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. Mantenere auto-rinnovamento nelle celle IMOP <ol><li> Preparare IMOP cultura dei media: DMEM / F12, 1X B27 supplemento, 25 mg / ml carbenecillin e 20 ng / ml bFGF. Fai 50 ml di IMOP coltura utilizzando reagenti sterili. Warm up 49 ml di DMEM / F12 in una conica da 50 ml in un bagno d&#39;acqua a 37 °. <ol><li> Disgelo 50X B27 supplemento e 100 mg / ml carbenecillin aliquote sterilizzate per filtrazione, per 5 minuti in un bagno d&#39;acqua a 37 ° C. Scongelare 100 mg / ml bFGF aliquota a temperatura ambiente. Aggiungere 1 ml 50X B27, 10 pl di 100 ug / ml bFGF e 12,5 ml di 100 mg / ml in DMEM carbenecillin / F12. </li></ol></li><li> Utilizzare 3 ml di media in un piatto di coltura di tessuti 60 mm per le cellule di coltura IMOP. Aggiungere mezzi freschi a culture ogni altro giorno raddoppiando il volume del supporto. Assicurarsi che la concentrazione di bFGF non scenda sotto i 5 ng / ml. <ol><li> Cultura cellule IMOP a 37 ° C con 5% di CO 2. Le cellule Passage dopo 5 - 7 giorni in coltura elencati nella procedura riportata di seguito. Cultura Trasferire in un 15ml conica utilizzando un puntale 10 ml. </li></ol></li><li> Rendere EDTA 1 mM in HBSS diluendo 0,1 ml di 0,5 M EDTA pH 8,0 in 50 ml di HBSS. Preriscaldare 1 mM EDTA fatto in HBSS in un bagno d&#39;acqua a 37 ° C. </li><li> Celle di raccolta di sedimentazione per gravità o centrifugazione a 200 xg per 5 min. a RT. Per gravità sedimentazione, posizionare la conica 15 ml contenente le culture del C incubatore 37 ° per 5-10 minuti. Dopo tale tempo, osservare le otospheres raccolgono sul fondo del cono 15 ml. NOTA: la forza centrifuga eccessiva può causare danni alle cellule. </li><li> Attentamente aspirare trascorso multimediale utilizzando un 2 ml aspiranti pipetta senza disturbare il pellet. Aggiungere 0,5 ml di soluzione di pre-riscaldato 1 mM EDTA HBSS alla cella pellet. Usando una pipetta P1000, delicatamente pipettare su e giù 2 - 3 volte. Posizionare conica a 37 ° C e incubare per &lt;5 min a facilitare la dissociazione in cellule singole. </li><li> Agitare delicatamente la soluzione di cellule per determinare se otospheres sono dissociate. Se no otosfere sedimentare sul fondo del cono, le cellule sono dissociate. Il tempo di incubazione può variare. NOTA: prolungata incubazione con EDTA provoca la morte delle cellule eccessivo. </li><li> Rimuovere le cellule da 37 ° C, aggiungere 2 ml di mezzi per neutralizzare e diluire l&#39;EDTA. Raccogliere le cellule per centrifugazione a 200 xg per 5 minuti a temperatura ambiente. Aspirare diluito EDTA utilizzando un 2 ml aspirazione pipetta e aggiungere 5 ml di PBS 1X per lavare le cellule. </li><li> Spin giù le cellule a 200 xg per 5 minuti a temperatura ambiente e aspirare fuori 1X PBS usando una pipetta 2 ml aspirazione. Risospendere le cellule utilizzando una pipetta P1000 in 0,5 ml di IMOP cultura mediatica pipettando gentilmente su e giù 2 - 3 volte. </li><li> Contare le cellule utilizzando un contatore di particelle microfluidica con le cassette appropriate 28. Un piatto confluenti di cellule IMOP conterrà ~ 6 X10 6 celle. Diluire le cellule 1: 100 in media e aggiungere 75 ul di soluzione di cellule nel cassetto per il conteggio. Piastra 1 x 10 6 cellule in un piatto di 6 cm di IMOP culture dei media (~ 1: 10 diluizione). IMOPs Passage ogni 5 - 7 giorni. NOTA: Le cellule che formano grandi otospheres o si attaccano al fondo del piatto di coltura tissutale saranno differenziare. Le cellule saranno anche morire se le culture sono sopra confluenti. </li></ol> 2. gelo e disgelo Celle IMOP <ol><li> Preparare medio di congelamento scongelamento e sintetico, per equilibrare la soluzione a 4 ° C prima di cellule congelamento. </li><li> Raccogliere le cellule da un cm piatto di cellule IMOP confluenti 6. Utilizzare una pipetta da 10 ml e cellule di trasferimento (~ 6 - 8 x 10 6 celle) in una conica da 15 ml. </li><li> Celle di raccolta di sedimentazione per gravità o centrifugazione a 200 xg per 5 minuti a temperatura ambiente. Se i otospheres sono piccole, le cellule non si depositano per gravità sedimentazione. Opzionale: Contare le cellule utilizzando passo 1.9 per determinare il numero di cellule totali. </li><li> Aspirare trascorso multimediale utilizzando un 2ml aspirazione pipetta lasciando il pellet sciolto dietro. </li><li> Aggiungere 0,25 ml di media congelamento sintetici di riattivare cells ad una densità di 5 x 10 ~ 5 a 3 x 10 6 cellule / ml. Pipettare delicatamente le cellule con una pipetta P1000. Trasferire la sospensione cellulare di fiale