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><tbody><tr><td>CoolCell LX Alcohol-Free Cell Freezing Containers</td><td>BioCision</td><td>BCS-405</td><td></td></tr><tr><td>Cryogenic Vials (2 ml)&amp;nbsp;</td><td>Corning</td><td>430654</td><td></td></tr><tr><td>1.5 Thickness Glass Coverslip (Round 12 mm)</td><td>Electron Microscopy Sciences</td><td>72230-01</td><td></td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>11320-082</td><td></td></tr><tr><td>Neurobasal Medium</td><td>Life Technologies</td><td>21103</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS) pH 7.4</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Life Technologies</td><td>14025-092</td><td></td></tr><tr><td>B27 Supplement (50X) Serum Free</td><td>Life Technologies</td><td>17504-044</td><td>Stored as 1 ml aliquots</td></tr><tr><td>L-Glutamine(200 mM)&amp;nbsp;</td><td>Life Technologies</td><td>25030-081</td><td>Stored as 5 ml aliquots</td></tr><tr><td>Natural Mouse Laminin</td><td>Life Technologies</td><td>23017-015</td><td>Stored as 1 mg/ml aliquots</td></tr><tr><td>Click-iT EdU Alexa Fluor 488&amp;nbsp;</td><td>Life Technologies</td><td>C10337</td><td></td></tr><tr><td>Synth-A-Freeze Cryopreservation Media</td><td>Life Technologies</td><td>A12542-01</td><td></td></tr><tr><td>Prolong Gold Antifade Mountant</td><td>Life Technologies</td><td>47743-736</td><td>Stored as 10 mg/ml 100 &amp;micro;l aliquots</td></tr><tr><td>Moxi Z Mini Automated Cell Counter</td><td>Orflo&amp;nbsp;</td><td>MXZ001</td><td></td></tr><tr><td>Moxi Z Cassette Type S</td><td>Orflo&amp;nbsp;</td><td>MXC002</td><td></td></tr><tr><td>Recombinant Murine Fibroblast Growth Factor, basic (bFGF)</td><td>Peprotech</td><td>450-33</td><td>Resuspended in 0.1% BSA in H20 and stored as 20 mg/ml aliquots</td></tr><tr><td>Poly-D-Lysine</td><td>Sigma</td><td>P7886</td><td>Resuspended in 1X PBS and stored as 10 mg/ml 100 &amp;micro;l aliquots</td></tr><tr><td>Carbenicillin, Disodium Salt</td><td>Thermo Fisher Scientific</td><td>BP2648-1</td><td>Resuspended in 10 mM Hepes pH 7.4 and stored as 100 mg/ml aliquots</td></tr><tr><td>5 ml pipet individually wrapped paperback (200/case)</td><td>Thermo Fisher Scientific</td><td>1367811D</td><td></td></tr><tr><td>10 ml pipet individually wrapped paperback (200/case)</td><td>Thermo Fisher Scientific</td><td>1367811E</td><td></td></tr><tr><td>Tissue Culture Treated Biolite 24-Well Plate</td><td>Thermo Fisher Scientific</td><td>130188</td><td></td></tr><tr><td>Tissue Culture Treated Biolite 6-Well Plate</td><td>Thermo Fisher Scientific</td><td>130184&amp;nbsp;</td><td></td></tr><tr><td>Tissue Culture Treated 6 cm Dish</td><td>Thermo Fisher Scientific</td><td>130181&amp;nbsp;</td><td></td></tr><tr><td>EMD Millipore Millex Sterile Syringe PVDF Filter Pore size: 0.22 &amp;mu;m</td><td>Thermo Fisher Scientific</td><td>SLGV033RS</td><td></td></tr><tr><td>TipOne filter pipet tips 0.1 - 10 &amp;mu;l elongated filter tip</td><td>USA Scientific</td><td>1120-3810</td><td></td></tr><tr><td>TipOne filter pipet tips 1 - 20 &amp;mu;l filter tip</td><td>USA Scientific</td><td>1120-1810</td><td></td></tr><tr><td>TipOne filter pipet tips 1 - 200 &amp;mu;l&amp;nbsp; filter tip</td><td>USA Scientific</td><td>1120-8810</td><td></td></tr><tr><td>TipOne filter pipet tips 101 - 1000 &amp;mu;l filter tip</td><td>USA Scientific</td><td>1126-7810</td><td></td></tr><tr><td>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><td>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 ...

