1. Recommandations Avant de partir <ol><li> Ne pas utiliser les CSP contaminées par des mycoplasmes parce qu&#39;ils ne seront pas bien différencier en cellules du nœud sinusal. Test de la contamination par des mycoplasmes avant de commencer le protocole. Pour cela, utilisez un kit PCR pour la détection rapide et très sensible de mycoplasmes et de suivre le protocole manufacturer`s. </li><li> Pour chaque boîte de Pétri (étape 2.3.4), manteau une 10 cm de boîte de culture cellulaire avec deux stérile 7 ml 0,1% de gélatine de peau de poisson d&#39;eau froide pendant 1 heure à 37 ° C. Retirer la gélatine et laisser le plat sec dans des conditions stériles dans un banc stérile. </li><li> Avant de commencer le protocole de différenciation vous avez besoin d&#39;une souris transfectées stables ES clone de lignée cellulaire à double contenant les caractéristiques suivantes: i) constitutif surexpression de TBX3 utilisant un vecteur d&#39;expression de mammifère. Ii) le gène de résistance à G418 sous le contrôle du promoteur MYH6-5. </li><li> Les cellules indifférenciées PSC devraient être co-cultiverd avec des cellules nourricières irradiées dans des conditions normales comme décrit une. </li></ol> 2. Procédure Différenciation <ol><li> Deux jours avant Différenciation - Retirer les CSP des cellules nourricières. <ol><li> Aspirer le milieu, laver les cellules avec 10 ml de solution saline tamponnée au phosphate PBS, ajouter 7 ml de solution de collagénase IV et incuber les cellules pendant 10 min à 37 ° C. Placer un filtre 40 um stérile dans un tube de 50 ml. </li><li> Rincer soigneusement les colonies de la CFP (éviter d&#39;enlever les cellules nourricières) par pipetage la solution de collagénase monter et descendre 5 fois. </li><li> Transférer la suspension cellulaire à un filtre de 40 um, de rincer le filtre trois fois avec 8 ml de PBS. Tourner autour du filtre et placez-le à l&#39;envers dans une boîte de Pétri. Retirer les colonies de cellules par pipetage de la CFP 10 ml de PBS à la partie inférieure du filtre. </li><li> Transférer la suspension cellulaire à un tube de 15 ml et centrifuger pendant 5 min à 300 x g. </li><li> Retirez le PBS, les cellules en suspension dans 1 ml Accutase et incubate à 37 ° C pendant 5 min. </li><li> Ajouter 10 ml de PBS, mélanger la solution de cellules par pipetage de haut en bas 5 fois et centrifuger pendant 5 min à 300 x g. </li><li> Retirer le PBS, les cellules en suspension dans 10 ml de milieu de culture et déterminer le nombre de cellules. </li><li> Semences 15000 cellules / cm 2 sur un 75 cm ballon de 2 filtre et les cultiver pendant 2 jours à 37 ° C, 5% de CO2. Après 2 jours, la fiole doit être de 50 à 70% de confluence. </li></ol></li><li> Jour 0 - Début de la différenciation <ol><li> Retirez le moyen et laver les cellules avec 10 ml de PBS. </li><li> Aspirer le PBS, ajouter 2 ml Accutase et incuber les cellules à 37 ° C pendant 5 min. Placer un filtre 40 um stérile dans un tube de 50 ml. </li><li> Ajouter 10 ml de PBS, transférer la suspension de cellules au filtre 40 um, de rinçage du filtre par addition de 10 ml de PBS et centrifuger le flux continu pendant 5 minutes à 300 x g. </li><li> Suspendre les cellules dans 10 ml de milieu de différenciation et de déterminer le nombre de cellules. </li><li> Diluer til suspension cellulaire avec un milieu de différenciation pour une concentration finale de 20 000 cellules / ml. </li><li> Placer 20 ml d&#39;eau et 5 ml baisse (HD) solution dans un plat quadratique Petri pour éviter le dessèchement de la HD suspendus. NOTE: Pour chaque plaque contenant 24 iSABs ainsi (voir la section 2.8.6.4/2.8.7) commencer avec 16 boîtes de Pétri. </li><li> Pipeter jusqu&#39;à 50 ml de suspension de cellules dans un plateau. </li><li> Tourner autour du couvercle de la boîte de Pétri. Placez 180 HD contenant chacun 20 pi (400 cellules / HD) suspension de cellules sur le couvercle à l&#39;aide d&#39;une pipette 12 canaux. </li><li> Retourner délicatement autour du couvercle et le placer sur la boîte de Pétri. REMARQUE: La vitesse de rotation autour du couvercle est très important. Si le couvercle est tourné autour trop lent ou trop rapide, la tension superficielle de la suspension cellulaire ne est pas suffisamment élevée pour maintenir le rideau tombe sur le couvercle. Avant d&#39;essayer de produire des HD pour le premier entraînement de temps tournant autour du couvercle avec des gouttes de 20 ul