Nous tenons à remercier Marcelo Toro pour l&#39;aide à la cytométrie en flux. Cette étude a été soutenue par le Suédois coeur et du poumon Fondation, la Commission européenne FP7 &quot;CardioCell&quot;, Conseil de recherche suédois, AFA assurances et ALF. OB a été soutenu par la Deutsche Forschungsgemeinschaft.

1. Isolement des noyaux cardiaque <ol><li> Tubes ultracentrifugeuse Coat (Tubes à centrifuger Beckman # 363664) avec 10 ml de solution de revêtement de 1% de BSA / PBS. Reboucher les tubes et les laisser tourner pendant 30 min dans un rotateur tube. Retirer la solution de revêtement et de laisser la centrifugeuse tubes d&#39;air sec (un tube par cœur de la souris est nécessaire pour l&#39;analyse des cœurs de souris, en alternance jusqu&#39;à 5 cœurs de souris ou 1 g de tissu cardiaque d&#39;une espèce différente (par exemple l&#39;homme) peuvent être traitées dans un tube). </li><li> Toutes les étapes suivantes doivent être effectuées sur la glace. Disséquer le ventricule gauche du cœur de souris frais ou congelés composant logiciel enfichable avec un scalpel. Remarque, ce protocole est optimisé pour le coeur de la souris, mais peut également être adapté pour le rat ou le cœur humain. Vous pouvez également utiliser jusqu&#39;à 1 g de tissu cardiaque d&#39;une espèce différente. </li><li> Couper l&#39;échantillon dans de petites cabines avec un scalpel. </li><li> Transférer les morceaux de tissus dans un tube Falcon de 50 ml remplie avec 15 ml de tampon de lyse. </li><li> Homogénéiser letissu cardiaque avec un T-25 homogénéisateur Ultra-Turrax sonde (IKA) à 24.000 tours par minute pendant 10 sec. </li><li> Diluer l&#39;homogénat avec un volume égal de tampon de lyse à 30 ml. </li><li> Utilisez un verre (40 ml) de continuer à douncer homogénéiser le tissu et sans les noyaux. Effectuer huit coups avec un pilon grand débattement. </li><li> Passez les noyaux brut d&#39;isoler par le biais de 100 um et 70 um en maille de nylon cellule crépine (BD Biosciences), consécutivement. </li><li> Isoler les noyaux brut d&#39;isoler dans une centrifugeuse réfrigérée (4 ° C) à 700 xg pendant 10 min. </li><li> Eliminer le surnageant soigneusement en inversant les tubes et essuyez l&#39;intérieur du tube avec un papier absorbant. Veillez à ne pas perturber le culot des noyaux. </li><li> Dissoudre les noyaux brut isoler dans 5 ml de tampon de saccharose par la solution de pipetage plusieurs fois de haut en bas. Ajouter un nouveau 25 ml de tampon de saccharose à l&#39;dissous culot. </li><li> Ajouter 10 ml de tampon de saccharose fraîchement préparée à l&#39;enduit tube d&#39;ultracentrifugation (voir l&#39;étape 1.1). &lt;/ Li&gt; <li> Soigneusement superposer l&#39;ajoute 10 ml de tampon de saccharose, avec les noyaux culot remis en suspension dans un tampon de saccharose dissous de l&#39;étape 1.9. </li><li> Équilibrer les tubes à centrifuger avant de les placer dans un rotor oscillant JS13.1 libre et placer le rotor dans une centrifugeuse à grande vitesse (Beckman Avanti S-25). </li><li> Spin de l&#39;échantillon noyaux à 13.000 xg à 4 ° C pendant 60 min. </li><li> Lorsque la rotation est terminée, enlever les tubes soigneusement du rotor et jeter le surnageant en inversant les tubes et en essuyant le reste des débris de l&#39;intérieur des tubes avec une serviette en papier. </li><li> Dissoudre le culot dans 1 ml de noyaux tampon de stockage des noyaux (NSB, plus de tampon). Remarque: NSB, plus contient 1,5 mM de spermine comme stabilisant l&#39;ADN. </li><li> Passez à l&#39;étape 2.1, immunomarquage des noyaux des cardiomyocytes. </li></ol> 2. Immunocoloration de cytométrie en flux <ol><li> Préparer le contrôle négatif pour immunocoloration. Prélever une partie aliquote de 20 sur ul de l&#39;échantillon noyaux et unjj 980 ul de NSB, plus de mémoire tampon. </li><li> Ajouter anti-péricentriolaire matériau 1 anticorps (anticorps de lapin anti-PCM-1, Anticorps Atlas) à l&#39;échantillon des noyaux dans une dilution de 1:500 à immunolabel noyaux des cardiomyocytes. Ajouter l&#39;anticorps d&#39;isotype dans la même dilution que l&#39;anticorps anti-PCM-1 au témoin négatif, préparé à l&#39;étape 2.1. </li><li> Incuber contrôle négatif et le tube échantillon à 4 ° C pendant la nuit. </li><li> Laver l&#39;échantillon témoin négatif et au moins une fois avec NSB, plus de tampon (spin down tubes dans une centrifugeuse réfrigérée (4 ° C) à 700 xg pendant 10 min. Rejeter le surnageant et dissoudre le culot des noyaux dans 1 ml de NSB, plus de tampon). </li><li> Ajouter anticorps anti-lapin secondaire fluorescent (FITC ou APC) à un contrôle négatif et le tube échantillon dans une