1. Raccomandazioni Prima di iniziare <ol><li> Non utilizzare PSC contaminati da micoplasma perché non differenziarsi correttamente in cellule del nodo del seno. Test per la contaminazione micoplasma prima di iniziare il protocollo. Fate questo utilizzando un kit PCR per una rapida, rilevamento altamente sensibile di micoplasmi e seguire il protocollo costruttore `. </li><li> Per ogni piatto Petri (fase 2.3.4), cappotto una 10 cm 2 cellule piatto cultura con sterile 7 ml 0,1% di gelatina da acqua fredda pelle di pesce per 1 ora a 37 ° C. Togliere la gelatina e lasciare il piatto asciutto in condizioni sterili in un banco sterile. </li><li> Prima di avviare il protocollo di differenziazione è necessario un doppio transfettato del mouse stabile ES clone linea cellulare contenente le seguenti caratteristiche: i) costitutiva over-espressione di TBX3 utilizzando un vettore di espressione di mammifero. Ii) La resistenza G418 gene sotto il controllo del promotore MYH6-5. </li><li> Le cellule indifferenziate PSC devono essere co-coltivazioned con cellule feeder irradiate in condizioni standard, come descritto 1. </li></ol> 2. Differenziazione Procedura <ol><li> Due giorni prima di differenziazione - Togliere PSC da cellule di alimentazione. <ol><li> Aspirare il mezzo, lavare le cellule con 10 ml di tampone fosfato salino PBS, aggiungere 7 ml di soluzione collagenasi IV e incubare le cellule per 10 min a 37 ° C. Inserire un filtro sterile 40 micron in un tubo da 50 ml. </li><li> Risciacquare attentamente le colonie PSC (evitare di rimuovere le cellule feeder) pipettando la soluzione collagenasi su e giù 5 volte. </li><li> Trasferire la sospensione cellulare ad un filtro 40 micron, risciacquare il filtro tre volte con 8 ml di PBS. Girare intorno al filtro e metterlo a testa in giù in una capsula di Petri. Rimuovere le colonie di cellule PSC pipettando 10 ml di PBS al fondo del filtro. </li><li> Trasferire la sospensione cellulare in una provetta da 15 ml e centrifugare per 5 min a 300 x g. </li><li> Rimuovere la PBS, sospendere le cellule in 1 ml Accutase e incubate a 37 ° C per 5 min. </li><li> Aggiungere 10 ml di PBS, miscelare la soluzione di cellule pipettando su e giù per 5 volte e centrifugare per 5 min a 300 x g. </li><li> Rimuovere la PBS, sospendere le cellule in 10 ml mezzo di coltura e di determinare il numero di cellulare. </li><li> Seme 15.000 cellule / cm 2 su 75 cm 2 beuta filtro e coltivare per 2 giorni a 37 ° C, 5% CO2. Dopo 2 giorni la muffola dovrebbe essere 50-70% confluenti. </li></ol></li><li> Giorno 0 - Avvio di Differenziazione <ol><li> Rimuovere il supporto e lavare le cellule con 10 ml di PBS. </li><li> Aspirare il PBS, aggiungere 2 ml Accutase e incubare le cellule a 37 ° C per 5 min. Inserire un filtro sterile 40 micron in un tubo da 50 ml. </li><li> Aggiungere 10 ml di PBS, trasferire la sospensione cellulare al filtro 40 micron, risciacquare il filtro mediante aggiunta di 10 ml di PBS e centrifugare il flusso passante per 5 min a 300 x g. </li><li> Sospendere le cellule in 10 ml di terreno di differenziamento e determinare il numero di cellule. </li><li> Diluire tegli cellula sospensione con terreno di differenziamento ad una concentrazione finale di 20.000 cellule / ml. </li><li> Pipettare 20 ml di acqua e 5 ml di soluzione appesi goccia (HD) in una capsula Petri quadratica per evitare l&#39;essiccamento-out del HDs. NOTA: Per ogni 24 piastra contenente iSABs così (vedi sezione 2.8.6.4/2.8.7) iniziano con 16 piastre di Petri. </li><li> Pipettare fino a 50 ml di sospensione cellulare in un vassoio. </li><li> Girare intorno al coperchio della capsula di Petri. Mettere 180 HDs ciascuno contenente 20 ml (400 cellule / HD) sospensione cellulare sul coperchio con una pipetta a 12 canali. </li><li> Ruotare delicatamente intorno al coperchio e posizionarlo sulla piastra di Petri. NOTA: La velocità di rotazione attorno al coperchio è molto importante. Se il coperchio è girato troppo lento o troppo veloce, la tensione superficiale della sospensione cellulare non è sufficiente a trattenere l&#39;attaccatura cade sul coperchio. Prima di provare a produrre HDs per la prima volta la pratica girando intorno al coperchio con 20 gocce microlitri di media. </li><li> Coltivare le cellule