1. Recomendaciones Antes de empezar <ol><li> No utilice PSC contaminados con micoplasma porque no van a diferenciar adecuadamente en las células del nódulo sinusal. Prueba para la contaminación por micoplasmas antes de iniciar el protocolo. Para ello, utilice un kit de PCR para la detección rápida y altamente sensible de micoplasmas y seguir el protocolo del fabricante `. </li><li> Para cada placa de Petri (paso 2.3.4), capa uno 10 cm 2 de células placa de cultivo estéril con 7 ml de 0,1% de gelatina de piel de pescado de agua fría durante 1 hora a 37 ° C. Retire la gelatina y dejar el plato seco en condiciones estériles en un banco estéril. </li><li> Antes de poder iniciar el protocolo de diferenciación que necesita un ratón estable transfectadas ES clon línea celular doble que contiene las siguientes características: i) Constitutiva sobreexpresión de TBX3 utilizando un vector de expresión de mamífero. Ii) el gen de resistencia a G418 bajo el control del promotor MYH6-5. </li><li> Las células indiferenciadas PSC deben ser co-cultivard con células alimentadoras irradiados en condiciones estándar como se describe 1. </li></ol> 2. Procedimiento Diferenciación <ol><li> Dos días antes de la diferenciación - Quitar las ventanillas únicas de células alimentadoras. <ol><li> Aspirar el medio, se lavan las células con 10 ml de solución salina tamponada con fosfato PBS, añadir solución de 7 ml de colagenasa IV e incubar las células durante 10 min a 37 ° C. Coloque un filtro estéril de 40 micras en un tubo de 50 ml. </li><li> Enjuagar cuidadosamente las colonias PSC (evitar para eliminar las células alimentadoras) pipeteando la solución de colagenasa arriba y abajo 5 veces. </li><li> Transferir la suspensión celular a un filtro de 40 micras, enjuague el filtro tres veces con 8 ml de PBS. Da la vuelta al filtro y colocarlo boca abajo en una placa de Petri. Retire las colonias de células PSC pipeteando 10 ml de PBS a la parte inferior del filtro. </li><li> Transferir la suspensión celular a un tubo de 15 ml y centrifugar durante 5 min a 300 x g. </li><li> Retire la PBS, suspender las células en 1 ml Accutase y incubate a 37 ° C durante 5 min. </li><li> Añadir 10 ml de PBS, se mezcla la solución de células pipeteando arriba y abajo 5 veces y centrifugar durante 5 min a 300 x g. </li><li> Retire la PBS, suspender las células en medio de cultivo de 10 ml y determinar el número de células. </li><li> Semilla 15.000 células / cm 2 en un matraz de 75 cm 2 de filtro y cultivarlos durante 2 días a 37 ° C, 5% de CO2. Después de 2 días el matraz debería ser 50-70% de confluencia. </li></ol></li><li> Día 0 - Inicio de Diferenciación <ol><li> Retire el medio y se lavan las células con 10 ml de PBS. </li><li> Aspirar el PBS, añadir 2 ml Accutase e incubar las células a 37 ° C durante 5 min. Coloque un filtro estéril de 40 micras en un tubo de 50 ml. </li><li> Añadir 10 ml de PBS, transferir la suspensión celular a la del filtro 40 micras, enjuague el filtro mediante la adición de 10 ml de PBS y centrifugar el flujo a través durante 5 min a 300 x g. </li><li> Suspender las células en medio de diferenciación 10 ml y determinar el número de células. </li><li> Diluir tél de células en suspensión con medio de diferenciación a una concentración final de 20.000 células / ml. </li><li> Pipetear 20 ml de agua y 5 ml de solución gota (HD) en una placa de Petri cuadrática para evitar desecación del HDs colgantes. NOTA: Por cada 24 placas conteniendo así iSABs (ver sección 2.8.6.4/2.8.7) comenzar con 16 placas de Petri. </li><li> Pipetear hasta 50 ml de suspensión celular en una bandeja. </li><li> Girar alrededor de la tapa de la placa de Petri. Coloque 180 HDs que contienen cada uno 20 l (400 células / HD) suspensión de células en la tapa con una pipeta de 12 canales. </li><li> Con cuidado, gira alrededor de la tapa y colocarlo en la placa de Petri. NOTA: La velocidad de giro alrededor de la tapa es muy importante. Si la tapa se dio la vuelta demasiado lento o demasiado rápido, la tensión superficial de la suspensión de células no es lo suficientemente alta para retener el colgante gotas en la tapa. Antes de tratar de producir HDs por primera práctica momento de hacerlo girar la tapa con 20 l gotas de medio. </li><li> Cultivar las células durante 2 días a 37 ° C, 5% de CO 2 