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 border="0" cellpadding="0" cellspacing="0" width="1075">
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		<tr height="20">
			<td height="20" style="height:20px;width:455px;">CoolCell LX Alcohol-Free Cell Freezing Containers</td>
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			<td style="width:145px;">BCS-405</td>
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			<td height="20" style="height:20px;width:455px;">Cryogenic Vials (2 ml)&#160;</td>
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			<td style="width:145px;">72230-01</td>
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			<td height="20" style="height:20px;width:455px;">DMEM/F12</td>
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			<td style="width:145px;">11320-082</td>
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			<td height="20" style="height:20px;width:455px;">Neurobasal Medium</td>
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			<td style="width:145px;">10010-023</td>
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			<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>
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			<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>
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			<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>
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			<td height="20" style="height:20px;width:455px;">Natural Mouse Laminin</td>
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			<td style="width:145px;">23017-015</td>
			<td style="width:280px;">Stored as 1 mg/ml aliquots</td>
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			<td height="20" style="height:20px;width:455px;">Click-iT EdU Alexa Fluor 488&#160;</td>
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			<td height="20" style="height:20px;width:455px;">Moxi Z Mini Automated Cell Counter</td>
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			<td height="20" style="height:20px;width:455px;">Moxi Z Cassette Type S</td>
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			<td height="40" style="height:40px;width:455px;">Recombinant Murine Fibroblast Growth Factor, basic (bFGF)</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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		</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 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 ...

initiating-differentiation-immortalized-multipotent-otic-progenitor

Research

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

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.

Derivação de Cardiac células progenitoras de células estaminais embrionárias

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

Biologia do Desenvolvimento

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.

Indução direta de Neurais Human células-tronco de sangue periférico células progenitoras hematopoéticas

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.

Análise de Zebrafish larvas Músculo Esquelético Integrity com 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 ...

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.

Isolamento de células progenitoras Mesangiogenic (PPM) de humano Medula Óssea

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.

Dirigido a diferenciação de progenitores hematopoiéticos primitivo e definitiva de células pluripotentes 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.

Na diferenciação in vitro de pluripotentes humanas Células-tronco em 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 Diferenciação de células-tronco mesenquimais humanas em células casos funcionais

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.

Photoconversion de célula única no Zebrafish intacta 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.

Indução de diferenciação endotelial em células progenitoras cardíacas sob condições de baixo de soro

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.

Na geração de Vitro do Somite derivados de humanos induzida por células tronco pluripotentes

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