per 2 giorni a 37 ° C, 5% CO 2 per far loro formare EBS. NOTA: Posizionare piatto Petri uno riempito con acqua sulla sommità di una pila di 5 piatti Petri. In caso contrario, l&#39;HD nella capsula di Petri superiore si asciugherà. </li></ol></li><li> Day 2 <ol><li> Ruotare delicatamente attorno al coperchio del piatto quadratica Petri e trasferire i EBs derivati ​​da due piastre di Petri (360 EBS) in una provetta da 50 ml. </li><li> Attendere 10 minuti per consentire la EB si depositano sul fondo della provetta (Non centrifugare EBS). </li><li> Aspirare il più media possibile, sospendere le EBs in 10 ml di medio di differenziazione e trasferire la sospensione di una capsula di Petri 10 cm. </li></ol></li><li> Giorno cultura 2-6 Suspension <ol><li> Coltivare EBS in sospensione per 4 giorni a 37 ° C, 5% CO 2, cambiare media dopo 2 giorni. NOTA: Anche se la capsula di Petri non è rivestito (al contrario di un piatto di coltura cellulare) EBS spesso attaccare alla superficie. Controllare le capsule di Petri per EBs collegati ogni giorno e, se necessario, scollegare i EBsdalla superficie delicatamente pipettando. Optional: Agitare le piastre di Petri continuamente durante il periodo di coltura in sospensione. Fate attenzione con la velocità dello shaker. EBS non dovrebbero accumularsi nel mezzo, né galleggiare al confine delle piastre di Petri. </li></ol></li><li> Giorno 6 <ol><li> Trasferire i EBs da una piastra di Petri di un gelatina rivestite 10 centimetri 2 cellule piatto cultura. </li></ol></li><li> Day 7-12 <ol><li> Le cellule dovrebbero iniziare a battere dopo 8-12 giorni. Controllare battendo foci di cellule giornaliera al microscopio. Le cellule iniziano a formare uno strato. </li><li> Cambiare media giornaliera. NOTA: Dopo aver cambiato media, le cellule richiedono 3-4 ore per recuperare e per iniziare a battere di nuovo. </li></ol></li><li> Giorno 12-15 Selezione delle cellule nodali del seno. <ol><li> Tre giorni dopo che le cellule hanno iniziato battendo (intorno al giorno 12-15), aspirare il terreno e pipetta 10 ml di media differenziazione fresco contenenti 400 mg / ml G418 alle cellule. </li></ol></li><li&gt; Quattro giorni dopo l&#39;inizio della selezione dissocio lo strato di cellule. NOTA: E &#39;possibile che lo strato di cella è già staccato dalla superficie. <ol><li> Rimuovere accuratamente il mezzo senza aspirare lo strato di cellule. </li><li> Aggiungere 10 ml di PBS e rimuovere con attenzione il PBS senza aspirare lo strato di cellule. </li><li> Aggiungere 10 ml di soluzione di collagenasi IV, trasferire le cellule in una provetta da 50 ml e incubare le cellule a 37 ° C per 5 min. </li><li> Mescolare energicamente la sospensione pipettando su e giù 10 volte e incubare le cellule a 37 ° C per 5 min. Deve avvenire Una sospensione di piccoli gruppi. Se ci sono ancora enormi parti del livello di sinistra, quindi ripetere il passaggio 2.8.4 altre due volte. </li><li> Aggiungere 20 ml di PBS e centrifugare per 10 min a 300 x g. </li><li> Aspirare accuratamente il PBS. NOTA: Se iSABs devono essere generati procedere con passo 2.8.7. Se Isab seno derivato nodali singole cellule devono essere generati andare avanti con il punto 2.9. </li><li> Sospendere le cellulein 12 ml di mezzo di differenziazione contenente 400 mg / ml G418 e seminare su 6 pozzi rivestiti di gelatina 24 pozzi (2 ml / pozzetto). </li></ol></li><li> Generazione di singola cellula nodale. <ol><li> Sospendere le cellule dal punto 2.8.6 in 5 ml Accutase e incubare a 37 ° C per 5 minuti. </li><li> Mescolare energicamente la sospensione pipettando su e giù 10 volte e incubare le cellule a 37 ° C per 5 min. </li><li> Aggiungere 20 ml di PBS e centrifugare per 5 min a 300 x g. </li><li> Sospendere le cellule in 12 ml di mezzo di differenziazione contenente 400 mg / ml G418 e seminare su 6 pozzi di una gelatina rivestite 24 pozzi (2 ml / pozzetto). </li></ol></li><li> Day 2 dopo dissociazione, aspirare il terreno e lavare le cellule tre volte con PBS. Aggiungere 1 ml di media differenziazione / bene. </li><li> Day 4-8 dopo dissociazione, cambiare Medio ogni 2 ° giorno. Il giorno 10, dopo l&#39;uso di dissociazione iSABs o ISAB seno derivati ​​nodale celle singole sono disponibili per ulteriori analisi. </li></ol>

