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. Manter a auto-renovação em células IMOP <ol><li> Prepare IMOP meios de cultura: DMEM / F12, suplemento B27 1X, 25 ug / ml e carbenecillin 20 ng / ml de bFGF. Adicione 50 ml de meio de cultura IMOP utilizando reagentes estéreis. Aquecer-se 49 ml de DMEM / F12 numa cónico de 50 ml num banho de água a 37 C °. <ol><li> Descongelar 50X suplemento B27 e 100 mg / ml carbenecillin alíquotas, esterilizadas por filtração para 5 min num banho de água a 37 ° C. Descongelar a 100 ug / ml de bFGF aliquota à TA. Adicionar 1 ml de 50X B27, 10 ul de 100 ug / ml de bFGF e 12,5 uL de 100 mg / ml em DMEM carbenecillin / F12. </li></ol></li><li> Use 3 ml de mídia em uma placa de 60 mm de cultura de tecidos para as células de cultura IMOP. Adicionar meio fresco às culturas a cada dois dias, dobrando o volume de mídia. Certifique-se de que a concentração de bFGF não cai abaixo de 5 ng / ml. <ol><li> IMOP células de cultura a 37 ° C com 5% de CO 2. Células Passage após 5 - 7 dias em cultura como listadas nas etapas abaixo. Transferência de cultura até uma 15ml cônico com uma ponta de pipeta 10 ml. </li></ol></li><li> Adicione 1 mM de EDTA em HBSS por diluição de 0,1 ml de EDTA 0,5 M pH 8,0 em 50 ml de HBSS. Pré-quente EDTA 1 mM feita em HBSS em um banho de água a 37 ° C. </li><li> Células colheita por sedimentação gravidade ou centrifugação a 200 xg durante 5 minutos. à TA. Por sedimentação por gravidade, colocar o cónico de 15 ml contendo as culturas no incubador a 37 ° C durante 5 - 10 min. Após esse tempo, observar os otospheres se acumulem na parte inferior da cónico de 15 ml. NOTA: força centrífuga excessivo pode causar danos às células. </li><li> Cuidadosamente aspirado gasto de mídia usando uma pipeta 2 ml de aspiração sem perturbar o sedimento celular. Adicionar 0,5 ml de solução de pré-aquecido a 1 mM EDTA HBSS para a célula sedimento. Com uma pipeta P1000, pipetar suavemente para cima e para baixo 2 - 3 vezes. Coloque cónica a 37 ° C e incubar durante &lt;5 minutos para facilitar a dissociação em células individuais. </li><li> Agite suavemente a solução de células para determinar se otospheres são dissociados. Se nenhum otoesferas sedimentem no fundo do cone, as células são dissociadas. O tempo de incubação pode variar. NOTA: prolongada incubação com EDTA pode resultar em morte excessiva de células. </li><li> Remover as células a partir de 37 ° C, adiciona-se 2 ml de meio para neutralizar e diluir o EDTA. Recolher as células por centrifugação a 200 xg durante 5 min à temperatura ambiente. Aspirar diluída EDTA usando uma pipeta 2 ml de aspiração e adicionar 5 ml de 1X PBS para lavar as células. </li><li> Girar para baixo as células a 200 xg durante 5 min à temperatura ambiente e aspirar para fora 1X PBS utilizando uma pipeta de 2 ml de aspiração. Ressuspender as células utilizando uma pipeta P1000 em 0,5 ml de meio de cultura IMOP cuidado pipetando para cima e para baixo 2 - 3 vezes. </li><li> Contagem de células utilizando um contador de partículas microfluídico com as fitas apropriadas 28. Uma placa confluente de células IMOP conterá ~ 6 X10 6 células. Dilui-se as células de 1: 100 em meio e adicionar 75 ul de uma solução de células para a cassete para contagem. Placa 1 x 10 6 células em um prato de seis centímetros no IMOP culture meios (~ 1: 10 de diluição). IMOPs passagem a cada 5 - 7 dias. NOTA: As células que formam grandes otospheres ou se ligam à parte inferior do prato de cultura de tecidos irá diferenciar. As células também vão morrer se as culturas são mais confluentes. </li></ol> 2. congelamento e descongelamento IMOP Cells <ol><li> Preparar o meio de congelamento e descongelamento sintética por equilibrar a solução a 4 ° C antes de serem células de congelação. </li><li> Recolher as células a partir de um disco de 6 cm confluente de células IMOP. Usar uma pipeta de 10 mL e de transferência de células (~ 6 - 8 x 10 6 células) num cónico de 15 ml. </li><li> Células colheita por sedimentação por gravidade ou centrifugação a 200 xg durante 5 min à temperatura ambiente. Se os otospheres são pequenas, as células não vai resolver por gravidade sedimentação. Opcional: Contagem de células usando passo 1,9 para determinar o número de células totais. </li><li> Aspirar gasto de mídia usando uma pipeta 2 ml aspiração, deixando o sedimento de células solto para trás. </li><li> Adicionar 0,25 ml de meio de congelamento sintéticos para voltar a suspender ceLLS a uma densidade de 5 x 10 ~ 5 a 3 x 10 6 células / ml. Pipeta