para hacerles forman EBS. NOTA: Coloque un plato de petri llena de agua en la parte superior de una pila de 5 placas de Petri. De lo contrario la HD en el plato superior de Petri se secará. </li></ol></li><li> Día 2 <ol><li> Con cuidado, gira alrededor de la tapa del plato Petri cuadrática y transferir los EBS derivados de dos placas de Petri (360 EBS) a un tubo de 50 ml. </li><li> Espere 10 minutos para dejar que el EB se depositan en el fondo del tubo (No centrifugar la EB). </li><li> Aspirar tanto medio como sea posible, suspender los EBS en 10 ml de medio de diferenciación y transferir la suspensión a un plato de 10 cm Petri. </li></ol></li><li> Día de la cultura 2.6 Suspensión <ol><li> Cultivar los EBS en suspensión durante 4 días a 37 ° C, 5% de CO 2, cambie medio después de 2 días. NOTA: A pesar de que la placa de Petri no está recubierta (en oposición a una placa de cultivo celular) de los OC menudo se adhieren a la superficie. Controlar las placas de Petri para EB unidos cada día y si es necesario, retire los EBSde la superficie por pipeteo suave. Opcional: Agitar las placas de Petri de forma continua durante el período de cultivo en suspensión. Tenga cuidado con la velocidad del agitador. Las EB ni deben acumular en el medio ni flotar a la frontera de las placas de Petri. </li></ol></li><li> Día 6 <ol><li> Transfiera los EB de un placa de Petri con una gelatina recubiertas 10 cm placa de cultivo celular 2. </li></ol></li><li> Día 12/07 <ol><li> Las células deben comenzar a batir después de 8-12 días. Compruebe por golpear a los focos de las células diariamente bajo un microscopio. Las células comienzan a formar una capa. </li><li> Cambio de medio día. NOTA: Después de cambiar medio, las células requieren 3-4 horas para recuperarse y comenzar a latir de nuevo. </li></ol></li><li> Día 12-15 selección de las células ganglionares de los senos paranasales. <ol><li> Tres días después de que las células han empezado a golpear (alrededor del día 12-15), aspirar las medianas y pipeta 10 ml de medio de diferenciación fresca que contienen 400 mg / ml de G418 a las células. </li></ol></li><li&gt; Cuatro días después del comienzo de la selección disociar la capa de células. NOTA: Es posible que la capa de células ya está separado de la superficie. <ol><li> Retire el medio con cuidado, sin aspirar la capa de células. </li><li> Añadir 10 ml de PBS y retirar el PBS con cuidado, sin aspiración de la capa de células. </li><li> Añadir 10 ml de solución de colagenasa IV, transferir las células a un tubo de 50 ml y se incuban las células a 37 ° C durante 5 min. </li><li> Mezclar vigorosamente la suspensión por la pipeta hacia arriba y hacia abajo 10 veces y se incuban las células a 37 ° C durante 5 min. Debe producirse una suspensión de pequeñas agrupaciones. Si aún hay grandes partes de la capa de la izquierda, a continuación, repita el paso 2.8.4 dos veces más. </li><li> Añadir 20 ml de PBS y centrifugar durante 10 min a 300 x g. </li><li> Aspirar el PBS con cuidado. NOTA: Si iSABs se deben generar continúe con el paso 2.8.7. Si las células individuales nodales isab sinusales derivado deben generarse continuar con el paso 2.9. </li><li> Suspender las célulasen 12 ml de medio de diferenciación que contiene 400 mg / ml de G418 y semillas de ellos en 6 pozos de gelatina recubiertas 24 pocillos (2 ml / pocillo). </li></ol></li><li> Generación de células ganglionares sola. <ol><li> Suspender las células de la etapa 2.8.6 en 5 ml Accutase y se incuba a 37 ° C durante 5 min. </li><li> Mezclar vigorosamente la suspensión por la pipeta hacia arriba y hacia abajo 10 veces y se incuban las células a 37 ° C durante 5 min. </li><li> Añadir 20 ml de PBS y centrifugar durante 5 min a 300 x g. </li><li> Suspender las células en 12 ml de medio de diferenciación que contienen 400 mg / ml de G418 y semillas de ellos en 6 pocillos de una gelatina recubiertos 24 pocillos (2 ml / pocillo). </li></ol></li><li> Día 2 después de la disociación, aspirar el medio y lavar las células tres veces con PBS. Añadir 1 ml de medio de diferenciación / pocillo. </li><li> Día 4-8 después de la disociación, cambio de medio cada 2º día. En el día 10 después de iSABs uso disociación o isab seno derivada nodal células individuales están disponibles para su posterior análisis. </li></ol>