dilution de 1:1000. </li><li> Incuber témoin négatif et le tube d&#39;échantillon à 4 ° C pendant 1 h. </li><li> Laver l&#39;échantillon témoin négatif et au moins une fois avec NSB, plus de tampon (spin down tubes dans une centrifugeuse réfrigérée (4 ° C) à 700 xg pendant 10 min. Rejeter le surnageant et dissoudre le culot des noyaux dans 1 ml de NSB, plus de tampon). </li><li> Procéder à l&#39;analyse par cytométrie en flux et tri. </li></ol> 3. Cytométrie en flux <ol><li> Tubes Coat noyaux de collecte (Falcon 15 ml) avec 1% de BSA / PBS solution avant de commencer le tri par cytométrie en flux, comme décrit dans l&#39;étape 1.1. </li><li> Filtrer l&#39;échantillon et le témoin négatif à travers un tamis de cellules um 30 et charger la première témoin négatif à l&#39;cytomètre de flux (Afflux BD). Définir la grille des premier et second à définir les noyaux et les maillots (noyaux unique), sur la base de diffusion vers l&#39;avant (FSC), avant de largeur d&#39;impulsion de dispersion (FS largeur d&#39;impulsion) et dispersion latérale (SSC) (fig. 2a et b). Ajout d&#39;un ADN tache (DRAQ5 (1:500)) à l&#39;échantillon peut aider à identifier la population des noyaux initialement. </li><li> Chargez l&#39;échantillon immunomarquées et de définir la troisième porte d&#39;isoler les noyaux des cardiomyocytes (PCM-1-positve) de non-cardiomyocytes noyaux (PCM-1-négative). Démarrez le tri(Fig. 2c et d). </li></ol> Facultatif: Dans le but d&#39;analyser la teneur en ADN nucléaire (ploïdie) et d&#39;effectuer une analyse du cycle cellulaire ajouter un ADN approprié tache pour les noyaux (par exemple Hoechst 33342 ou DRAQ5) (Fig. 2e). <ol start="4"><li> Après le tri par cytométrie en flux, placer les noyaux sur la glace et ré-analyser pour déterminer la pureté de tri (Fig. 3a et b). </li><li> Isoler les noyaux triés dans les tubes de prélèvement à 1500 xg dans une centrifugeuse réfrigérée pendant 15 min. </li><li> Dissoudre l&#39;extrait noyaux dans un tampon compatible avec l&#39;application en aval. </li></ol> 4. Les résultats représentatifs La morphologie des noyaux et l&#39;intégrité peut être évaluée par des taches d&#39;ADN et visualisés par microscopie (Fig. 1). Succès PCM-1 étiquetage peut être évaluée par microscopie à épifluorescence et par cytométrie en flux (Fig. 1 et Fig. 2c et d). PCM-1-positive et les populations négatives devraient être bien séparés les uns des autres (Fig. 2c et d). Dans ventricule gauche murin environ 30% de tous les noyaux doit être noyaux cardiomyocytes (figure 2d). Tri pureté peut être évaluée par une nouvelle analyse des noyaux triés (Fig. 3a et b). Les deux populations des noyaux doit avoir une pureté supérieure à 95% de tri. <img alt="Figure 1" src="/files/ftp_upload/4205/4205fig1.jpg" /> Figure 1. PCM-1 identifie les noyaux des cardiomyocytes. Noyaux cardiaque (a) sont colorées avec des anticorps à PCM-1 (b) et à Nkx2.5 (c) dans un coeur de souris adultes. (D) PCM-1-étiquetés noyaux sont entourés par le cytoplasme des cardiomyocytes (chaîne lourde de myosine (MHC)) et d&#39;exprimer le facteur de transcription Nkx2.5, documenter l&#39;identification exacte des noyaux des cardiomyocytes par PCM-1 coloration (barres d&#39;échelle de 20 um et 10 um (d, en médaillon)). (E) des isolats noyaux cardiaques visualisés avec le colorant ADN DRAQ5. (F et g) Cardiomyocnoyaux YTE sont étiquetés avec des anticorps contre MIC-1 (échelle de barre de 10 um). Remarque, le schéma de coloration epinuclear de PCM-1 dans les noyaux des myocytes dans la section des tissus et dans les noyaux isolés (flèches). <img alt="Figure 2" src="/files/ftp_upload/4205/4205fig2.jpg" /> Figure 2. Tri par cytométrie en flux des noyaux des cardiomyocytes. (A) des noyaux cardiaque sont identifiés par diffusion vers l&#39;avant (FSC) et sur ​​le côté de dispersion (SSC). (B) Un deuxième porte identifie noyaux unique par le FSC et FS de largeur d&#39;impulsion 5. (C, d) de déclenchement fluorescent permet la séparation des noyaux des cardiomyocytes (PCM-1-positif) et non-cardiomyocytes (PCM-1-négative) des noyaux de tissu cardiaque. (E) des cardiomyocytes de souris sont pour la plupart (&gt; 80%) diploïde (2n), seul un petit sous-ensemble est tétraploïde (4n) 6. Remarque, les cardiomyocytes humains contiennent une fréquence plus élevée de noyaux polyploïdie (&gt; 2n) 7,8. <img alt="Figure 3" src="/files/ftp_upload/4205/4205fig3.jpg" /> <strong&gt; Figure 3. Analyse de la pureté des noyaux des cardiomyocytes et non des cardiomyocytes triés. Re-analyse des noyaux non-cardiomyocytes (a) et des cardiomyocytes triés (b). Les deux populations montrent une pureté supérieure à 99% de tri.