de milieu. </li><li> Cultiver les cellules pendant 2 jours à 37 ° C, 5% de CO 2 de les laisser forment EBS. REMARQUE: Lieu plat de Pétri une remplie d&#39;eau sur le dessus d&#39;une pile de 5 boîtes de Pétri. Sinon, la HD dans la boîte de Pétri supérieure va sécher. </li></ol></li><li> Jour 2 <ol><li> Retourner délicatement autour du couvercle de la boîte de Pétri quadratique et transférer les EB provenant de deux boîtes de Pétri (360 EBS) à un tube de 50 ml. </li><li> Attendez 10 min pour laisser les EB se déposent au fond du tube (ne pas centrifuger EBS). </li><li> Aspirer autant que possible à moyen, de suspendre les EBS 10 milieu de différenciation ml et transférer la suspension dans un plat de 10 cm Petri. </li></ol></li><li> Journée de la culture 2-6 Suspension <ol><li> Cultiver les EB en suspension pendant 4 jours à 37 ° C, 5% de CO 2, changement de milieu après deux jours. REMARQUE: Même si la boîte de Pétri ne est pas revêtue (par opposition à une boîte de culture cellulaire) EBS attacher souvent à la surface. Contrôler les boîtes de Pétri pour EBS attachés chaque jour et si nécessaire, détachez les EBde la surface par pipetage doux. Facultatif: Agiter les boîtes de Pétri en continu pendant la période de culture en suspension. Soyez prudent avec la vitesse de l&#39;agitateur. EBS ne devraient ni se accumuler dans le milieu, ni flotter à la frontière des boîtes de Pétri. </li></ol></li><li> Jour 6 <ol><li> Transférez les EB d&#39;une boîte de Petri à une gélatine recouvertes de 10 cm de boîte de culture cellulaire 2. </li></ol></li><li> Jour 7-12 <ol><li> Les cellules devraient commencer à battre après 8-12 jours. Vérifiez pour battre foyers des cellules quotidienne sous un microscope. Les cellules commencent à se former une couche. </li><li> Changer milieu tous les jours. NOTE: Après avoir changé moyenne, les cellules ont besoin 3-4 heures pour récupérer et commencer à battre. </li></ol></li><li> Jour 12-15 Sélection des cellules ganglionnaires de sinus. <ol><li> Trois jours après que les cellules ont commencé à battre (environ 12-15 jours), aspirer les moyennes et pipette 10 ml de milieu de différenciation frais contenant 400 ug / ml de G418 aux cellules. </li></ol></li><li&gt; Quatre jours après le début de la sélection dissocier la couche cellulaire. NOTE: Il est possible que la couche de cellules est déjà détachée de la surface. <ol><li> Retirer soigneusement le milieu sans aspiration de la couche cellulaire. </li><li> Ajouter 10 ml de PBS et retirer délicatement le PBS sans aspiration de la couche cellulaire. </li><li> Ajouter 10 ml de solution de collagénase IV, transférer les cellules dans un tube de 50 ml et incuber les cellules à 37 ° C pendant 5 min. </li><li> Mélanger vigoureusement la suspension par pipetage de haut en bas 10 fois et incuber les cellules à 37 ° C pendant 5 min. Une suspension de petites grappes devrait se produire. Se il ya encore d&#39;énormes parties de la couche à gauche, puis répétez l&#39;étape 2.8.4 deux fois de plus. </li><li> Ajouter 20 ml de PBS et centrifuger pendant 10 min à 300 x g. </li><li> Aspirer le PBS attentivement. NOTE: Si iSABs doivent être générés passez à l&#39;étape 2.8.7. Si ISAB sinus dérivée des cellules individuelles nodaux doivent être généré, allez directement à l&#39;étape 2.9. </li><li> Suspendre les cellulesdans 12 ml de milieu de différenciation contenant 400 ug / ml de G418 et épépiner sur 6 puits revêtues de gélatine 24 puits (2 ml / puits). </li></ol></li><li> Génération de cellule unique nodal. <ol><li> Suspendre les cellules de l&#39;étape 2.8.6 dans 5 ml Accutase et incuber à 37 ° C pendant 5 min. </li><li> Mélanger vigoureusement la suspension par pipetage de haut en bas 10 fois et incuber les cellules à 37 ° C pendant 5 min. </li><li> Ajouter 20 ml de PBS et centrifuger pendant 5 min à 300 x g. </li><li> Suspendre les cellules dans 12 ml de milieu de différenciation contenant 400 ug / ml de G418 et épépiner sur six puits d&#39;une gélatine revêtues 24 puits (2 ml / puits). </li></ol></li><li> Jour 2 après dissociation, aspirer le milieu et laver les cellules trois fois avec du PBS. Ajouter 1 ml de milieu de différenciation / puits. </li><li> Jour 4-8 après dissociation, changement de milieu tous les 2 ème jour. Au jour 10 après iSABs d&#39;utilisation de dissociation ou ISAB sinus nodal dérivée des cellules individuelles sont disponibles pour une analyse plus approfondie. </li></ol>