<ol>
	<li>Wobus, A. M., Wallukat, G., Hescheler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+mouse+embryonic+stem+cells+are+able+to+differentiate+into+cardiomyocytes+expressing+chronotropic+responses+to+adrenergic+and+cholinergic+agents+and+Ca2++channel+blockers.">Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers.</a> Differentiation. 48 (3), 173-182 (1991).</li><li>David, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MesP1+drives+vertebrate+cardiovascular+differentiation+through+Dkk-1-mediated+blockade+of+Wnt-signalling.">MesP1 drives vertebrate cardiovascular differentiation through Dkk-1-mediated blockade of Wnt-signalling.</a> Nat Cell Biol. 10, 338-345 (2008).</li><li>David, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+programming+of+pluripotent+stem+cells+towards+distinct+cardiovascular+cell+types.">Forward programming of pluripotent stem cells towards distinct cardiovascular cell types.</a> Cardiovasc Res. 84 (2), 263-272 (2009).</li><li>Hoogaars, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tbx3+controls+the+sinoatrial+node+gene+program+and+imposes+pacemaker+function+on+the+atria.">Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria.</a> Genes Dev. 21 (9), 1098-1112 (2007).</li><li>Klug, M. G., Soonpaa, M. H., Koh, G. Y., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+selected+cardiomyocytes+from+differentiating+embronic+stem+cells+form+stable+intracardiac+grafts.">Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts.</a> J Clin Invest. 98 (1), 216-224 (1996).</li><li>Marger, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pacemaker+activity+and+ionic+currents+in+mouse+atrioventricular+node+cells.">Pacemaker activity and ionic currents in mouse atrioventricular node cells.</a> Channels (Austin). 5 (3), 241-250 (2011).</li><li>Jung, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programming+and+isolation+of+highly+pure+physiologically+and+pharmacologically+functional+sinus-nodal+bodies+from+pluripotent+stem+cells).">Programming and isolation of highly pure physiologically and pharmacologically functional sinus-nodal bodies from pluripotent stem cells).</a> Stem cell reports. 2 (5), 592-605 (2014).</li><li>Maltsev, V. A., Wobus, A. M., Rohwedel, J., Bader, M., Hescheler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocytes+differentiated+in+vitro+from+embryonic+stem+cells+developmentally+express+cardiac-specific+genes+and+ionic+currents.">Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents.</a> Circulation research. 75 (2), 233-244 (1994).</li><li>He, J. Q., Ma, Y., Lee, Y., Thomson, J. A., Kamp, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cells+develop+into+multiple+types+of+cardiac+myocytes:+action+potential+characterization.">Human embryonic stem cells develop into multiple types of cardiac myocytes: action potential characterization.</a> Circ Res. 93 (1), 32-39 (2003).</li><li>Yanagi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarization-activated+cyclic+nucleotide-gated+channels+and+T-type+calcium+channels+confer+automaticity+of+embryonic+stem+cell-derived+cardiomyocytes.">Hyperpolarization-activated cyclic nucleotide-gated channels and T-type calcium channels confer automaticity of embryonic stem cell-derived cardiomyocytes.</a> Stem cells. 25 (11), 2712-2719 (2007).</li><li>Barbuti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+composition+and+functional+properties+of+f-channels+in+murine+embryonic+stem+cell-derived+pacemaker+cells.">Molecular composition and functional properties of f-channels in murine embryonic stem cell-derived pacemaker cells.