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 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>
			<td style="width:196px;">BioCision</td>
			<td style="width:145px;">BCS-405</td>
			<td style="width:280px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Cryogenic Vials (2 ml)&#160;</td>
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			<td style="width:145px;">430654</td>
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			<td height="20" style="height:20px;width:455px;">1.5 Thickness Glass Coverslip (Round 12 mm)</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;">21103</td>
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			<td height="20" style="height:20px;width:455px;">Phosphate Buffered Saline (PBS) pH 7.4</td>
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			<td style="width:145px;">10010-023</td>
			<td style="width:280px;"></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>
			<td style="width:280px;"></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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		<tr height="20">
			<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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		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Natural Mouse Laminin</td>
			<td style="width:196px;">Life Technologies</td>
			<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>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">C10337</td>
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			<td style="width:145px;">47743-736</td>
			<td style="width:280px;">Stored as 10 mg/ml 100 &#181;l aliquots</td>
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			<td height="20" style="height:20px;width:455px;">Moxi Z Mini Automated Cell Counter</td>
			<td style="width:196px;">Orflo&#160;</td>
			<td style="width:145px;">MXZ001</td>
			<td style="width:280px;"></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>
			<td style="width:196px;">Peprotech</td>
			<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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		<tr height="40">
			<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>
			<td></td>
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		<tr height="20">
			<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>
			<td></td>
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		<tr height="20">
			<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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		<tr height="20">
			<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>
			<td style="width:280px;"></td>
		</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 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

Research

JoVE Journal

Developmental Biology

8.4K 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

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.

Ableitung von Herzvorläuferzellen aus embryonalen Stammzellen

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

Forschung

Entwicklungsbiologie

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.

Direkte Induktion von humanen neuralen Stammzellen aus peripherem Blut hämatopoetischen Vorläuferzellen

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.

Analyse von Zebrafisch-Larven Skelettmuskel Integrität mit 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.

Isolieren von Mesangiogenic Progenitorzellen (MPL) aus menschlichem Knochenmark

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.

Differenzierung der Primitive und Definitive hämatopoetischen Vorläuferzellen aus menschlichen pluripotente Stammzellen gerichtet

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.

In vitro Differenzierung von humanen pluripotenten Zellen in Trophoblastische Stammzellen

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- Differenzierung humaner mesenchymaler Stammzellen in funktionellen Cardiomyocyte-ähnlichen Zellen

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.

Einzellige Ladungszustand im lebendigen intakten Zebrafisch

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.

Induktion der endothelialen Differenzierung in kardialen Vorläuferzellen unter niedrigen Serum-Bedingungen

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.

In vitro-Generation der Somiten Derivate aus menschlichen induzierten Zellen pluripotenten Stammzellen

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