The work was supported in part by the Duncan and Nancy MacMillan Faculty Development Chair Endowment Fund (K.Y.K.), Busch Biomedical Research Grant (K.Y.K.) and the Rutgers Faculty Development Grant (K.Y.K.).

1. El mantenimiento de auto-renovación en células PIMO <ol><li> Preparar PIMO medios de cultivo: DMEM / F12, 1X suplemento B27, 25 mg / ml y carbenecillin 20 ng / ml bFGF. Hacer 50 ml de PIMO medios de cultivo utilizando reactivos estériles. Calentar 49 ml de DMEM / F12 en una cónica de 50 ml en un baño de agua a 37 ° C. <ol><li> Suplemento Descongele 50X B27 y 100 mg / carbenecillin alícuotas de filtro esterilizado ml durante 5 minutos en un baño de agua a 37 ° C. Descongelar 100 g / ml bFGF alícuota a RT. Añadir 1 ml de 50X B27, 10 l de 100 mg / ml bFGF y 12,5 l de 100 mg / ml carbenecillin en DMEM / F12. </li></ol></li><li> Utilice 3 ml de medio en un 60 mm placa de cultivo tisular de células PIMO cultivo. Añadir medio fresco a los cultivos cada dos días al duplicar el volumen de medios de comunicación. Asegúrese de que la concentración de bFGF no cae por debajo de 5 ng / ml. <ol><li> Células PIMO cultivo a 37 ° C con 5% de CO 2. Las células Passage después de 5 - 7 días en cultivo que se enumeran en los siguientes pasos. La cultura de transferencia a un 15ml cónica con una punta de pipeta de 10 ml. </li></ol></li><li> Hacer EDTA 1 mM en HBSS mediante la dilución de 0,1 ml de 0,5 M EDTA pH 8,0 en 50 ml de HBSS. Pre-caliente EDTA 1 mM hizo en HBSS en un 37 ° C baño de agua. </li><li> Cosecha células por sedimentación por gravedad o centrifugación a 200 xg durante 5 min. a TA. Para sedimentación por gravedad, coloque el cónico de 15 ml que contiene las culturas en el 37 ° C incubadora durante 5 a 10 min. Después de ese tiempo, observan los otospheres recogen en la parte inferior de la cónica 15 ml. NOTA: la fuerza centrífuga excesiva puede causar daño celular. </li><li> Cuidadosamente aspirado pasado multimedia usando una pipeta 2 ml de aspiración sin alterar el sedimento celular. Añadir 0,5 ml de la solución de pre-calentado 1 mM EDTA HBSS a la celda de pellets. Con una pipeta P1000, pipetear suavemente hacia arriba y hacia abajo 2 - 3 veces. Coloque cónica a 37 ° C y se incuba durante &lt;5 min para facilitar la disociación en células individuales. </li><li> Agite suavemente la solución de células para determinar si se disocian otospheres. Si no otoesferas de sedimentos para la parte inferior de la cónica, las células se disocian. El tiempo de incubación puede variar. NOTA: La prolongada incubación con EDTA puede provocar la muerte celular excesiva. </li><li> Retire las células de 37 ° C, añadir 2 ml de medio para neutralizar y diluir el EDTA. Recoger las células por centrifugación a 200 xg durante 5 min a TA. Aspirar diluyó EDTA utilizando una pipeta de 2 ml de aspiración y añadir 5 ml de 1X PBS para lavar las células. </li><li> Girar las células hacia abajo a 200 xg durante 5 min a RT y aspirar a cabo 1X PBS usando una pipeta de 2 ml de aspiración. Resuspender las células por medio de una pipeta P1000 en 0,5 ml de medio de cultivo PIMO pipeteando suavemente hacia arriba y hacia abajo 2 - 3 veces. </li><li> Contar las células utilizando un contador de partículas de microfluidos con los cassettes apropiados 28. Una placa confluente de células PIMO contendrá ~ 6 X10 6 células. Diluir las células 1: 100 en los medios de comunicación y añadir 75 ul de solución de células en el cassette para el conteo. Plate 1 x 10 6 células en una placa de 6 cm en PIMO culture medios de comunicación (~ 1: 