suavemente as células com uma pipeta P1000. Transferir a suspensão de células para frascos criogénicos usando uma pipeta P1000 com 1 ml filtrados ponta. </li><li> Colocar os tubos em um recipiente de congelamento de álcool livre e colocar o recipiente de congelação a -80 ° C para reduzir a temperatura de 1 ° C por minuto até a temperatura atingir -80 ° C. </li><li> Para armazenamento a longo prazo de células, transferir os tubos de ensaio para a fase de vapor de um tanque de armazenamento de azoto líquido. Para descongelar células para a cultura, equilibrar criotubo contendo as células congelados a -80 ° C. </li><li> Pré-quente meios de cultura IMOP em um banho de água a 37 ° C. </li><li> Descongelar o frasco congelado rapidamente por agitação na parte inferior do frasco num banho de água a 37 ° C. Adicionar 1 mL de meios de cultura pré-aquecido a IMOP células descongeladas uma vez que o último cristal de gelo desaparece. Transferência de células para a 15 ml cônico e adicionar um adicional de 4 ml de meio de cultura IMOP </li><li> Gire a 15 ml conica durante 5 min. a 200 xg e aspirar o meio gasto. Ressuspender as células em 2 ml de meio de cultura de células e IMOP placa num prato de 6 cm. Incubar as culturas a 37 ° C com 5% de CO 2 para a expansão. </li></ol> 3. diferenciação de células IMOP em Sensorial Epithelia <ol><li> Prepare 50 ml de IMOP media diferenciação epitélio sensorial: DMEM / F12, suplemento 1X B27, 25 ug / ml carbenecillin. Faça 50 ml de IMOP sensorial media diferenciação do epitélio. Aquecer-se 49 ml de DMEM / F12 numa cónico de 50 ml num banho de água a 37 C °. Descongelar 50X suplemento B27 e 100 mg / ml carbenecillin alíquotas para 5 min. num banho de água a 37 ° C. Adicionar 1 ml de 50X B27 e 12,5 uL de 100 mg / ml em DMEM carbenecillin / F12. </li><li> Para a colheita, se dissociam, ressuspender e contagem de células repetir os passos 1,4-1,9 </li><li> Placa 1 X 10 6 células num prato de 6 cm no dia -3 IMOP utilizando meios de cultura. No Dia 0, as culturas de transferência utilizando uma pipeta de 10 ml para uma cónico de 15 ml. Recolher o otoesferas por gravidade sedimentação como indicado na etapa 1.6. Aspirar gasto de mídia usando uma pipeta 2 ml de aspiração e deixar as otospheres na parte inferior do cone. </li><li> Delicadamente adicionar em 2 ml de diferenciação do epitélio sensorial mídia. Transferência otospheres para um prato de 6 cm, usando um grande furo pipeta de 10 mL. NOTA: corte mecânica de pipetagem dura pode dissociar as células dos otospheres. </li><li> Adicionar 2 ml de meios sensoriais diferenciação epitelial fresco às culturas a cada dois dias. Se necessário, as células podem ser coletadas por sedimentação gravidade e mídia substituído. </li><li> Recolha otospheres no dia 10, transferindo para a 15 ml cônico e permitindo que os otospheres para sedimentar como indicado na etapa 1.6.Aspirate os meios gastos usando uma pipeta 2 ml de aspiração e deixar as otospheres imperturbável. </li><li> Fix otospheres por incubação em 4% de formaldeído em PBS 1X durante 15 min à TA. Remover solução de formaldeído, lavar otospheres com tampão de lavagem (1X PBS contendo 0,1% de Triton X-100) antes da incubação num tampão de bloqueio (1X PBS contendo 10% de soro normal de cabra e 0,1% de Triton X-100) durante 1 h. <ol><li> Substituir tampão e incubar as amostras em tampão de bloqueio com anticorpo diluído. Incubar a 4 ° C. Lavar as amostras com 1X PBS contendo 0,1% de Triton X-100 sujeita a otosphere para imunocoloração 27. Transferência otospheres em 1X PBS e colocar otospheres em meio de montagem numa lâmina de vidro usando uma pipeta P1000. Coloque uma lamela sobre a amostra e permitir a montagem de meios para secar a 4 ° C. </li></ol></li><li> Adquirir imagens de epifluorescência, utilizando uma configuração de microscópio invertido equipado com uma câmera CCD de 16 bits e quer um ar 20X 0,75 ou um NA objetivo de imersão em óleo de 40X 1.3. Recolha de fluorescência de diferentes canais de cores usando a comprimentos de onda listado (excitação e emissão): azul (377 ± 25 nm / 447 ± 30 nm), verde (475 ± 25 nm / 540 ± 25 nm), vermelho (562 ± 20nm / e 625 ± 20 nm) e infravermelhos (628 ± 20nm / e 692 ± 20nm). </li></ol> 4. Assaying EdU Incorporação <ol><li> No Dia -3, placa 1 x 10 6 células IMOP em um disco de 6 cm. Três dias mais tarde, no dia 0, colheita, dissociar, ressuspender e contar as células, repetindo os passos 1,4-1,9. </li><li> Placa de 2,5 x 10 5 células em meio de cultura e IMOP 5 x10 5 células em diferenciação