<ol>
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La capacità di produrre staminali cellulari derivate cellule pacemaker cardiaco può consentire la ricostituzione del corretto ritmo cardiaco nel senso di &quot;pacemaker biologici&quot;. Allo stesso modo, droga test in vitro potranno beneficiare di loro disponibilità. PSC può dare origine a qualsiasi tipo di cellula del corpo dei mammiferi tra cui cardiomiociti con proprietà delle cellule pacemaker 8,9,10,11,12,13. Tuttavia, in genere le popolazioni di cellule all&#39;interno di &quot;corpi embrionali&qu...

Il termine &quot;sindrome del seno malato&quot; riassume molteplici malattie che portano al deterioramento del sistema di pacemaker cardiaco. Si compone di patologico, bradicardia sintomatica sinusale, blocco seno-atriale, arresto sinusale e la sindrome di tachicardia-bradicardia. In tal modo, una &quot;sindrome del nodo del seno&quot; è spesso accompagnata da malattie cardiache generali, come cardiopatia ischemica, cardiomiopatia o miocardite. Attualmente, gli approcci terapeutici sono basati sul impianto di pacemaker elettrici. Tuttavia, questo va insieme a una serie di rischi come infezioni e guasto della batteria. Nel complesso, l&#39;incidenza di complicazioni è ancora molto elevato in pazienti che hanno impiantato un pacemaker. Inoltre, al contrario di pacemaker endogena, questi dispositivi non rispondono alla stimolazione ormonale. Un futuro alternativa può contare sulla disponibilità di &quot;pacemaker biologico&quot; per la quale PSC potrebbero servirecome fonte cellulare adeguato e che sarebbe anche di grande valore per i test anti-droga in vitro. Eppure, un grave problema sta nella rarissima comparsa di cellule nodali del seno entro corpi embrionali (EBS) - questo genere non supera ~ 0.5% 1. In precedenza, è stato dimostrato che &quot;la programmazione in avanti&quot; verso specifici sottotipi cardiomiociti è fattibile tramite la sovraespressione di diversi primi fattori di trascrizione cardiovascolari, come fattore 1 (MesP1) specifici-posteriore-mesoderma e NK2 trascrizione correlati, locus 5 (Nkx2.5) 2, 3. Per dimensioni e la funzione del nodo senoatriale (SAN) normale, la T-box fattore di trascrizione TBX3 è cruciale, che ha dimostrato di avvio del programma di gene pacemaker e controllare differenziazione della SAN 4. Mentre questo migliorato l&#39;aspetto delle cellule pacemaker funzionali, il contenuto ancora non ha superato il 40% ~ all&#39;interno dell&#39;intera popolazione di cellule cardiomyocytic. Pertanto, una selezione di antibiotico passo basato ulteriore MYH6-promoter 5 è stato introdotto da noi. Questo conduce infine a aggregati cardiomiociti ancora inosservate (&quot;indotte corpi seno-atriale,&quot; iSABs &quot;) che presentano altamente aumento delle frequenze battendo (&gt; 400 bpm) in vitro, per la prima volta si avvicinano a quelli di un cuore murino e paragonabile a coltivati ​​in vitro seno cellule nodali isolate da un cuore murino 6. Sotto l&#39;amministrazione Isoprenalina anche frequenze battendo di 550 bpm vengono raggiunti. In particolare, iSABs consistono in oltre l&#39;80% funzionali cellule nodali come evidente dalla vasta fisiologico analisi 7. Recentemente, diversi approcci per la generazione di cellule del seno nodali utilizzando riprogrammazione diretta 19, superficie marcatori 14 o trattamento farmacologico con piccole molecole 16,17 sono stati descritti. Tuttavia, nessuno di questi metodi ha portato ad una tale elevata purezza delle cellule pacemaker e battendo frequenze vicine alla murino chearte come osservato in iSABs. Inoltre, in un ex vivo modello coltivati ​​topo adulto fette ventricolari che hanno perso la loro attività battere spontanea, i iSABs sono in grado di integrare nel tessuto fetta, rimanendo così spontaneamente attivo e robusto stimolazione le fette di cuore a contrazioni 7. Un protocollo dettagliato per la generazione di questi iSABs è descritto in questo documento.