criogeniche con una pipetta P1000 con 1 ml di filtrati punta. </li><li> Porre le fiale in un contenitore di congelamento senza alcool e posizionare il contenitore di congelamento a -80 ° C per ridurre la temperatura di 1 ° C al minuto fino a quando la temperatura raggiunge i -80 ° C. </li><li> Per la conservazione a lungo termine delle cellule, trasferire i flaconi alla fase vapore di un serbatoio di stoccaggio azoto liquido. Per scongelare le cellule per la cultura, equilibrare fiala criogenico contenente cellule congelate a -80 ° C. </li><li> Terreni di coltura IMOP Pre-caldo in un bagno di 37 ° C Acque. </li><li> Scongelare il flacone congelato rapidamente agitando il fondo della provetta in un bagno di acqua 37 ° C. Aggiungere 1 ml di terreni di coltura pre-riscaldato IMOP alle cellule scongelate una volta che il cristallo glaciale scompare. Trasferire le cellule in un ml conica 15 e aggiungere un ulteriore 4 ml di IMOP cultura mediatica </li><li> Gira la 15 ml conico per 5 min. a 200 xg e aspirare i media spesi. Risospendere le cellule in 2 ml di terreni di coltura e le cellule IMOP piastra in un piatto 6 cm. Incubare culture a 37 ° C con 5% di CO 2 per l&#39;espansione. </li></ol> 3. differenziazione delle cellule IMOP in epiteli sensoriali <ol><li> Preparare 50 ml di IMOP supporti differenziazione epiteli sensoriali: DMEM / F12, 1X B27 supplemento, 25 mcg / ml carbenecillin. Fai 50 ml di IMOP supporti differenziazione epiteli sensoriali. Warm up 49 ml di DMEM / F12 in una conica da 50 ml in un bagno d&#39;acqua a 37 °. Supplemento B27 disgelo 50X e 100 mg / ml carbenecillin aliquote per 5 min. in un bagno d&#39;acqua a 37 ° C. Aggiungere 1 ml 50X B27 e 12,5 ml di 100 mg / ml carbenecillin in DMEM / F12. </li><li> Per raccogliere, dissociarsi, risospendere e contare le cellule ripetere i passaggi 1,4-1,9 </li><li> Piastra 1 x 10 6 cellule in una capsula di 6 cm in Giorno -3 utilizzando IMOP coltura. Il giorno 0, culture di trasferimento con una pipetta 10 ml in una conica da 15 ml. Raccogliere il otosfere di gravità sedimentazione a quanto dichiarato nella fase 1.6. Aspirare trascorso multimediale utilizzando un 2 ml aspiranti pipetta e lasciare i otospheres sul fondo del cono. </li><li> Aggiungere delicatamente in 2 ml di mezzi differenziazione sensoriale epiteli. Trasferimento otospheres a un piatto 6 centimetri con un grande foro da 10 ml pipetta. NOTA: tosatura meccanica da duro pipettaggio può dissociarsi cellule dagli otospheres. </li><li> Aggiungere 2 ml di fresco supporti differenziazione epiteliale sensoriale culture ogni altro giorno. Se necessario, le cellule possono essere raccolte mediante sedimentazione gravità e supporti sostituito. </li><li> Raccogliere otospheres al giorno 10 con il trasferimento ad un ml conica 15 e permettendo ai otospheres di sedimentare, come indicato al punto 1.6.Aspirate media spesi utilizzando un 2 ml aspiranti pipetta e lasciare i otospheres indisturbati. </li><li> Fissare otospheres incubando a 4% formaldeide in PBS 1X per 15 minuti a RT. Rimuovere la soluzione di formaldeide, lavare otospheres con tampone di lavaggio (1X PBS contenente 0,1% TritonX-100) prima incubazione in tampone di bloccaggio (1X PBS contenente 10% siero di capra normale e 0,1% Triton X-100) per 1 ora. <ol><li> Sostituire buffer e incubare i campioni in tampone di bloccaggio con l&#39;anticorpo diluito. Incubare a 4 ° C. Lavare i campioni con 1X PBS contenente 0,1% Triton X-100 soggetti la otosphere dell&#39;immunocolorazione 27. Trasferire otospheres in PBS 1X e luogo otospheres in mezzo di montaggio su un vetrino con una pipetta P1000. Mettere un vetrino sul campione e permettono mezzi di montaggio a secco a 4 ° C. </li></ol></li><li> Acquisire immagini epifluorescenza utilizzando una configurazione microscopio rovesciato dotato di una fotocamera CCD a 16 bit e di un aereo o di un 20X 0,75 NA obiettivo 40X 1.3 immersione in olio. Raccogliere la fluorescenza da diversi canali di colore utilizzando la elencati (di eccitazione e di emissione) le lunghezze d&#39;onda: blu (377 ± 25 nm / 447 ± 30 nm), verde (475 ± 25 nm / 540 ± 25 nm), rosso (562 ± 20 nm / e 625 20nm ±) e infrarossi (628 ± 20 nm / e 692 ± 20nm). </li></ol> 4. Saggio EdU Costituzione <ol><li> Il giorno -3, piastra 1 x 10 6 cellule IMOP in un piatto di 6 centimetri. Tre giorni dopo il giorno 0, vendemmia, dissociarsi, risospendere e contare le cellule ripetendo i passi 1.4-1.9. </li><li> Piatto 2,5 x 10 5 cellule in mezzi di coltura IMOP e 5 x10 5 cellule in sensoriale mezzi differenziazione epiteli in diversi pozzetti di un piatto 6 