epitélio sensorial meios diferentes em poços de uma placa de 6 poços. </li><li> Determinar a percentagem de células em fase S por EdU incorporação no dia 3 utilizando um ensaio de incorporação de EdU <ol><li> Adicionar estoque EdU directamente às culturas IMOP para se obter uma concentração final de 1 uM EdU no meio de cultura. </li><li> Incubar EdU em culturas IMOP durante 2 horas e incubar a 37 ° C com 5% de CO 2. Colheita, dissociam e coletar células repetir os passos 1,4-1,9. Adicionar em 4% de formaldeído em PBS 1X durante 15 min. à TA para fixar as células. Células colheita por centrifugação a 200 xg durante 5 min à temperatura ambiente. Remoção da solução de formaldeído e properly dispor. </li><li> Núcleos do rótulo de células com Hoechst e incorporada EdU com verde fluorescente dye-azida de acordo com o protocolo do fabricante. NOTA: Todas as lavagens são feitas com 1X PBS, BSA a 3% e 0,1% de células Tween 20. Lavar com 1X PBS duas vezes. </li><li> Células de montagem em um slide usando antifade reagente e coloque um copo 1,5 tampa sobre a amostra. Adquirir imagens fluorescentes de células marcadas utilizando um microscópio de epifluorescência. </li></ol></li></ol> 5. Diferenciar neurônios derivados-IMOP <ol><li> Adicione 50 ml de meio de diferenciação neuronal: meios Neurobasal, 1X suplemento B27, 2 mM de L-Glutamina. Descongelar garrafa de 50X B27 e 200 mM de L-glutamina em 37 ° C num banho de água durante 5 min. Adicionar 1 ml de 50X B27 e 0,5 ml de 200 mM de L-glutamina, para 48,5 ml de meio neurobasal. </li><li> Brasão lamela, colocando 12 mm redondas 1,5 lamelas de vidro em um estéril placa de 10 cm e adicionar 70% EtOH a placa para esterilizar e limpar as lamelas. </li><li> Agite suavemente a enseadarslips para garantir que eles estão cobertos de etanol. Deixar a placa durante 10 minutos à TA. Lavar as lamelas 3 vezes com estéril 1X PBS para lavar o etanol remanescente. Lavar as lamelas uma vez com H 2 0 estéril para lavar restante 1X PBS. </li><li> Aspirar o H 2 0 usando a 2 ml de aspiração pipeta e deixar as lamelas seco. Expor as lamelas a luz UV na capa de tecido de cultura durante 15 min. Loja lamelas em um ambiente estéril se não for usado imediatamente. </li><li> Coloque uma lamela 12 milímetros rodada em cada poço de um prato de 24 poços. Agitar placa suavemente para assegurar que as lamelas ficar na posição horizontal na parte inferior do poço. </li><li> Descongelar 2 mg / ml de poli-D-lisina da solução à TA e diluir para uma concentração de 10 ug de poli-D-lisina / ml em 1X PBS. Adicionar 0,25 ml de 10 ug / ml de poli-D-lisina para os poços. Deixar a placa na incubadora a 37 ° C durante 1 h. </li><li> Lavar os poços 3 vezes com PBS estéril 1X. Descongelar / solução de estoque de 10 mg mL laminina à RT e dilui-se a 10 ug / ml em PBS 1X. Adicionar 0,25 ml de 10 ug / ml de laminina solução de trabalho em um único poço de uma 24 multi-poços prato. Incubar a placa a 37 ° C incubadora. Aspirar a solução laminina utilizando uma pipeta de 2 ml de aspiração. </li><li> Lava-se a lamela 3 vezes com PBS 1X, adicionando em 1 ml de PBS 1X com uma pipeta P1000 e remover o PBS 1X por aspiração com uma pipeta de 2 ml de aspiração. Deixar 1X PBS a partir última lavagem no poço até que as células estão prontas a ser revestida. Iniciado diferenciação neuronal pela colheita, dissociando e contagem de células em passos 1.4-1.9.Plate 1 x 10 6 células em um prato de seis centímetros no Dia -3. </li><li> No Dia 0, colheita, dissociar e contar as células repetindo 1,4-1,9. Semente de 1 x 10 5 - 1,5 x 10 5 células em 0,5 ml IMOP media diferenciação neuronal pré-aquecido por cavidade em um 24 multi-poços prato. Aspirar e adicionar meios diferenciação neuronal pré-aquecido para culturas em dias alternados. </li><li> Corrigir neurônios IMOP derivados em formaldeído 4% por 15 min. umat RT no dia 7. Remover solução de formaldeído, lavar neurónios IMOP derivados com tampão de lavagem (1X PBS contendo 0,1% Triton X-100) antes da incubação num tampão de bloqueio (1X PBS contendo 10% de soro normal de cabra e 0,1% de Triton X-100) durante 1 h. <ol><li> Substituir tampão e incubar as amostras em tampão de bloqueio com anticorpo diluído. Incubar a 4 ° C. Lavar as amostras com PBS 1X contendo 0,1% de Triton X-100 As células sujeitas a imunocoloração 27. Lavar a lamela com 1X PBS, antes de colocar em suportes de montagem. Permitir que os meios de montagem para secar a 4 ° C antes de adquirir imagens de epifluorescência como listado no passo 3.8. </li></ol></li></ol>