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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

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Progenitor derivado de sistema de cultivo de oligodendrocitos de cerebro fetal humano

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Investigación

Neurociencias

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

La diferenciación de células madre embrionarias en los precursores de oligodendrocitos

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Biología

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

Diferenciación y caracterización de osteoclastos a partir de células madre pluripotentes inducidas humanas

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

Biología del Desarrollo

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

La diferenciación de ratón recién nacido piel derivados de las células madre en células germinales-como<em&gt; In vitro</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

Generación de neuronas humanas y oligodendrocitos a partir de células madre pluripotentes para modelar las interacciones neuronal-oligodendrocitos

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Differentiating Human Embryonic Stem Cells into Neural Progenitors

Source: Jeon, K. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/60571">Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells.</a> J. Vis. Exp.&#160;(2020)
This video demonstrates the differentiation of human embryonic stem cells, or hESCs, directly into neural progenitor cells without using feeder cells or any intermediate steps. The selective inhibition of bone morphogenetic protein or BMP and transforming growth factor-beta, or TGF-&#946;, signaling pathways leads to hESCs differentiation into neural progenitor cells, or NPCs.

Diferenciación de células madre embrionarias humanas en progenitores neurales

1. Preparation of human embryonic stem cells (hESC)-qualified Basement Membrane Matrix-coated Plates

	Slowly thaw the hESC-qualified basement membrane ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

A Co-culture Method to Model Neuron-Oligodendrocyte Interactions

Source: Assetta, B.,et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/61778">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions.</a> J. Vis. Exp. (2020).
This video demonstrates the establishment of a co-culture of human induced neurons (iNs) and induced oligodendrocyte precursor cells (iOPCs). iNs are mixed with iOPCs in a co-culture and then incubated to support the maturation of both iNs into neurons and iOPCs into oligodendrocytes. Neurons form synaptic connections with other neurons and oligodendrocytes, while oligodendrocytes develop myelin sheaths around the neurons&#39; axons.

Un método de cocultivo para modelar las interacciones neurona-oligodendrocito

1. Human neuron induction from human pluripotent stem cells

	Lentivirus preparation (~5 days)
	
		Plate ~1 million Human embryonic kidney 293T (HEK293T) ...

Co-culture and Interaction Studies

Differentiation of Neural Progenitor Cells into Neurons

Source: Mao, X., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
The video showcased a cell culture method for differentiating neural progenitor cells into neurons. By incubating cells in a nutrient-rich medium supplemented with growth factors, the progenitor cells matured into neurons with developed axons and dendrites. A stabilizing agent ensured cell viability and protected against oxidative damage throughout the differentiation process.

Diferenciación de Células Progenitoras Neurales en Neuronas

1. Differentiation from NPCs to neurons (Phase II)

	Seed about 5 x 105 mouse embryonic stem cells or mESC derivatives within 2 mL of basal ...

Differentiating Otic Progenitor Cells Into Sensory Epithelial Cells Followed by Immunostaining

Source: Azadeh, J. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/53692">Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells.</a>&#160;J. Vis. Exp. (2016).
This video demonstrates the differentiation of immortalized multipotent otic progenitor (iMOP) cells into sensory epithelial cells. It outlines the steps involved in culturing iMOP cells, forming otospheres, inducing differentiation, and visualizing cellular components and differentiation markers through fluorescence microscopy to confirm successful cell differentiation.

Diferenciación de células progenitoras óticas en células epiteliales sensoriales seguidas de inmunotinción

1. Maintaining Self-renewal in IMOP Cells

	Prepare iMOP culture media: DMEM/F12, 1X B27 supplement, 25 &#181;g/ml carbenicillin and 20 ng/ml basic ...

Differentiating Immortalized Multipotent Otic Progenitors into Spiral Ganglion Neurons and Evaluating the Differentiation

Source: Azadeh, J., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53692">Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells.</a> J. Vis. Exp. (2016).
The video demonstrates the differentiation of immortalized multipotent otic progenitors (iMOPs) into spiral ganglion neurons (SGNs) and immunofluorescence-based confirmation of the differentiation. The iMOPs are plated onto culture substrate-coated coverslips in a neuronal differentiation medium for differentiation into SGNs. The differentiated cells are labeled with antibodies specific for neuronal differentiation markers and analyzed under a microscope to confirm differentiation.

Diferenciación de progenitores óticos multipotentes inmortalizados en neuronas ganglionares espirales y evaluación de la diferenciación

1. Differentiating iMOP-Derived Neurons 

	Make 50 ml of neuronal differentiation media: Neurobasal media, 1X B27 supplement, 2 mM L-Glutamine. Thaw ...