<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

Trattamento della &quot;sindrome del seno malato&quot; si basa su pacemaker artificiali. Questi portano pericoli come guasto della batteria e le infezioni. Inoltre, mancano di ormone reattività e la procedura generale è costosa. &quot;Pacemaker biologico&quot; generati dal PSC possono diventare un&#39;alternativa, ma il contenuto tipico di cellule pacemaker a corpi embrionali (EBS) è estremamente bassa. Il protocollo descritto combina &quot;programmazione in avanti&quot; del PSC murini tramite il nodo del seno induttore TBX3 con la selezione antibiotica a base MYH6-promotore. Questo produce aggregati cardiomiociti coerenti di&gt; 80% cellule pacemaker fisiologicamente funzionali. Questi &quot;indotte-seno-atriale-corpi&quot; (&quot;iSABs&quot;) sono spontaneamente contraenti a frequenze ancora non raggiunte (400-500 bpm) corrispondenti alle cellule nodali isolate da cuori di topo e sono in grado di camminare miocardio murine ex vivo. Utilizzando il protocollo descritto seno altamente purosingole cellule nodali possono essere generati, che può essere ad esempio utilizzato per il test della droga in vitro. Inoltre, i iSABs generati in base a questo protocollo può diventare un passo fondamentale verso l&#39;ingegneria dei tessuti del cuore.

Il protocollo descritto consente la generazione di iSABs con una frequenza pestaggio di circa 450 bpm dal PSC (mostrati nel film) che si trova vicino alla frequenza battito del cuore del mouse. Dopo dissociazione iSABs (step 2.8.8) le osservati singole cellule mostrano la forma tipica di cellule del nodo del seno (cellule fusiformi e ragno) come mostrato in Figura 1. Queste cellule proteine ​​altamente espresso che sono noti per essere essenziali per la funzione del nodo del seno, come iperpolarizza...

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

Questo protocollo descrive come produrre funzionale del seno tessuto nodale da cellule murine staminali pluripotenti (PSC). T-box3 (TBX3) sovraespressione più cardiaca miosina pesante catena (MYH6) selezione antibiotica promoter porta a aggregati altamente puri di cellule pacemaker. Questi &quot;indotte-seno-atriale-corpi&quot; (&quot;iSABs&quot;) contengono più di 80% di cellule pacemaker, mostrano fortemente aumentato i tassi di battere e sono in grado di camminare miocardio ex vivo.

Generazione di Murine Cardiac pacemaker aggregati di cellule Sulla base ES-Cell-programmazione in combinazione con MYH6-Promoter-Selection

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 Views.  University of Rostock.  Questo protocollo descrive come produrre funzionale del seno tessuto nodale da cellule murine staminali pluripotenti (PSC). T-box3 (TBX3) sovraespressione più cardiaca miosina pesante catena (MYH6) selezione antibiotica promoter porta a aggregati altamente puri di cellule pacemaker. Questi "indotte-seno-atriale-corpi" ("iSABs") contengono più di 80% di cellule pacemaker, mostrano fortemente aumentato i tassi di battere e sono in grado di camminare m...

Video: 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.

Umano cellule staminali pluripotenti Basato Tossicità per lo sviluppo saggi per Chemical Screening e Safety Systems Biology generazione dati

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

Ricerca

Biologia dello sviluppo

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.

Generazione di aggregati di cellule staminali embrionali di topo che Mostra rottura di simmetria, Polarizzazione e Emergent Comportamento Collettivo<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 ottimizzazione Dimensione in pozzetti per sospensione a base cardiaco differenziazione delle cellule staminali umane pluripotenti

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.

Una tecnica novel di trapianto di grassi del grasso mammario per visualizzare la generazione di vascelli delle cellule staminali endoteliali vascolari

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.

Analisi quantitativa intero-monta immunofluorescenza delle popolazioni di cellule progenitrici cardiache in embrioni di topo

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.

Generazione di insulina senza impalcatura, tridimensionale esprimendo Pancreatoids dal Mouse progenitori pancreatico 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.

Analisi della camera cardiaca sviluppo durante l'embriogenesi del Mouse utilizzando l'intero Monte epifluorescenza

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.

Generazione in vitro di cellule progenitrici cardiache specifiche del campo del cuore del mouse

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.

Generazione di organoidi mammari mosaici per provepsinizzazione differenziale

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.

Generazione di un chip di barriera emato-encefalica basata sull'iPSC umano

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 ...