Name: initiating differentiation in immortalized multipotent otic progenitor cells
Uploaded: 2016-01-02T13:00:00.000+00:00
Duration: 12 min 17 s
Description: Watch this Scientific Journal Video about Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells at JoVE.com

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. Die Aufrechterhaltung der Selbsterneuerung in IMOP Cells <ol><li> Bereiten iMOP Kulturmedien: DMEM / F12, 1X B27 ergänzen, 25 ug / ml Carbenecillin und 20 ng / ml bFGF. Machen Sie 50 ml iMOP Kulturmedien unter Verwendung von sterilen Reagenzien. Warm up 49 ml DMEM / F12 in einem 50 ml konischen in einem 37 ° C Wasserbad. <ol><li> Auftau 50X B27 Supplement und filtersterilisiert 100 mg / ml Carbenecillin Aliquots für 5 min in einem 37 ° C Wasserbad. Auftauen 100 ug / ml bFGF Aliquot bei RT. 1 ml 50X B27, 10 ul 100 ug / ml bFGF und 12,5 &amp; mgr; l von 100 mg / ml Carbenecillin in DMEM / F12. </li></ol></li><li> Verwenden Sie 3 ml Medium in einem 60 mm-Gewebekulturschale zur Züchtung iMOP Zellen. In frischem Medium, um Kulturen jeden zweiten Tag durch eine Verdoppelung des Volumens der Medien. Sicherzustellen, dass die Konzentration von bFGF nicht unter 5 ng / ml abfiel. <ol><li> Kultur iMOP Zellen bei 37 ° C mit 5% CO 2 Passage-Zellen nach 5-7 Tagen in Kultur, wie in den unten aufgeführten Schritte. Übertragungs Kultur in einen 15ml konischen Verwendung einer 10 ml Pipettenspitze. </li></ol></li><li> Stellen 1 mM EDTA in HBSS durch Verdünnen von 0,1 ml 0,5 M EDTA pH 8,0 in 50 ml HBSS. Pre-warmen 1 mM EDTA in HBSS in einem 37 ° C Wasserbad gestellt. </li><li> Ernten der Zellen durch Schwerkraftsedimentation oder durch Zentrifugieren bei 200 xg für 5 min. bei RT. Für Schwerkraftsedimentation, legen Sie die 15-ml-Erlen mit den Kulturen in der 37 ° C Inkubator für 5 bis 10 min. Nach dieser Zeit wird, beobachten die otospheres sammeln sich am Boden des konischen 15 ml. HINWEIS: Übermäßiger Zentrifugalkraft können Zellschäden verursachen. </li><li> Saugen Sie vorsichtig den verbrauchten Medien mit einer 2 ml Ansaugpipette ohne das Zellpellet zu stören. 0,5 ml vorgewärmtes, 1 mM EDTA HBSS Lösung Pellet Zelle. Mit Hilfe eines P1000 Pipette vorsichtig pipettieren nach oben und unten 2 - 3-mal. Zeigen konischen bei 37 ° C und Inkubation für &lt;5 min zur Dissoziation in Einzelzellen zu erleichtern. </li><li> Vorsichtig schwenken die Zell-Lösung zu bestimmen, ob otospheres dissoziiert sind. Wenn keine otoSphären zu sedimentieren zu dem Boden des konischen werden die Zellen dissoziiert. Die Inkubationszeit kann variieren. HINWEIS: Längerer Inkubation mit EDTA in übermäßigen Zelltod führen. </li><li> Entfernen Zellen von 37 ° C, 2 ml Medium zu neutralisieren und zu verdünnen das EDTA. Sammeln der Zellen durch Zentrifugation bei 200 xg für 5 Minuten bei RT. Saugen Sie verdünnten EDTA unter Verwendung einer 2 ml Ansaugpipette und 5 ml 1X PBS, um die Zellen zu waschen. </li><li> Spin Zellen nach unten bei 200 xg für 5 Minuten bei Raumtemperatur und saugen aus 1X PBS mit einem 2 ml Ansaugpipette. Resuspendieren der Zellen unter Verwendung eines P1000 Pipetten in 0,5 ml iMOP Kulturmedium durch sanftes Auf- und Abpipettieren 2-3 mal. </li><li> Graf Zellen unter Verwendung eines mikrofluidischen Partikelzähler mit den entsprechenden Kassetten 28. Ein konfluenten Platte iMOP Zellen ~ 6 X10 enthalten 6 Zellen. Zellen verdünnt 1: 100 in Medium und füge 75 ul der Zell-Lösung in die