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La capacité à produire des cellules souches du stimulateur cardiaque cellulaires dérivées peuvent permettre la reconstitution du rythme cardiaque approprié dans le sens de &quot;stimulateurs biologiques&quot;. De même, les tests de médicaments in vitro bénéficiera de leur disponibilité. PSC peut donner lieu à ne importe quel type de l&#39;organisme d&#39;un mammifère y compris cardiomyocytes avec des propriétés de cellules pacemaker 8,9,10,11,12,13 cellulaire. Cependant, généralement les popul...

Le terme «maladie du sinus» résume plusieurs maladies conduisant à la détérioration du système de stimulateur cardiaque. Il comprend pathologique, bradycardie symptomatique des sinus, bloc sino-auriculaire, arrêt sinusal ainsi que le syndrome de tachycardie-bradycardie. Ainsi, une «maladie du sinus&quot; est souvent accompagnée par des maladies cardiaques généraux comme une maladie cardiaque ischémique, cardiomyopathies ou une myocardite. À l&#39;heure actuelle, les approches thérapeutiques basées sur l&#39;implantation de stimulateurs électriques. Toutefois, cela va de pair avec un certain nombre de risques tels que les infections et une panne de batterie. Dans l&#39;ensemble, l&#39;incidence des complications est encore très élevé chez les patients ayant implanté un stimulateur artificiel. En outre, par opposition au stimulateur endogène, ces dispositifs ne répondent pas à la stimulation de l&#39;hormone. Une alternative avenir peut compter sur la disponibilité de «pacemakers biologiques&quot; pour lequel PSC pourraient servirune source cellulaire appropriée et qui serait aussi très utile pour les tests in vitro de médicament. Pourtant, un problème majeur réside dans l&#39;apparition très rare de cellules ganglionnaires de sinus dans les corps embryoïdes (EBS) - ce généralement ne dépasse pas ~ 0,5% 1. Auparavant, il a été montré que la &quot;programmation avant&quot; vers des sous-types spécifiques de cardiomyocytes est réalisable via la surexpression des facteurs premiers distincts cardiovasculaires de transcription tels que le facteur 1 (MesP1) spécifiques à-mésoderme postérieur et NK2 transcription liée, locus 5 (Nkx2.5) 2, 3. Pour la taille et la fonction du noeud sino-auriculaire (SAN) normale, la T-box facteur de transcription TBX3 est crucial, qui a été montré pour lancer le programme génique du stimulateur et à contrôler la différenciation de la SAN 4. Bien que cette renforcée l&#39;apparition de cellules de stimulateur cardiaque fonctionnelle, la teneur encore ne dépasse pas environ 40% dans l&#39;ensemble de la population de cellules cardiomyocytic. Par conséquent, une étape de sélection par antibiotique à base supplémentaire MYH6-5 promoteur a été introduit par nous. Cela conduit finalement à des agrégats de cardiomyocytes encore non observées (&quot;organismes induits sino-auriculaire;&quot; iSABs ») qui présentent des fréquences battant fortement augmenté (&gt; 400 bpm) in vitro, pour se rapprochant de celles d&#39;un cœur murin et comparable à cultivées in vitro la première fois sinus cellules ganglionnaires isolées d&#39;un cœur murin 6. Sous l&#39;administration Isoprénaline même fréquences battant de 550 bpm sont atteints. Notamment, iSABs se composent de plus de 80% de cellules nodales fonctionnels évidents de la vaste physiologique analyses 7. Récemment, plusieurs approches pour générer des sinus cellules nodales utilisant reprogrammation directe 19, marqueurs de surface 14 ou traitement pharmacologique avec de petites molécules 16,17 ont été décrits. Pourtant, aucune de ces méthodes conduit à une telle pureté élevé de cellules pacemaker et en battant des fréquences proches de la souris, ill&#39;art comme observé dans iSABs. De plus, dans un modèle ex vivo de cultivées tranches ventriculaires de souris adultes qui ont perdu leur activité de battements spontanés, les iSABs sont capables de se intégrer dans le tissu de la tranche, restant ainsi spontanément actif et robuste stimulation les tranches cardiaques à des contractions 7. Un protocole détaillé pour la génération de ces iSABs est décrite dans le présent document.