</a> J Mol Cell Cardiol. 46 (3), 343-351 (2009).</li><li>Ma, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+purity+human-induced+pluripotent+stem+cell-derived+cardiomyocytes:+electrophysiological+properties+of+action+potentials+and+ionic+currents.">High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents.</a> American journal of physiology. Am J Physiol Heart Circ Physiol. 301 (5), H2006-H2017 (2011).</li><li>David, R., Franz, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+pluripotency+to+distinct+cardiomyocyte+subtypes.">From pluripotency to distinct cardiomyocyte subtypes.</a> Physiology (Bethesda). 27 (3), 119-129 (2012).</li><li>Scavone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell-derived+CD166++precursors+develop+into+fully+functional+sinoatrial-like+cells).">Embryonic stem cell-derived CD166+ precursors develop into fully functional sinoatrial-like cells).</a> Circ Res. 113 (4), 389-398 (2013).</li><li>Morikawa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification,+isolation+and+characterization+of+HCN4-positive+pacemaking+cells+derived+from+murine+embryonic+stem+cells+during+cardiac+differentiation.">Identification, isolation and characterization of HCN4-positive pacemaking cells derived from murine embryonic stem cells during cardiac differentiation.</a> Pacing Clin Electrophysiol. 33 (33), 290-303 (2010).</li><li>Wiese, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+sinus+node+head+and+differentiation+of+sinus+node+myocardium+are+independently+regulated+by+Tbx18+and+Tbx3.">Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3.</a> Circ Res. 104 (3), 388-397 (2009).</li><li>Kleger, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+calcium-activated+potassium+channels+induces+cardiogenesis+of+pluripotent+stem+cells+and+enrichment+of+pacemaker-like+cells.">Modulation of calcium-activated potassium channels induces cardiogenesis of pluripotent stem cells and enrichment of pacemaker-like cells.</a> Circulation. 122 (18), 1823-1836 (2010).</li><li>Bakker, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-box+transcription+factor+TBX3+reprogrammes+mature+cardiac+myocytes+into+pacemaker-like+cells.">T-box transcription factor TBX3 reprogrammes mature cardiac myocytes into pacemaker-like cells.</a> Cardiovasc Res. 94 (3), 439-449 (2012).</li><li>Kapoor, N., Liang, W., Marban, E., Cho, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+conversion+of+quiescent+cardiomyocytes+to+pacemaker+cells+by+expression+of+Tbx18.">Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18.</a> Nat Biotechnol. 31 (1), 54-62 (2013).</li><li>Johns, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+expression+of+a+voltage-gated+potassium+channel+in+vitro+(rat+cardiac+myocytes)+and+in+vivo+(rat+liver).+A+novel+strategy+for+modifying+excitability.">Adenovirus-mediated expression of a voltage-gated potassium channel in vitro (rat cardiac myocytes) and in vivo (rat liver). A novel strategy for modifying excitability.</a> J Clin Invest. 96 (2), 1152-1158 (1995).</li><li>Nuss, H. B., Marban, E., Johns, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+a+human+potassium+channel+suppresses+cardiac+hyperexcitability+in+rabbit+ventricular+myocytes.">Overexpression of a human potassium channel suppresses cardiac hyperexcitability in rabbit ventricular myocytes.</a> J Clin Invest. 103 (6), 889-896 (1999).</li></ol>