The authors have nothing to disclose.

Monitoramento culturas IMOP Um protocolo para a manutenção de auto-renovação e promover a diferenciação de uma linhagem celular IMOP romance é descrito e formatos chapeamento adicionais são incluídos (Tabela 1). Vários passos críticos que ajudam com a expansão e diferenciação de células IMOP rotina são anotados. Semelhante a culturas de células estaminais pluripotentes, células IMOP tem, teoricamente, um tempo de vida indeterminado. Para ga...

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 retirada diminui a proliferação em células IMOP Para diminuir a capacidade proliferativa de células IMOP e iniciar a diferenciação de células IMOP, bFGF foi retirado das culturas. Para confirmar que o fator de crescimento retirada diminui a proliferação, EdU incorporação foi empregado como um ensaio de proliferação. Foi comparada com a percentagem de células que incorporaram EdU de otosphere...

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 A diferenciação em células multipotentes imortalizadas 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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Universidad de Cartagena UDEC

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JoVE Journal

Developmental Biology

8.4K visualizações.  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.

Vídeo: 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 Sistema de Cultura oligodendroglial Cérebro 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 ...

Neurociência

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.

Diferenciação de células-tronco embrionárias em precursores oligodendroglial

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

Biologia

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.

Diferenciação e Caracterização de Osteoclastos de Células-Tronco Pluripotentes Induzidas Humanas

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

Biologia do Desenvolvimento

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.

Diferenciação de camundongos neonatos pele derivadas de células-tronco em células germinativas semelhantes<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.

Geração de neurônios humanos e oligodendrócitos a partir de células-tronco pluripotentes para modelagem de interações neurônio-oligodendrócitos

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.

Diferenciando células-tronco embrionárias humanas em progenitores neurais

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.

Um método de co-cultura para modelar interações neurônio-oligodendrócitos

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.

Diferenciação de células progenitoras neurais em neurônios

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.

Diferenciação de células progenitoras óticas em células epiteliais sensoriais seguidas de imunomarcação

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.

Diferenciando progenitores óticos multipotentes imortalizados em neurônios ganglionares espirais e avaliando a diferenciação

1. Differentiating iMOP-Derived Neurons 

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