Kassette für die Zählung. Platte 1 x 10 6 Zellen in einer 6-cm-Schale in iMOP culture Medien (~ 1: 10 Verdünnung). Passage iMOPs alle 5 - 7 Tage die. Hinweis: Zellen, die große otospheres bilden oder sich an der Unterseite der Gewebekulturschale wird unterscheiden. Die Zellen werden auch sterben, wenn die Kulturen über konfluent. </li></ol> 2. Einfrieren und Auftauen IMOP Cells <ol><li> Vorbereitung synthetischen Einfriermedium durch Auftauen und Äquilibrieren der Lösung auf 4 ° C vor dem Gefrierzellen. </li><li> Sammeln Sie Zellen aus einer konfluenten 6 cm Schüssel iMOP Zellen. Verwenden Sie eine 10 ml Pipette und Übertragungszellen (~ 6 - 8 x 10 6 Zellen) in einen 15-ml-Erlen. </li><li> Ernten der Zellen durch Schwerkraftsedimentation oder durch Zentrifugieren bei 200 xg für 5 Minuten bei RT. Wenn die otospheres sind klein, werden Zellen, die nicht durch die Schwerkraft Sedimentation absetzen. Optional: Zählen von Zellen mit Schritt 1,9 bis Gesamtzellzahl zu bestimmen. </li><li> Saugen Sie verbrauchte Medium mit einem 2 ml Ansaugpipette während die losen Zellpellet hinter. </li><li> In 0,25 ml synthetischen Einfrieren Medien ce resuspendierenlls in einer Dichte von ~ 5 × 10 5 bis 3 × 10 6 Zellen / ml. Gently Pipette Zellen mit einem P1000 Pipette. Übertragen Sie die Zellsuspension in Kryoröhrchen mit einem P1000 Pipette mit 1 ml gefilterte Spitze. </li><li> Legen Sie die Fläschchen in einem Alkohol frei Gefrierbehälter und setzen Sie den Gefrierbehälter bei -80 ° C, die Temperatur 1 ° C pro Minute zu verringern, bis die Temperatur erreicht 80 ° C. </li><li> Für langfristige Lagerung von Zellen, übertragen die Vials in die Dampfphase aus einem Flüssigstickstofflagertank. Auftauen Zellen für die Kultur, ins Gleichgewicht Kryo-Fläschchen mit gefrorenen Zellen bei -80 ° C. </li><li> Pre-warmen iMOP Kulturmedien in einem 37 ° C Wasserbad. </li><li> Auftauen gefrorenen Fläschchen schnell von Wirbeln den Boden des Fläschchens in einem 37 ° C Wasserbad. 1 ml der vorgewärmten iMOP Kulturmedien, um aufgetauten Zellen, sobald die letzten Eiskristall verschwindet. Übertragungszellen in einem 15 ml konischen und fügen zusätzliche 4 ml iMOP Nährmedien </li><li> Drehen Sie das 15 ml Conische für 5 min. bei 200 xg und saugen die verbrauchte Medien. Resuspendieren der Zellen in 2 ml iMOP Kulturmedien und Zellen in einer Platte 6 cm Schale. Kulturen bei 37 ° C mit 5% CO 2 für die Expansion. </li></ol> 3. Differenzierung IMOP Cells in Sinnesepithelien <ol><li> Bereiten Sie 50 ml iMOP Sinnesepithelien Differenzierungsmedium: DMEM / F12, 1X B27 ergänzen, 25 ug / ml Carbenecillin. Machen Sie 50 ml iMOP Sinnesepithelien Differenzierung Medien. Warm up 49 ml DMEM / F12 in einem 50 ml konischen in einem 37 ° C Wasserbad. Auftau 50X B27 Supplement und 100 mg / ml Carbenecillin Aliquots für 5 min. in einem 37 ° C Wasserbad. 1 ml 50X B27 und 12,5 &amp; mgr; l von 100 mg / ml Carbenecillin in DMEM / F12. </li><li> Zu ernten, zu distanzieren, resuspendieren und zählen Zellen Wiederholen Sie die Schritte 1,4-1,9 </li><li> Platte 1 x 10 6 Zellen in einer 6-cm-Schale an Tag -3 Verwendung iMOP Kulturmedien. Am Tag 0 Transfer Kulturen mit einer 10 ml Pipette in ein 15-ml-Erlen. Sammeln Sie die otoKugeln durch die Schwerkraft der Sedimentation, wie in Schritt 1.6 angegeben. Aspirat aus verbrauchten Medien unter Verwendung einer 2 ml Ansaugpipette und lassen die otospheres auf der Unterseite des konischen. </li><li> Sanft