Los autores no tienen nada que revelar.

La capacidad de producir tallo de células derivadas de células marcapasos cardíacos puede permitir la reconstitución del ritmo cardíaco adecuado en el sentido de &quot;marcapasos biológicos&quot;. Del mismo modo, las pruebas de drogas in vitro se beneficiará de su disponibilidad. PSC puede dar lugar a cualquier tipo de célula del cuerpo de un mamífero incluyendo cardiomiocitos con propiedades de células marcapasos 8,9,10,11,12,13. Sin embargo, por lo general las poblaciones de células dentro de &qu...

El término &quot;síndrome del seno enfermo&quot; resume múltiples enfermedades que conducen al deterioro del sistema de marcapasos cardíaco. Comprende patológica, bradicardia sintomática sinusal, bloqueo sinoauricular, paro sinusal, así como el síndrome de taquicardia-bradicardia. De este modo, un &quot;síndrome del seno enfermo&quot; suele ir acompañada de enfermedades cardíacas generales tales como la cardiopatía isquémica, las miocardiopatías o miocarditis. En la actualidad, los enfoques terapéuticos se basan en la implantación de marcapasos eléctricos. Sin embargo, esto va junto con una serie de riesgos tales como infecciones y fallo de la batería. En general, la incidencia de complicaciones es todavía muy alta en pacientes que han implantaron un marcapasos artificial. Además, a diferencia de la marcapasos endógeno, estos dispositivos no responden a la estimulación hormonal. Una alternativa de futuro puede depender de la disponibilidad de &quot;marcapasos biológicos&quot; para el que PSC podrían servircomo fuente celular adecuado y que también sería de gran valor para vitro pruebas de drogas en. Sin embargo, un problema importante reside en la aparición muy rara de células nodales de los senos dentro de cuerpos embrioides (EBS) - esto normalmente no excede de ~ 0,5% 1. Anteriormente, se demostró que la &quot;programación hacia adelante&quot; hacia los subtipos específicos de los cardiomiocitos es factible a través de la sobreexpresión de factores de transcripción distintos cardiovasculares precoces tales como el factor-mesodermo específica posterior 1 (MesP1) y NK2 de transcripción relacionados, locus 5 (Nkx2.5) 2, 3. Para el tamaño y la función del nódulo sinoauricular (SAN) normal, la T-box factor de transcripción Tbx3 es crucial, que se ha demostrado para iniciar el programa de genes marcapasos y para controlar la diferenciación de la SAN 4. Si bien esto mejora la apariencia de las células marcapasos funcionales, el contenido todavía no excedió de ~ 40% dentro de toda la población de células cardiomyocytic. Por lo tanto, una etapa de selección de antibiótico basado MYH6-promotor adicional 5 fue introducido por nosotros. Esto conduce en última instancia a los agregados de cardiomiocitos todavía no observados (&quot;cuerpos inducidos sinoauricular;&quot; iSABs &quot;) que exhiben altamente aumento de la frecuencia golpeando (&gt; 400 bpm) in vitro, por primera vez se aproxima a las de un corazón murino y comparable a cultivado in vitro las células ganglionares de los senos aisladas de un corazón murino 6. Bajo la administración Isoprenalina se logran incluso superando frecuencias de 550 lpm. Notablemente, iSABs consisten en más del 80% de las células ganglionares funcionales como se desprende de un extenso análisis fisiológico 7. Recientemente, varios enfoques para generar seno células ganglionares mediante la reprogramación directa 19, superficie marcadores 14 o el tratamiento farmacológico con pequeñas moléculas 16,17 fueron descritos. Sin embargo, ninguno de estos métodos dio lugar a una alta pureza de las células marcapasos tal y golpeando a frecuencias cercanas a la murino élarte como se observa en iSABs. Por otra parte, en un modelo ex vivo de las rebanadas ventriculares ratón adulto cultivadas que han perdido su actividad paliza espontánea, los iSABs son capaces de integrarse en el tejido rebanada, por lo que permanece activa espontáneamente y robustamente paseo por las rebanadas del corazón a las contracciones 7. Un protocolo detallado para la generación de estos iSABs se describe en este 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