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+content,+ploidy+level+and+number+of+nuclei+in+the+human+heart+after+myocardial+infarction.">DNA content, ploidy level and number of nuclei in the human heart after myocardial infarction.</a> Cardiovascular Research. 36, 45-51 (1997).</li><li>Adler, C. P., Friedburg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+DNA+content.+ploidy+level+and+cell+number+in+geriatric+hearts:+postmortem+examinations+of+human+myocardium+in+old+age.">Myocardial DNA content. ploidy level and cell number in geriatric hearts: postmortem examinations of human myocardium in old age.</a> Mol. Cell Cardiol. 18, 3953-39 (1986).</li><li>Dubois, N. 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Vis. Exp. (20), e914 (2008).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

L&#39;identification précise des noyaux des cardiomyocytes est crucial pour l&#39;analyse des processus de régénération dans le myocarde 2,3. Les techniques conventionnelles pour isoler les cardiomyocytes des tissus frais sont principalement basées sur la digestion enzymatique des protéines de la matrice extracellulaire et la purification ultérieure à partir de cellules interstitielles par centrifugation à faible vitesse. Une purification supplémentaire des cardiomyocytes vivant à partir de cellule...

<table border="1"><tbody><tr><td> 1. Tampon de lyse </td></tr><tr><td> Nom du réactif </td></tr><tr><td> 0,32 M de saccharose </td></tr><tr><td> 10 mM de Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM de CaCl 2 </td></tr><tr><td> 5 mM d&#39;acétate de magnésium </td></tr><tr><td> 2,0 mM d&#39;EDTA </td></tr><tr><td> 0,5 mM EGTA </td></tr><tr><td> DTT 1 mM </td></tr></tbody></table><table border="1"><tbody><tr><td> 2. Tampon saccharose </td></tr><tr><td> Nom du réactif </td></tr><tr><td> 2,1 M de saccharose </td></tr><tr><td> 10 mM de Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM d&#39;acétate de magnésium </td></tr><tr><td> DTT 1 mM </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. Tampon de stockage des noyaux (NSB plus) </td></tr><tr&gt; <td> Nom du réactif </td></tr><tr><td> 0,44 M de saccharose </td></tr><tr><td> 10 mM de Tris-HCl (pH = 7,2) </td></tr><tr><td> 70 mM de KCl </td></tr><tr><td> 10 mM MgCl 2 </td></tr><tr><td> 1,5 mM de spermine </td></tr></tbody></table><table border="1"><tbody><tr><td> Réactifs et appareillage </td><td> Entreprise </td></tr><tr><td> Isotype IgG de lapin-ChIP Grade, # ab37415 </td><td> Abcam </td></tr><tr><td> De lapin anti-PCM-1 anticorps, # HPA023374 </td><td> Anticorps Atlas </td></tr><tr><td> Ane sec. un anticorps anti-lapin Alexa 488 Fluor, # A-21206 ou l&#39;équivalent sec. immunofluorescence </td><td> Life Technologies </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> crépines de cellules de 30 um, 70 um et 100 um </td><td> BD Biosciences </td></tr><tr><td> Verre douncer (40 ml) etpilon &quot;L&quot; </td><td> VWR (Wheaton Industries Inc) </td></tr><tr><td> T-25 Ultra-Turrax Homogénéisateur </td><td> IKA Allemagne </td></tr><tr><td> Outil de dispersion S25 N-18 G </td><td> IKA Allemagne </td></tr><tr><td> Beckman Avanti Centrifugeuse </td><td> Beckman Coulter </td></tr><tr><td> Falcon tubes 15 ml et 50 ml </td><td> VWR </td></tr><tr><td> Tubes à centrifuger Beckman # 363664 </td><td> Beckman Coulter </td></tr><tr><td> JS13.1 rotor oscillation libre </td><td> Beckman Coulter </td></tr><tr><td> Afflux cytomètre </td><td> Beckman Coulter </td></tr><tr><td> Rotator Tube </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