10 de dilución). IMOPs Passage cada 5 - 7 días. NOTA: Las células que forman grandes otospheres o se adhieren a la parte inferior de la placa de cultivo de tejido va a diferenciar. Las células también se mueren si las culturas son más confluentes. </li></ol> 2. congelación y descongelación células PIMO <ol><li> Preparar medio de congelación sintética de descongelación y equilibrar la solución a 4 ° C antes de células de congelación. </li><li> Recoger las células de un confluente placa de 6 cm de células PIMO. Usar una pipeta de 10 ml y las células de transferencia (~ 6 - 8 x 10 6 células) en un cónica 15 ml. </li><li> Cosecha células por sedimentación por gravedad o centrifugación a 200 xg durante 5 min a TA. Si los otospheres son pequeñas, las células no se asientan por sedimentación por gravedad. Opcional: Recuento de células utilizando el paso 1.9 para determinar el número de células totales. </li><li> Aspirar pasó multimedia usando una pipeta 2 ml de aspiración, dejando el sedimento celular suelta atrás. </li><li> Añadir 0,25 ml de medio de congelación sintéticos de volver a suspender ceLLS a una densidad de 5 x 10 ~ 5 a 3 x 10 6 células / ml. Pipetear suavemente las células con una pipeta P1000. Transferir la suspensión celular a viales criogénicos utilizando una pipeta P1000 con 1 ml filtradas punta. </li><li> Colocar los viales en un recipiente de congelación libre de alcohol y coloque el recipiente de congelación a -80 ° C para reducir la temperatura de 1 ° C por minuto hasta que la temperatura alcanza -80 ° C. </li><li> Para el almacenamiento a largo plazo de las células, la transferencia de los viales a la fase de vapor de un tanque de almacenamiento de nitrógeno líquido. Para descongelar las células para la cultura, equilibre vial criogénico que contiene células congeladas a -80 ° C. </li><li> Pre-caliente medios de cultivo PIMO en un 37 ° C baño de agua. </li><li> Descongele el vial congelado rápidamente agitando el fondo del vial en un 37 ° C baño de agua. Añadir 1 ml de medios de cultivo pre-calentado PIMO a las células descongeladas una vez que el cristal de hielo pasada desaparece. La transferencia de células en un ml cónica 15 y añadir otros 4 ml de PIMO medios de cultivo </li><li> Gira la 15 ml conica durante 5 min. a 200 xg y aspirar los medios gastados. Resuspender las células en 2 ml de medio de cultivo PIMO y células de la placa en una placa de 6 cm. Incubar los cultivos a 37 ° C con 5% de CO 2 para la expansión. </li></ol> 3. Diferenciar las células PIMO en sensorial epitelios <ol><li> Preparar 50 ml de PIMO medio de diferenciación epitelios sensoriales: DMEM / F12, suplementos 1X B27, 25 mg / ml carbenecillin. Hacer 50 ml de medio de diferenciación PIMO sensorial epitelios. Calentar 49 ml de DMEM / F12 en una cónica de 50 ml en un baño de agua a 37 ° C. Suplemento de B27 Descongelar 50X y 100 mg / ml para carbenecillin alícuotas de 5 min. en un 37 ° C baño de agua. Añadir 1 ml de 50X B27 y 12,5 l de 100 mg / ml carbenecillin en DMEM / F12. </li><li> Para cosechar, disociar, resuspender y contar células Repita los pasos 1,4 a 1,9 </li><li> Plate 1 x 10 6 células en una placa de 6 cm en el Día -3 utilizando PIMO medios de cultivo. En el Día 0, los cultivos de transferencia utilizando una pipeta de 10 ml en un cónica 15 ml. Recoger el otoesferas de sedimentación por gravedad como se indica en el paso 1.6. Aspirar pasó multimedia usando un 2 ml de aspiración pipeta y dejar las otospheres en la parte inferior de la cónica. </li><li> Añadir suavemente en 2 ml de medio de