<table border="0" cellpadding="0" cellspacing="0" width="597">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Name of the Material/Equipment</td>
			<td style="width:132px;">Company</td>
			<td style="width:131px;">Catalog Number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iscove&#39;s liquid medium with stable glutamine</td>
			<td>Biochrom AG</td>
			<td>FG 0465</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM liquid medium without Na-pyruvate, with stable glutamine</td>
			<td>Biochrom AG</td>
			<td>FG 0435</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CLS type IV, CLS IV</td>
			<td>Biochrom AG</td>
			<td>C4-22</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Non-essential amino acids</td>
			<td>Biochrom AG</td>
			<td>K 0293</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS Superior</td>
			<td>Biochrom AG</td>
			<td>S 0615</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium pyruvate (100 mM)</td>
			<td>Biochrom AG</td>
			<td>L 0473</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">G 418-BC liquid (ready-to-use solution), sterile</td>
			<td>Biochrom AG</td>
			<td>A 2912</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagent reservoir PP f.multichannel pip. 60ml,sterile</td>
			<td>Brand</td>
			<td>703409</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accutase</td>
			<td>eBioscience,Inc.</td>
			<td>00-4555-56</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorf Xplorer/Xplorer plus, electronic pipette</td>
			<td>Eppendorf</td>
			<td>4861000155</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon Cell Strainer 40&#181;m</td>
			<td>Falcon</td>
			<td>352340</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin&#160;</td>
			<td>GE Healthcare</td>
			<td>P11-010</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri Dishes</td>
			<td>Greiner BioOne</td>
			<td>663102</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS without Ca and Mg</td>
			<td>PAN-Biotech</td>
			<td>P04-36500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>PAN-Biotech</td>
			<td>P30-3302</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leukemia inhibitory factor</td>
			<td>Phoenix Europe GmbH</td>
			<td>LIF-250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quadratic petri dishes</td>
			<td>Roth</td>
			<td>PX67.1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin from cold water fish skin</td>
			<td>SigmaAldrich</td>
			<td>G7765</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-Mercaptoethanol</td>
			<td>SigmaAldrich</td>
			<td>M3148&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Thioglycerol</td>
			<td>SigmaAldrich</td>
			<td>M6145</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue culture dishes</td>
			<td>TPP</td>
			<td>93100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue culture flask</td>
			<td>TPP</td>
			<td>90076</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue culture test plates (24 well)</td>
			<td>TPP</td>
			<td>92424</td>
		</tr>
	</tbody>
</table>

generation of murine cardiac pacemaker cell aggregates based on es-cell-programming in combination with myh6-promoter-selection