fügen in 2 ml Sinnesepithelien Differenzierung Medien. Transfer otospheres zu einer 6 cm-Schale mit einer großen Bohrung 10 ml Pipette. HINWEIS: Mechanische Scher vor rauen Pipettieren können Zellen aus den otospheres distanzieren. </li><li> 2 ml frischem sensorischen Epitheldifferenzierung Medien Kulturen jeden zweiten Tag. Falls erforderlich, können die Zellen durch Schwerkraftsedimentation und Medien ersetzt gesammelt werden. </li><li> Sammeln otospheres am Tag 10 durch die Übertragung von einer 15-ml-Erlen und es den otospheres sedimentieren, wie in Schritt angegeben 1.6.Aspirate die verbrauchten Medien unter Verwendung einer 2 ml-Pipette und verlassen Ansaugen der otospheres ungestört. </li><li> Fix otospheres durch Inkubation in 4% Formaldehyd in 1 × PBS für 15 min bei RT. Entfernen Formaldehydlösung, waschen otospheres mit Waschpuffer (1X PBS, das 0,1% Triton X-100) vor der Inkubation in Blockierungspuffer (1 × PBS, das 10% normales Ziegenserum und 0,1% Triton X-100) für 1 Stunde. <ol><li> Ersetzen Puffer und Inkubation Proben in Blocking-Puffer mit verdünnter Antikörper. Inkubieren bei 4 ° C. Wasch Proben mit 1X PBS, enthaltend 0,1% Triton X-100 unterliegt die otosphere der Immunofärbung 27. Transfer otospheres in 1X PBS und Ort otospheres in die Montagetechnik auf einen Glasobjektträger mit einem P1000 Pipette. Setzen Sie ein Deckglas über die Probe und lassen Sie die Montage Medien bei 4 ° C zu trocknen. </li></ol></li><li> Erwerben Epifluoreszenz Bildern unter Verwendung eines Umkehrmikroskops Setup mit einem 16-Bit-CCD-Kamera und entweder 20X 0,75 Luft oder 40X 1,3 NA -Ölimmersionsobjektiv ausgestattet. Sammeln Fluoreszenz von verschiedenen Farbkanälen unter Verwendung der genannten (Anregung und Emission) Wellenlängen: blau (377 ± 25 nm / 447 ± 30 nm), Grün (475 ± 25 nm / 540 ± 25 nm), rot (562 ± 20 nm / und 625 ± 20 nm) und Infrarot (628 ± 20 nm / und 692 ± 20nm). </li></ol> 4. Assaying EdU Incorporation <ol><li> Am Tag -3, Platte 1 x 10 6 iMOP Zellen in einer 6 cm Schale. Drei Tage später, am Tag 0, ernte, distanzieren, resuspendieren und zählen Sie die Zellen durch Wiederholen der Schritte von 1,4 bis 1,9. </li><li> Platte 2,5 x 10 5 Zellen in iMOP Nährmedien und 5 x10 5 Zellen in Sinnesepithelien Differenzierungsmedium in verschiedenen Vertiefungen einer 6 Well-Platte. </li><li> Bestimmen den Prozentsatz an Zellen in S-Phase durch EdU Einarbeitung am Tag 3 durch Verwendung eines EdU Inkorporationstest <ol><li> Hinzufügen EdU Lager direkt an die iMOP Kulturen bis zu einer Endkonzentration von 1 uM EdU in dem Kulturmedium zu erhalten. </li><li> Inkubieren EdU in iMOP Kulturen für 2 Stunden und bei 37 ° C mit 5% CO 2. Harvest, distanzieren und sammeln Zellen wiederholen Sie die Schritte 1,4-1,9. Hinzufügen in 4% Formaldehyd in 1 × PBS für 15 min. bei RT, um Zellen zu reparieren. Ernten der Zellen durch Zentrifugation bei 200 xg für 5 Minuten bei RT. Entfernen Formaldehydlösung und properly entsorgen. </li><li> Etikett Kerne von Zellen mit Hoechst und einge EdU mit grünem Fluoreszenzfarbstoff-Azid gemß dem Protokoll des Herstellers. HINWEIS: Alle Waschungen werden mit 1X PBS, 3% BSA und 0,1% Tween 20. Wasche die Zellen mit 1 × PBS zweimal durchgeführt. </li><li> Berg-Zellen auf einem Objektträger mit Antifade-Reagenz und legen Sie eine 1,5 Abdeckglas über die Probe. Erwerben Fluoreszenzbilder der markierten Zellen mit Hilfe eines Epifluoreszenz-Mikroskopie. </li></ol></li></ol> 