diferenciación sensorial epitelios. Transferencia otospheres a un plato de 6 cm utilizando un gran ml pipeta de diámetro 10. NOTA: esquila mecánica de duras pipeteo puede disociar las células de los otospheres. </li><li> Añadir 2 ml de los medios de comunicación sensorial diferenciación epitelial fresco a culturas cada dos días. Si es necesario, las células pueden ser recogidos por sedimentación por gravedad y los medios reemplazado. </li><li> Recoger otospheres en el día 10 mediante la transferencia a un ml cónica 15 y permitiendo que los otospheres que sedimentan como se indica en el paso 1.6.Aspirate los medios de comunicación dedicado al uso de 2 ml de aspiración pipeta y dejan los otospheres sin ser molestados. </li><li> Fijar otospheres incubando en 4% de formaldehído en PBS 1X durante 15 min a RT. Retire solución de formaldehído, lavar otospheres con tampón de lavado (1X PBS que contenía 0,1% Triton X-100) antes de incubar en tampón de bloqueo (1X PBS que contenía 10% de suero normal de cabra y 0.1% de Triton X-100) durante 1 hora. <ol><li> Reemplazar tampón y se incuba las muestras en tampón de bloqueo con anticuerpo diluido. Se incuba a 4 ° C. Lávese las muestras con 1X PBS que contenía 0,1% de Triton X-100 sujetos del otosphere a inmunotinción 27. Otospheres transferencia en 1X PBS y otospheres lugar en los medios de montaje sobre un portaobjetos de vidrio usando una pipeta P1000. Ponga un cubreobjetos sobre la muestra y permitir que los medios de montaje se sequen a 4 ° C. </li></ol></li><li> Adquirir imágenes de epifluorescencia utilizando una configuración de microscopio invertido equipado con una cámara CCD de 16 bits y, o bien un aire 20X 0.75 o una NA objetivo de inmersión en aceite 40X 1,3. Recoger fluorescencia de canales de color diferentes utilizando el longitudes de onda que aparece (excitación y emisión): azul (377 ± 25 nm / 447 ± 30 nm), verde (475 ± 25 nm / 540 ± 25 nm), rojo (562 ± 20 nm / y 625 20 nm ±) e infrarrojo (628 ± 20 nm / 692 ± 20 yNuevo Méjico). </li></ol> 4. Ensayo de EdU Incorporación <ol><li> En el Día -3, placa 1 x 10 6 células PIMO en un plato de 6 cm. Tres días más tarde en el día 0, cosecha, disociar, resuspender las células y contar por pasos 1,4-1,9 repetir. </li><li> Placa de 2,5 x 10 5 células en medios de cultivo PIMO y 5 x10 5 células en medio de diferenciación sensorial epitelios en diferentes pocillos de una placa de 6 pocillos. </li><li> Determinar el porcentaje de células en fase S mediante la incorporación EdU en día 3 mediante el uso de un ensayo de incorporación de EdU <ol><li> Añadir EdU acciones directamente a las culturas PIMO para obtener una concentración final de 1 mM EdU en los medios de cultivo. </li><li> Incubar EdU en cultivos PIMO durante 2 horas y se incuba a 37 ° C con 5% de CO 2. Cosecha, disociar y recoger células repita los pasos 1.4-1.9. Añadir en 4% de formaldehído en PBS 1X durante 15 min. a temperatura ambiente para fijar las células. Cosecha células por centrifugación a 200 xg durante 5 min a TA. Retire solución de formaldehído y properly deseche. </li><li> Núcleos de la etiqueta de células con Hoechst y incorporados EdU con el verde de fluorescencia tinte azida de acuerdo con el protocolo del fabricante. NOTA: Todos los lavados se realizan con 1X PBS, BSA al 3% y 0,1% de células Tween 20. Lave con 1X PBS dos veces. </li><li> Células de montaje en una diapositiva utilizando antifade reactivo y coloque un vaso 1,5 cubierta sobre la muestra. Adquirir imágenes fluorescentes de células marcadas usando un microscopio de