réseau α-Actinine dans les cardiomyocytes dérivés de l’iPSC à l’aide d’une microscopie de localisation à molécule unique

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