5. Differenzierung IMOP abgeleiteten Neuronen <ol><li> Machen Sie 50 ml der neuronalen Differenzierung Medien: Neurobasal Medien, 1X B27 ergänzen, 2 mM L-Glutamin. Thaw Flasche 50X B27 und 200 mM L-Glutamin in 37 ° C Wasserbad für 5 min. 1ml 50X B27 und 0,5 ml 200 mM L-Glutamin 48,5 ml Neurobasal Medium. </li><li> Coat Deckglas, indem 12 mm rund 1,5 Deckgläser in einer sterilen 10 cm Platte und fügen Sie 70% EtOH an der Platte, die sterilisiert und reinigen Sie die Deckgläser. </li><li> Rühren Sie vorsichtig die Buchtrslips um sicherzustellen, dass sie in Ethanol abgedeckt sind. Lassen Sie die Platte für 10 Minuten bei RT. Mit steriler 1X PBS spülen Sie die Deckgläschen 3 mal zum Auswaschen der verbleibenden Ethanol. Spülen Sie das Deckglas einmal mit sterilem H 2 0 zu waschen restlichen 1X PBS. </li><li> Saugen Sie das H 2 0 mit einem 2 ml Ansaugpipette und lassen Sie die Deckgläser trocken. Setzen Sie die Deckgläser mit UV-Licht in der Gewebekultur Haube für 15 min. Speicher Deckgläschen in einer sterilen Umgebung, wenn sie nicht sofort verwendet. </li><li> Legen Sie eine 12 mm runde Deckglas in jede Vertiefung einer 24-Well-Schale. Schütteln Sie die Platte vorsichtig, um sicherzustellen, dass die Deckgläser liegen flach auf dem Boden des Bohrlochs. </li><li> Tau-2 mg / ml Poly-D-Lysin-Stammlösung bei RT gelöst und auf eine 10 ug / ml Poly-D-Lysin-Konzentration in 1X PBS. Zugabe von 0,25 ml von 10 ug / ml Poly-D-Lysin an die Vertiefungen. Lassen Sie die Platte in der 37 ° C-Inkubator für 1 Stunde. </li><li> 3-mal mit steriler 1X PBS Waschen Sie die Vertiefungen. Tau-10 mg / ml Laminin Stammlösung bei RT und 10 &amp; mgr; g / m verdünntenl in 1 × PBS. Zugabe von 0,25 ml von 10 ug / ml Laminin Arbeitslösung in eine einzige Vertiefung einer 24-Well-Platte mit mehreren. Inkubieren Sie die Platte bei 37 ° C Inkubator. Saugen Sie die Laminin-Lösung unter Verwendung eines 2 ml Ansaugpipette. </li><li> 3-mal mit 1X PBS waschen des Deckglases durch Zugabe von in 1 ml 1X PBS mit einer P1000-Pipette und Entfernen des 1X PBS durch Absaugen mit einer 2 ml Ansaugpipette. Lassen 1X PBS von den letzten Wasch im Brunnen, bis die Zellen sind bereit, überzogen werden. Einzuleiten neuronale Differenzierung durch Ernten, Dissoziieren und Zählen der Zellen in den Schritten 1.4-1.9.Plate 1 x 10 6 Zellen in einer 6-cm-Schale an Tag -3. </li><li> Am Tag 0, ernte, distanzieren und zählen Zellen durch Wiederholen von 1,4 bis 1,9. Seed 1 x 10 5 bis 1,5 x 10 5 iMOP Zellen in 0,5 ml vorgewärmten neuronalen Differenzierung Medium pro Well in einer 24 Multi-Well-Schale. Saugen Sie und fügen vorgewärmten neuronalen Differenzierung Medien Kulturen jeden zweiten Tag. </li><li> Fix iMOP abgeleiteten Neuronen in 4% Formaldehyd für 15 min. eint RT an Tag 7. Entfernen Formaldehydlösung, Waschen iMOP abgeleiteten Neuronen mit Waschpuffer (1 × PBS, enthaltend 0,1% Triton X-100) vor der Inkubation in Blockierungspuffer (1 × PBS, das 10% normales Ziegenserum und 0,1% Triton X-100) für 1 Stunde. <ol><li> Ersetzen Puffer und Inkubation Proben in Blocking-Puffer mit verdünnter Antikörper. Inkubieren bei 4 ° C. Wasch Proben mit 1X PBS, enthaltend 0,1% Triton X-100 unterworfen Zellen an Immun 27. Vor der Platzierung auf Montagemedien Waschen Sie die Deckgläser mit 1X PBS. Lassen Sie die Montage Medien bei 4 ° C vor dem Erwerb Epifluoreszenz Bilder wie in Schritt 3.8 aufgeführten trocknen. </li></ol></li></ol>