epifluorescencia. </li></ol></li></ol> 5. Diferenciar neuronas PIMO-Derivado <ol><li> Hacer 50 ml de medio de diferenciación neuronal: medios Neurobasal, 1X suplemento B27, 2 mM L-glutamina. Botella Deshielo de 50X B27 y 200 mM de L-glutamina en 37 ° C baño de agua durante 5 minutos. Añadir 1 ml de 50X B27 y 0,5 ml de 200 mM de L-glutamina a 48,5 ml de medio Neurobasal. </li><li> Escudo mediante la colocación de cubreobjetos redondos de 12 mm de 1,5 cubreobjetos de vidrio en una placa de 10 cm estéril y añadir 70% de EtOH a la placa para esterilizar y limpiar los cubreobjetos. </li><li> Agitar suavemente la calarslips para asegurarse de que están cubiertos en etanol. Deja la placa durante 10 minutos a temperatura ambiente. Enjuague los cubreobjetos 3 veces con 1X PBS estéril para lavar el etanol restante. Enjuague el portaobjetos una vez con H 2 0 estéril para lavar restante 1X PBS. </li><li> Aspirar el H 2 0 utilizando un 2 ml de aspiración pipeta y dejar que los cubreobjetos seco. Exponga los cubreobjetos a la luz UV en la campana de cultivo de tejidos de 15 min. Tienda cubreobjetos en un ambiente estéril si no se utiliza de inmediato. </li><li> Coloque cubreobjetos uno 12 mm redondo en cada pocillo de una placa de 24 pocillos. Agitar suavemente la placa para asegurarse de que los cubreobjetos se acueste en el fondo del pozo. </li><li> Descongelar 2 mg / ml de poli-D-lisina solución madre a TA y se diluye a un / ml de concentración de poli-D-lisina 10 mg en 1X PBS. Añadir 0,25 ml de 10 ug / ml de poli-D-lisina a los pocillos. Deja la placa en la incubadora a 37ºC durante 1 hora. </li><li> Lavar los pocillos 3 veces con 1X PBS estéril. Descongele 10 mg solución de laminina / ml a temperatura ambiente y diluir a 10 g / ml en 1X PBS. Añadir 0,25 ml de 10 mg / ml de laminina solución de trabajo en un solo pocillo de una placa de 24 pocillos múltiples. Incubar la placa a 37 ° C incubadora. Aspirar la solución laminina usando una pipeta 2 ml de aspiración. </li><li> Lavar el cubreobjetos 3 veces con PBS 1X mediante la adición de 1 ml de PBS 1X con una pipeta P1000 y retirar el PBS 1X por aspiración con una pipeta de 2 ml de aspiración. Deja 1X PBS del último lavado en el pozo hasta que las células están listas para ser plateado. Iniciar la diferenciación neuronal por la cosecha, disociando y contando las células en los pasos células 1.4-1.9.Plate 1 x 10 6 en una placa de 6 cm en el Día -3. </li><li> En el Día 0, cosecha, disociar y contar las células mediante la repetición de 1,4 a 1,9. Seed 1 x 10 5 - 1.5 x 10 5 células PIMO en 0,5 ml pre-calentado medio de diferenciación neuronal por pocillo en una placa de 24 pocillos múltiples. Aspirar y añadir precalentado medio de diferenciación neuronal de culturas cada dos días. </li><li> Fijar neuronas derivadas de PIMO en formaldehído al 4% durante 15 min. lat RT en el Día 7. Retire solución de formaldehído, lavar neuronas derivadas de PIMO con tampón de lavado (1X PBS que contenía 0,1% Triton X-100) antes de incubar en tampón de bloqueo (1X PBS que contenía 10% de suero normal de cabra y 0.1% de Triton X-100) durante 1 h. <ol><li> Reemplazar tampón y se incuba las muestras en tampón de bloqueo con anticuerpo diluido. Se incuba a 4 ° C. Lávese las muestras con 1X PBS que contenía 0,1% de Triton X-100 células sometidas a inmunotinción 27. Lave el cubreobjetos con PBS 1X antes de colocar en soportes de montaje. Permita que los medios de montaje se sequen a 4 ° C antes de la adquisición de imágenes de epifluorescencia que se enumeran en el paso 3.8. </li></ol></li></ol>