bene. </li><li> Determinare la percentuale di cellule in fase S di Edu incorporazione il giorno 3 utilizzando un saggio di incorporazione EdU <ol><li> Aggiungere EdU magazzino direttamente alle culture IMOP per ottenere una concentrazione finale di 1 micron EdU nei mezzi di coltura. </li><li> Incubare EdU nelle culture IMOP per 2 ore e incubare a 37 ° C con 5% di CO 2. Harvest, dissociare e raccogliere le cellule ripetere i punti 1.4-1.9. Aggiungere a 4% formaldeide in PBS 1X per 15 min. a RT per riparare le cellule. Celle di raccolta per centrifugazione a 200 xg per 5 minuti a temperatura ambiente. Rimuovere la soluzione di formaldeide e properly smaltire. </li><li> Etichetta nuclei di cellule con la Hoechst e incorporati EdU con verde fluorescente dye-azide secondo il protocollo del produttore. NOTA: Tutti i lavaggi sono fatti con PBS 1X, 3% BSA e lo 0,1% di cellule Tween 20. Lavare con PBS 1X due volte. </li><li> Montare le cellule su un vetrino utilizzando antifade reagenti e posizionare un bicchiere di 1,5 copertura sul campione. Acquisire le immagini fluorescenti di cellule marcate con un epifluorescenza. </li></ol></li></ol> 5. Differenziare neuroni IMOP-Derived <ol><li> Fai 50 ml di media differenziamento neuronale: Neurobasal multimediali, 1X B27 supplemento, 2 mM L-glutammina. Bottiglia disgelo di 50X B27 e 200 mm L-glutammina in 37 ° C bagnomaria per 5 min. Aggiungere 1 ml 50X B27 e 0,5 ml 200 mm L-glutammina a 48,5 ml di mezzi Neurobasal. </li><li> Cappotto coprioggetti mettendo 12 mm rotonde 1,5 vetrini in un piatto sterile, 10 centimetri e aggiungere il 70% EtOH al piatto per sterilizzare e pulire le lamelle. </li><li> Agitare delicatamente la baiarslips per assicurarsi che sono coperti in etanolo. Lasciare la piastra per 10 minuti a RT. Lavare i coprioggetti 3 volte con sterili 1X PBS per lavare l&#39;etanolo residuo. Sciacquare il vetrino una volta con H 2 0 sterili per lavare rimanente PBS 1X. </li><li> Aspirare il H 2 0 utilizzando un 2 ml aspiranti pipetta e lasciare i coprioggetti asciutto. Esporre i coprioggetti alla luce UV nella cappa coltura tissutale per 15 min. Conservare coprioggetto in un ambiente sterile, se non utilizzato immediatamente. </li><li> Posizionare vetrino uno 12 millimetri rotonda in ogni pozzetto di un piatto 24 bene. Agitare gentilmente piastra per garantire che i coprioggetti sdraiarsi sul fondo del pozzo. </li><li> Scongelare 2 mg / ml di soluzione di lisina poli-D-at RT e diluire a 10 ug / ml concentrazione poli-D-lisina in 1X PBS. Aggiungere 0,25 ml di 10 ug / ml di poli-D-lisina ai pozzetti. Lasciare la piastra nel 37 ° C incubatore per 1 ora. </li><li> Lavare i pozzetti 3 volte con sterili 1X PBS. Thaw / ml di soluzione laminina magazzino 10 mg a temperatura ambiente e portare a 10 mg / ml in PBS 1X. Aggiungere 0,25 ml di 10 mg / ml soluzione di lavoro laminina in un singolo pozzetto di un piatto 24 multi-pozzo. Incubare la piastra a 37 ° C incubatore. Aspirare la soluzione laminina utilizzando un 2 ml aspirazione pipetta. </li><li> Lavare il coprioggetto 3 volte con PBS 1X aggiungendo in 1 ml di PBS 1X con una pipetta P1000 e rimuovendo il 1X PBS aspirando con 2 ml aspiranti pipetta. Lasciare PBS 1X da ultimo lavaggio del bene fino a quando le cellule sono pronte per essere placcato. Avviare la differenziazione neuronale per la raccolta, dissociandosi e il conteggio delle cellule nei passaggi 1.4-1.9.Plate 1 x 10 6 cellule in una capsula di 6 cm in Day -3. </li><li> Il giorno 0, vendemmia, dissociarsi e contare le cellule ripetendo 1,4-1,9. Seed 1 x 10 5-1,5 x 10 5 cellule IMOP in 0,5 ml di pre-riscaldato multimediali differenziamento neuronale per bene in un piatto multi-pozzo 24. Aspirare e aggiungere pre-riscaldato supporti differenziamento neuronale a culture ogni altro giorno. </li><li> Fissare neuroni IMOP derivati ​​a 4% di formaldeide per 15 min. unt RT il giorno di soluzione di formaldeide 7. Rimuovere, lavare i neuroni IMOP derivati ​​con tampone di lavaggio (1X PBS contenente 0,1% TritonX-100) prima di incubazione in tampone di bloccaggio (1X PBS contenente il 10% di siero normale di capra e lo 0,1% Triton X-100) per 1 ora. <ol><li> Sostituire buffer e incubare i campioni in tampone di bloccaggio con l&#39;anticorpo diluito. Incubare a 4 ° C. Lavare i campioni con 1X PBS contenente 0,1% Triton X-100 cellule soggette a immunocolorazione 27. Lavare il coprioggetti con PBS 1X prima di mettere su un supporto di montaggio. Lasciare i supporti di montaggio a secco a 4 ° C prima di acquisire immagini epifluorescenza come indicato al punto 3.8. </li></ol></li></ol>