The authors have nothing to disclose.

Überwachung IMOP Kulturen Ein Protokoll zum Aufrechterhalten der Selbsterneuerung und die Förderung der Differenzierung einer neuartigen iMOP Zellinie beschrieben und zusätzliche Plattierung Formate enthalten sind (Tabelle 1). Mehrere wichtige Schritte, die mit Routine Expansion und Differenzierung von Zellen iMOP helfen, werden zur Kenntnis genommen. Ähnlich wie pluripotente Stammzellkulturen, iMOP Zellen haben theoretisch eine unbegrenzte Lebensdauer. ...

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 Widerrufs Verringert Proliferation in IMOP Cells Um die proliferative Kapazität von iMOP Zellen zu verringern und zu initiieren Differenzierung iMOP Zellen wurde bFGF aus den Kulturen entnommen. Um zu bestätigen, dass die Wachstumsfaktorentzug verringert Proliferation wurde EdU Einarbeitung als Proliferationstest verwendet. Der Prozentsatz von Zellen, die aus EdU otospheres mit iMOP Kulturmedium (enthal...

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.

Initiieren Differenzierung in Immortalisierte multipotenten Vorläuferzellen Otic

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

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

8.5K Aufrufe.  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.

Serie: 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-abgeleiteten Oligodendrozyten Culture System von Human Fetal Gehirn

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

Forschung

Neurowissenschaften

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.

Differenzierung von embryonalen Stammzellen in Oligodendrozyten Precursors

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

Biologie

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.

Differenzierung und Charakterisierung von Osteoklasten aus humanen induzierten pluripotenten Stammzellen

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

Entwicklungsbiologie

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.

Differenzierung von Newborn Mäusehaut Derived Stem Cells in Germ-like Cells<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.

Generierung humaner Neuronen und Oligodendrozyten aus pluripotenten Stammzellen zur Modellierung von Neuron-Oligodendrozyten-Interaktionen

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

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

High-Content-Screening-Differenzierungs- und Reifungsanalyse von fetalen und adulten neuralen Stammzellen-abgeleiteten Oligodendrozyten-Vorläuferzellkulturen

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...

Working with Auditory HEI-OC1 Cells

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, these cells have been used to investigate drug-activated apoptotic pathways, autophagy, senescence, mechanism of cell protection, inflammatory responses, cell differentiation, genetic and epigenetic effects of pharmacological drugs, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. Among other several important markers of cochlear hair cells, HEI-OC1 cells endogenously express prestin, the paradigmatic motor protein of outer hair cells. Thus, they can be very useful to elucidate novel functional aspects of this important auditory protein. HEI-OC1 cells are very robust, and their culture usually does not present big complications. However, they require some special conditions such as avoiding the use of common anti-bacterial cocktails containing streptomycin or other antibiotics as well as incubation at 33 &#176;C to stimulate cell proliferation and incubation at 39 &#176;C to trigger cell differentiation. Here, we describe how to culture HEI-OC1 cells and how to use them in some typical assays, such as cell proliferation, viability, death, autophagy and senescence, as well as how to perform patch-clamp and non-linear capacitance measurements.

House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins.

Arbeiten mit Auditory HEI-OC1-Zellen

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic ...

Differentiating Human Embryonic Stem Cells into Oligodendrocytes

Source: Assetta, B., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/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 process of generating oligodendrocyte precursor cells (OPCs) from human embryonic stem cells, followed by the maturation of OPCs to oligodendrocytes. This process of generating oligodendrocytes opens numerous avenues to improve research and treatment of neurodegenerative diseases, including Alzheimer&#39;s.

Differenzierung humaner embryonaler Stammzellen in Oligodendrozyten

1. Human oligodendrocyte precursor cell (OPC) induction from pluripotent stem cells and oligodendrocyte maturation

	Neural Progenitor Cell (NPC) ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Differentiating Otic Progenitor Cells Into Sensory Epithelial Cells Followed by Immunostaining

Source: Azadeh, J. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/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.

Differenzierung von otischen Vorläuferzellen in sensorische Epithelzellen mit anschließender Immunfärbung

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.bdigitaluss.remotexs.co/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.

Differenzierung immortalisierter multipotenter otischer Vorläuferzellen in spiralförmige Ganglienneuronen und Bewertung der Differenzierung

1. Differentiating iMOP-Derived Neurons 

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