The authors have nothing to disclose.

Monitoreo de Cultivos PIMO Un protocolo para el mantenimiento de auto-renovación y promoción de la diferenciación de una novela línea celular PIMO se describe y formatos de chapado adicionales se incluyen (Tabla 1). Se observan varios pasos críticos que ayudan con la expansión de la rutina y la diferenciación de las células PIMO. Al igual que en cultivos de células madre pluripotentes, células PIMO teóricamente tienen una vida útil indefinida. Pa...

The organs of the inner ear, the cochlea, utricle, saccule and three semicircular canals, mediate the ability to hear and balance. Within the cochlea, hair cells convert sounds into electrical signals that are relayed to the spiral ganglion neurons (SGN). The SGNs fire action potentials to propagate neural signals through the auditory circuit. Genetic mutations, ototoxic drugs and exposure to loud sounds contribute to hair cell and SGN death that result in hearing loss1-4. Once lost, these cells are not replaced. Use of iPSC and ESC to generate nascent hair cells or SGNs holds great promise for inner ear cell replacement therapies5-8. A flurry of progress has shown that pluripotent stem cells and inner ear derived progenitors can differentiate into hair cells and SGNs at various stages of maturity. Mammalian embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can be used to generate functional hair cells and SGNs9-11. Stem cells and progenitor cells derived from the mammalian inner ear have also been shown to form hair cells and neurons with properties of their in vivo cellular counterparts12-16. 
Use of iPSC or ESC-derived otic progenitors to replace lost hair cells and SGNs requires efficient differentiation. Improper differentiation or continued proliferation of engrafted stem-derived progenitors in the inner ear can exacerbate inner ear function and pose a tumorigenic risk such as teratomas formation in the inner ear17. There is a clear need for developing culture conditions and understanding differentiation of otic progenitors. One strategy in developing these methods is to recapitulate cell fate decisions made by neurosensory progenitors during inner ear development. Protocols that prevent proliferation and direct otic progenitors into hair cells or SGNs will help improve safety as well as efficacy of replacement therapies. 
During development, the inner ear begins with the thickening of surface ectoderm in a restricted region between rhombomeres 5 and 6 to become the otic placode. As the otic placode invaginates to form an otic cup, a collection of cells in the anterior region of the otic cup gives rise to the neural-sensory-competent domain (NSD), which contains precursors of hair cells and neurons of the inner ear18. Fate mapping studies from mouse, chicken and zebrafish developing inner ear suggest multiple populations of neurosensory progenitors that give rise to the sensory hair cells, surrounding supporting cells and otic neurons19-22. The high mobility group transcription factor, Sox2, has been implicated in sensory cell specification and used as a marker for inner ear progenitors23,24. Hypomorphic mutations that decrease Sox2 expression levels in the inner ear result in the loss of the hair cells, supporting cells and SGNs in the cochlea25,26. 
To study otic progenitor cells undergoing cell fate decisions, a fate restricted immortalized multipotent otic progenitor (iMOP) cell line from Sox2 expressing cochlear progenitors was previously established. iMOP cells were originally derived from embryonic E12.5-13.5 cochlea and infected with a c-Myc retrovirus27. iMOP cells can continually proliferate as colony forming cells known as otospheres and have the capacity to differentiate into hair cells, supporting cells and SGNs27. Understanding the capacity of iMOP cells to differentiate into distinct otic lineages allows application of these findings to efficiently generate iPSC or ESC-derived hair cells and SGNs. Efficient differentiation protocols will open new avenues for cell replacement therapies of inner ear diseases that are recalcitrant to conventional treatments. A crucial issue in generating otic cells by in vitro cell culture is to have differentiation markers that help determine if cells are undergoing differentiating. Cdkn1b (p27KIP) has been extensively used as an early marker for differentiation in developing inner ear, however, expression of Cdkn1b in iMOP cells and how it correlates to differentiation has not been addressed. In this study, the current culture conditions and how Cdkn1b expression correlates to other markers of iMOP differentiation are described.