Traitement de la &quot;maladie du sinus&quot; est basé sur les stimulateurs cardiaques artificielles. Ceux-ci portent des risques tels que l&#39;insuffisance et les infections batterie. En outre, ils ne ont pas la réponse hormonale et la procédure globale est coûteux. &quot;Pacemakers biologiques&quot; générés par les CSP peuvent devenir une alternative, mais le contenu typique des cellules pacemaker en corps embryoïdes (EBS) est extrêmement faible. Le protocole décrit combine «programmation avant» du PSC murins via le nœud sinusal inducteur TBX3 avec la sélection à base d&#39;antibiotiques MYH6-promoteur. Cela donne agrégats de cardiomyocytes cohérentes&gt; 80% de cellules pacemaker physiologiquement fonctionnels. Ces «induits-sino-auriculaire-organismes&quot; (&quot;iSABs&quot;) sont contractant spontanément à des fréquences encore non atteints (400-500 bpm) correspondant à des cellules ganglionnaires isolées de coeurs de souris et sont capables d&#39;arpenter myocarde murin ex vivo. En utilisant le protocole décrit des sinus de haute puretécellules individuelles nodaux peuvent être générés qui par exemple peut être utilisé pour les tests in vitro de médicaments. En outre, les iSABs générés selon ce protocole peuvent devenir une étape cruciale vers l&#39;ingénierie du tissu cardiaque.

Le protocole décrit permet de générer des iSABs avec une fréquence de battement d&#39;environ 450 bpm du PSC (indiquées dans le film) qui est près du cœur de la souris fréquence de battement. Après dissociation de iSABs (étape 2.8.8), les cellules individuelles observées montrent la forme typique de cellules du noeud sinusal (cellules fusiformes et araignées), comme illustré sur la figure 1. Ces cellules protéines hautement express qui sont connues pour être essentielles pour la fonction ...

Watch this Scientific Journal Video about Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection at JoVE.com

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Ce protocole décrit comment produire fonctionnel sinus nodal tissu de cellules souches pluripotentes (PSC) murins. T-Box3 (TBX3), plus la surexpression cardiaque myosine-chaîne lourde (MYH6) sélection par antibiotique promoteur conduit à des agrégats de cellules de stimulateur de haute pureté. Ces &quot;induit-sino-auriculaire-organismes&quot; (&quot;iSABs») contiennent plus de 80% de cellules de stimulateur cardiaque, montrent des taux de battement très augmenté et sont capables d&#39;arpenter myocarde ex vivo.

Génération de stimulateur cardiaque murin agrégats de cellules fondées sur la ES-Cell-programmation en combinaison avec MYH6-promoteur-sélection

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

generation-murine-cardiac-pacemaker-cell-aggregates-based-on-es-cell

Research

JoVE Journal

Developmental Biology

9.6K vues.  University of Rostock.  Ce protocole décrit comment produire fonctionnel sinus nodal tissu de cellules souches pluripotentes (PSC) murins. T-Box3 (TBX3), plus la surexpression cardiaque myosine-chaîne lourde (MYH6) sélection par antibiotique promoteur conduit à des agrégats de cellules de stimulateur de haute pureté. Ces "induit-sino-auriculaire-organismes" ("iSABs») contiennent plus de 80% de cellules de stimulateur cardiaque, montrent des taux de battement très augment?...

Vidéo: Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

Human cellules souches pluripotentes Basé Toxicité pour le développement dosages pour Chemical dépistage et la biologie des systèmes de sécurité de génération de données

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

Recherche

Biologie du développement

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/&#946;-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol to generate aggregates of mouse embryonic stem cells that display self-organization, symmetry breaking and elongation paralleling axial development. This technique allows the study of axial developmental processes and the generation of cell types that are otherwise difficult to perform in monolayer culture.

Génération d&#39;agrégats de souris de cellules souches embryonnaires qui montrent la brisure de symétrie, la polarisation et le comportement collectif émergent<em&gt; In Vitro</em

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation ...

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

Aggregate Optimization Taille en micropuits pour base Suspension-cardiaque Différenciation des pluripotentes humaines Cellules souches

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Une nouvelle technique de transplantation de graisse mammaire mammaire pour visualiser la génération de vaisseaux de cellules souches endothéliales vasculaires

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

Analyse quantitative Immunofluorescence entier-montent des Populations de progéniteurs cardiaques chez des embryons de souris

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

Génération d’insuline exempte d’échafaudage, en trois dimensions exprimant Pancreatoids de souris progéniteurs pancréatique In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

Analyse du développement de la chambre cardiaque durant l’embryogenèse de souris à l’aide de toute monture épifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

In Vitro Generation of Mouse Heart Field-specific Cardiac Progenitor Cells In Vitro Generation of Mouse Heart Field-specific Cardiac Progenitor Cells In Vitro Generation of Mouse Heart Field-specific Cardiac Progenitor Cells In Vitro

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Génération d’organoïdes mammaires mosaïques par trypsinisation différentielle

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

Génération d’une puce de barrière de sang-brain basée sur iPSC

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...