Identification des noyaux des cardiomyocytes a été difficile dans des coupes de tissus comme la plupart des stratégies de compter uniquement sur ​​les protéines marqueurs cytoplasmiques 1. Les événements rares dans les myocytes cardiaques tels que la prolifération et l&#39;apoptose nécessite une identification précise des noyaux des myocytes cardiaques pour analyser le renouvellement cellulaire dans l&#39;homéostasie et dans des conditions pathologiques 2. Ici, nous fournissons une méthode pour isoler les noyaux de cardiomyocytes post-mortem des tissus par la sédimentation de densité et immunomarquage avec des anticorps contre le matériel péricentriolaire 1 (PCM-1) et après le tri par cytométrie en flux. Cette stratégie permet une analyse à haut débit et l&#39;isolement avec l&#39;avantage de travailler aussi bien sur les tissus frais et congelés documents d&#39;archives. Cela permet d&#39;étudier des documents déjà recueillis dans des biobanques. Cette technique est applicable et testé dans un large éventail d&#39;espèces et adapté à de multiples applications en aval tels que le carbone-14 datant 3, cellule-cyarticle analyse 4, la visualisation des analogues de la thymidine (BrdU par exemple et les UDI) 4, l&#39;analyse du transcriptome et épigénétiques.

Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Noyaux cardiaque sont isolés par sédimentation de densité et immunomarquées avec des anticorps contre matériel péricentriolaire 1 (PCM-1) d&#39;identifier et de trier les noyaux des cardiomyocytes par cytométrie de flux.

Isolement des noyaux de cardiomyocytes post-mortem des tissus

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

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16.9K vues.  University of Lund. Noyaux cardiaque sont isolés par sédimentation de densité et immunomarquées avec des anticorps contre matériel péricentriolaire 1 (PCM-1) d'identifier et de trier les noyaux des cardiomyocytes par cytométrie de flux.

Vidéo: Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

L&#39;isolement des îlots humains à partir des patients partiellement pancréatectomisés

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

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Médecine

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolement de cellules souches du cancer à partir d&#39;échantillons cancer de la prostate humaine

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

Isolement des myocytes auriculaires des souris adultes

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques.

Évaluation électromécanique de l’activité cardiomyocyte modulée d’optogenetically

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

Modèle de cardiopathie ischémique et comparaison vidéo de la contraction cardiomyocyte à l’aide de cardiomyocytes dérivés de hiPSC

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Isolement d’adipocytes viables et d’une fraction vasculaire stromale à partir de tissu adipeux viscéral humain adapté à l’analyse de l’ARN et au phénotypage des macrophages

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

Implantation de cardiomyocytes induite par ultrasons dérivée de cellules souches pluripotentes chez des souris infarctus du myocarde

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more than half of the total cardiac cell population, and they contribute to adaptive compensations upon myocardial injury, including inflammatory responses, tissue repair, and scar formation. To study the post-MI cardiac microenvironment, single-cell RNA sequencing (scRNA-seq) is widely used to identify different cardiac cell types and intercellular communications. Among the procedures of scRNA-seq sample preparation, preparing the cell suspension is one of the most critical steps, because the cell viability can affect the quality of the scRNA-seq results. Therefore, we designed an experimental protocol for preparing a non-cardiomyocyte cell suspension from post-MI mouse hearts with an extra focus on improving the cell viability by choosing mild digestive enzymes, controlling the digestion time, and applying fluorescence-activated cell sorting (FACS). Finally, we isolated CD45+ cells from the non-cardiomyocyte cell suspension obtained through this protocol, and then we performed scRNA-seq.

Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

Préparation d’une suspension cellulaire non cardiomyocytaire pour le séquençage d’ARN unicellulaire à partir d’un cœur de souris adulte post-infarctus du myocarde

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

Isolement et culture de cellules souches humaines dérivées du tissu adipeux à l’aide d’une méthode innovante sans xénogène pour la thérapie humaine

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

Isolement, culture et caractérisation des fibroblastes cutanés primaires à partir de tissus chéloïdes humains

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...