<table border="0" cellpadding="0" cellspacing="0" width="1075">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">CoolCell LX Alcohol-Free Cell Freezing Containers</td>
			<td style="width:196px;">BioCision</td>
			<td style="width:145px;">BCS-405</td>
			<td style="width:280px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Cryogenic Vials (2 ml)&#160;</td>
			<td style="width:196px;">Corning</td>
			<td style="width:145px;">430654</td>
			<td style="width:280px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:455px;">1.5 Thickness Glass Coverslip (Round 12 mm)</td>
			<td style="width:196px;">Electron Microscopy Sciences</td>
			<td style="width:145px;">72230-01</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">DMEM/F12</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">11320-082</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Neurobasal Medium</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">21103</td>
			<td style="width:280px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Phosphate Buffered Saline (PBS) pH 7.4</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">10010-023</td>
			<td style="width:280px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">14025-092</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">B27 Supplement (50X) Serum Free</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">17504-044</td>
			<td style="width:280px;">Stored as 1 ml aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">L-Glutamine(200 mM)&#160;</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">25030-081</td>
			<td style="width:280px;">Stored as 5 ml aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Natural Mouse Laminin</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">23017-015</td>
			<td style="width:280px;">Stored as 1 mg/ml aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Click-iT EdU Alexa Fluor 488&#160;</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">C10337</td>
			<td style="width:280px;"></td>
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			<td height="20" style="height:20px;width:455px;">Synth-A-Freeze Cryopreservation Media</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">A12542-01</td>
			<td style="width:280px;"></td>
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			<td height="20" style="height:20px;width:455px;">Prolong Gold Antifade Mountant</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">47743-736</td>
			<td style="width:280px;">Stored as 10 mg/ml 100 &#181;l aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Moxi Z Mini Automated Cell Counter</td>
			<td style="width:196px;">Orflo&#160;</td>
			<td style="width:145px;">MXZ001</td>
			<td style="width:280px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Moxi Z Cassette Type S</td>
			<td style="width:196px;">Orflo&#160;</td>
			<td style="width:145px;">MXC002</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:455px;">Recombinant Murine Fibroblast Growth Factor, basic (bFGF)</td>
			<td style="width:196px;">Peprotech</td>
			<td style="width:145px;">450-33</td>
			<td style="width:280px;">Resuspended in 0.1% BSA in H20 and stored as 20 mg/ml aliquots</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:455px;">Poly-D-Lysine</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:145px;">P7886</td>
			<td style="width:280px;">Resuspended in 1X PBS and stored as 10 mg/ml 100 &#181;l aliquots</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:455px;">Carbenicillin, Disodium Salt</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">BP2648-1</td>
			<td style="width:280px;">Resuspended in 10mM Hepes pH 7.4 and stored as 100 mg/ml aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">5 ml pipet individually wrapped paperback (200/case)</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td>1367811D</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">10 ml pipet individually wrapped paperback (200/case)</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td>1367811E</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Tissue Culture Treated Biolite 24 -Well Plate</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">130188</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Tissue Culture Treated Biolite 6 -Well Plate</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">130184&#160;</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Tissue Culture Treated 6 cm Dish</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">130181&#160;</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EMD Millipore Millex Sterile Syringe PVDF Filter Pore size: 0.22&#956;m</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 0.1-10 ul elongated filter tip</td>
			<td>USA Scientific</td>
			<td>1120-3810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 1-20 ul filter tip</td>
			<td>USA Scientific</td>
			<td>1120-1810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 1-200 ul&#160; filter tip</td>
			<td>USA Scientific</td>
			<td>1120-8810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 101-1000 ul filter tip</td>
			<td>USA Scientific</td>
			<td>1126-7810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">15 ml conical tubes sterile 20 bags of 25 tubes (500 tubes)</td>
			<td>USA Scientific</td>
			<td>1475-0511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">50 ml conical tubes sterile 20 bags of 25 tubes (500 tubes)</td>
			<td>USA Scientific</td>
			<td>1500-1211</td>
			<td></td>
		</tr>
	</tbody>
</table>

initiating differentiation in immortalized multipotent otic progenitor cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

bFGF Retiro disminuye la proliferación de células PIMO Para disminuir la capacidad proliferativa de las células PIMO e iniciar la diferenciación de las células PIMO, bFGF fue retirado de los cultivos. Para confirmar que la retirada del factor de crecimiento disminuye la proliferación, la incorporación EdU fue empleado como un ensayo de proliferación. El porcentaje de células que incorporaba EdU de oto...

Watch this Scientific Journal Video about Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells at JoVE.com

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

Iniciando Diferenciación en inmortalizadas células pluripotentes Otic Progenitoras

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

initiating-differentiation-immortalized-multipotent-otic-progenitor

Research

JoVE Journal

Developmental Biology

8.4K vistas.  Rutgers University. The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

Video: Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Derivación de células cardiacas progenitoras a partir de células madre embrionarias

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Investigación

Biología del Desarrollo

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

La inducción directa de las células madre neurales humanas a partir de células de sangre periférica progenitoras hematopoyéticas

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Análisis de pez cebra larvas esquelético Integridad del músculo con el colorante azul de Evans

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

Células Progenitoras de aislamiento Mesangiogenic (PSM) de Humana de Médula Ósea

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

Dirige la diferenciación de progenitores hematopoyéticos primitivo y definitivo de las células madre Pluripotent humanas

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

Diferenciación in vitro de células madre pluripotentes humana en células trofoblásticas

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

In Vitro Diferenciación de células madre mesenquimales humanas en células funcionales en cardiomiocitos

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Sola célula Photoconversion en pez cebra intacto de la vida

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

Inducción de la diferenciación endotelial en las células cardiacas progenitoras en suero bajo condiciones

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

En la generación de Vitro del Somite derivados humanos inducida por células madre pluripotentes

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...