The authors have nothing to disclose.

Monitoraggio Culture IMOP Un protocollo per mantenere auto-rinnovamento e promuovere la differenziazione di una linea cellulare IMOP romanzo è descritto e formati placcatura aggiuntivi sono inclusi (Tabella 1). Diversi passaggi critici che aiutano con l&#39;espansione di routine e la differenziazione delle cellule IMOP sono noti. Simile a colture di cellule staminali pluripotenti, le cellule IMOP teoricamente hanno una durata indefinita. Per garantire che l...

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 Ritiro Decrementi proliferazione in cellule IMOP Per diminuire la capacità proliferativa delle cellule IMOP e avviare la differenziazione delle cellule IMOP, bFGF è stato ritirato dalle culture. Per confermare che il fattore di crescita ritiro diminuisce la proliferazione, EdU incorporazione è stato impiegato come un saggio di proliferazione. La percentuale di cellule che incorporava EdU da otospheres ...

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.

Avvio di differenziazione in cellule multipotenti immortalizzate Otic progenitrici

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

8.5K Views.  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.

Progenitore di derivazione Sistema Cultura Oligodendrocyte dal cervello umano fetale

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

Ricerca

Neuroscienze

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.

Differenziazione delle cellule staminali embrionali in precursori degli oligodendrociti

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.

Differenziamento e caratterizzazione di osteoclasti da cellule staminali pluripotenti indotte umane

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

Biologia dello sviluppo

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.

Differenziazione del neonato mouse pelle cellule staminali derivate in cellule germinali-simili<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.

Generazione di neuroni umani e oligodendrociti da cellule staminali pluripotenti per la modellazione delle interazioni neurone-oligodendrociti

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://app.jove.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.

Differenziazione delle cellule staminali embrionali umane in progenitori neurali

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://app.jove.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 metodo di co-coltura per modellare le interazioni neurone-oligodendrocita

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://app.jove.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.

Differenziazione delle cellule progenitrici neurali in neuroni

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://app.jove.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.

Differenziazione delle cellule progenitrici otiche in cellule epiteliali sensoriali seguita da immunocolorazione

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://app.jove.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.

Differenziazione di progenitori otici multipotenti immortalizzati in neuroni gangliari a spirale e valutazione della differenziazione

1. Differentiating iMOP-Derived Neurons 

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