El tratamiento del &quot;síndrome del seno enfermo&quot; se basa en los marcapasos artificiales. Estos llevan peligros tales como fallo de la batería y las infecciones. Por otra parte, carecen de la capacidad de respuesta de la hormona y el procedimiento general es costosa. &quot;Marcapasos biológicos&quot; generados a partir de las ventanillas únicas pueden llegar a ser una alternativa, sin embargo, el contenido típico de las células marcapasos en cuerpos embrionarios (EB) es extremadamente baja. El protocolo descrito combina &quot;programación futura&quot; del PSC murinos través del TBX3 inductor nodo sinusal con antibióticos de selección basado MYH6-promotor. Esto produce agregados cardiomiocitos consistentes de&gt; 80% de células marcapasos fisiológicamente funcionales. Estos &quot;inducida por sinoauricular-cuerpos&quot; (&quot;iSABs&quot;) se contraen espontáneamente a frecuencias aún no alcanzados (400-500 bpm) correspondientes a las células ganglionares aisladas de corazones de ratones y son capaces de caminar de miocardio murino ex vivo. Utilizando el protocolo descrito sinusal altamente puracélulas individuales nodales se pueden generar por ejemplo, que se puede utilizar para pruebas de drogas en vitro. Por otra parte, los iSABs generados de acuerdo con este protocolo puede llegar a ser un paso crucial hacia la ingeniería de tejidos del corazón.

El protocolo descrito permite la generación de iSABs con una frecuencia de latidos de alrededor de 450 ppm de PSC (que se muestran en la película) que está cerca de la frecuencia de los latidos del corazón del ratón. Después de la disociación de iSABs (etapa 2.8.8) las células individuales observados muestran la forma típica de las células del nodo sinusal (células fusiformes y de la araña) como se muestra en la Figura 1. Estas células proteínas altamente expresas que se sabe que son esenc...

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

Este protocolo describe cómo producir seno tejido ganglionar funcional a partir de células madre pluripotentes murinos (PSC). T-Caja3 (TBX3) sobreexpresión plus-cadena-miosina pesada cardiaca (MYH6) selección antibiótico promotor conduce a agregados de células marcapasos de alta pureza. Estos &quot;inducida sinoauricular-cuerpos&quot; (&quot;iSABs&quot;) contienen más de 80 células marcapasos%, muestran altamente aumento de las tasas de latir y se puede regular el ritmo miocardio ex vivo.

Generación de murino cardíaca marcapasos celulares Agregados Basado en ES-Cell-Programación en combinación con MYH6-promotor-Selección

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 vistas.  University of Rostock.  Este protocolo describe cómo producir seno tejido ganglionar funcional a partir de células madre pluripotentes murinos (PSC). T-Caja3 (TBX3) sobreexpresión plus-cadena-miosina pesada cardiaca (MYH6) selección antibiótico promotor conduce a agregados de células marcapasos de alta pureza. Estos "inducida sinoauricular-cuerpos" ("iSABs") contienen más de 80 células marcapasos%, muestran altamente aumento de las tasas de latir y se puede regular e...

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.

Humano madre pluripotentes células Basado Toxicidad para el desarrollo de ensayos de la seguridad química Detección y biología de sistemas de generación de datos

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

Investigación

Biología del Desarrollo

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.

Generación de agregados de células madre embrionarias de ratón que Muestre Symmetry Breaking, Polarización y Comportamiento Colectivo Emergente<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.

Agregada la optimización del tamaño de micropocillos para basada en suspensión cardiaca diferenciación de células madre pluripotentes

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 nueva técnica de trasplante de grasa mamaria para visualizar la generación de vasos de células madre endoteliales vasculares

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.

Análisis de la inmunofluorescencia de Whole-mount cuantitativa de las poblaciones progenitoras cardiacas en embriones de ratón

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.

Generación de insulina libre de andamios, tridimensional expresar Pancreatoids de progenitores pancreáticos de ratón 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.

Análisis del desarrollo de la cámara cardiaca durante la embriogénesis de ratón usando todo de Monte de epifluorescencia

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 Generación de Células Progenitoras Cardíacas específicas de Mouse Field

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.

Generación de organoides mamarios de mosaico por trippsinización diferencial

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.

Generación de un chip de barrera hematoencefálica basada en iPSC humana

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 ...