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. IMOP Hücrelerde Öz yenileme bakımı <ol><li> Kültür ortamı iMOP hazırlayın: DMEM / F12, 1X B27 ek, 25 ug / ml ve 20 carbenecillin ng / ml bFGF. Steril reaktifler kullanılarak iMOP kültür ortamı 50 ml sağlayın. 37 ° C bir su banyosu içinde 50 ml konik DMEM / F12 49 ml kadar ısıtın. <ol><li> Çözülme 50X B27 takviyesi ve 37 ° C su banyosu içinde 5 dakika süre ile filtre ile sterilize edilmiş 100 mg / ml carbenecillin alikotları. RT&#39;de 100 ug / ml bFGF kısım çözülme. Ekle 1 mi 50X B27, 100 ug / ml bFGF 10 ul DMEM / F12 içinde 100 mg / ml carbenecillin 12.5 ul. </li></ol></li><li> Kültürleme iMOP hücreleri için 60 mm doku kültürü çanağı içinde ortam 3 ml kullanın. Medya hacmini iki katına çıkararak her gün kültürlere taze medya ekleyin. BFGF o konsantrasyonu 5 ng / ml altına düşmeyecek emin olun. <ol><li> Aşağıdaki adımları listelenen kültürdeki 7 günden - 5 sonra% 5 CO 2. Geçiş hücreleri ile 37 ° C &#39;de kültür iMOP hücreleri. Bir 15 Aktarım kültürü10 ml&#39;lik bir pipet kullanarak ml konik. </li></ol></li><li> HBSS 50 ml 0,5 M EDTA, pH 8.0, 0.1 ml seyreltilmesi ile HBSS 1 mM EDTA sağlayın. Ön Sıcak 1 mM EDTA, 37 ° C su banyosu içinde HBSS içinde yapılır. </li><li> Yerçekimi çökelme veya 5 dakika boyunca 200 x g&#39;de santrifüj ile hasat hücreleri. oda sıcaklığında karıştırıldı. 10 dakika - yerçekimi çökelme için, 5 37 ° C kuluçka makinesi içinde kültürleri içeren 15 ml&#39;lik konik yerleştirin. Bu süreden sonra, otospheres 15 ml konik altında toplanır gözlemleyin. NOT: Aşırı merkezkaç kuvveti hücre hasarına neden olabilir. </li><li> Dikkatlice aspire hücre pelet bozmadan pipet aspirasyon bir 2 ml kullanarak ortam geçirdi. Pelet hücre önceden ısıtılmış 1 mM EDTA HBSS çözeltisi 0,5 ml ilave edilir. P1000 pipet kullanarak, yavaşça yukarı pipetlemeyin ve aşağı 2-3 kez. 37 ° C &#39;de konik yerleştirin ve tek hücre ayrışmasını kolaylaştırmak için &lt;5 dakika boyunca inkübe edin. </li><li> Yavaşça otospheres ayrışmış olup olmadığını belirlemek için hücre çözümü girdap. No Oto Eğerküreler konik altına tortu, hücreler ayrışmış edilir. Inkübasyon süresi değişebilir. Not: EDTA ile uzun süreyle kuluçkalanması aşırı hücre ölümüne neden olur. </li><li> , 37 ° C ila hücreleri çıkarın EDTA nötralize etmek ve sulandırmak için ortam 2 ml ekleyin. Oda sıcaklığında 5 dakika boyunca 200 x g&#39;de santrifüj ile hücreler toplanır. Aspire dışarı 2 ml aspirasyon pipet kullanarak ve hücreleri yıkamak için 1X PBS 5 ml ekleyin EDTA seyreltilmiş. </li><li> Oda sıcaklığında 5 dakika boyunca 200 xg&#39;de hücreleri aşağı Spin ve 2 ml aspire pipet kullanarak 1X PBS dışarı aspire. 3 kez - hafifçe aşağı 2 yukarı pipetleme tarafından iMOP kültür ortamı, 0.5 ml P1000 pipet kullanarak hücreleri tekrar. </li><li> Uygun kasetler 28 ile mikroakışkan parçacık sayacı kullanarak hücreleri saymak. IMOP hücrelerin birleşik plaka ~ 6 x10 6 hücre içerecektir. Medyada 100 ve sayım için kaset içine hücre çözeltisi 75 ul ekleyin: hücrelerini 1 seyreltilir. IMOP kültürlerdeki 6 cm tabak Plaka 1 x 10 6 hücreMedya yeniden (~ 1: 10 seyreltme). Passage iMOPs her 5-7 gün. NOT: Büyük otospheres oluşturacak veya ayırt edecek doku kültürü çanak alt eklemek Hücreler. Kültürler konfluent yaşın üzerinde iseniz hücreler de ölecektir. </li></ol> 2. Donma ve Çözülme IMOP Hücreleri <ol><li> Eritilmiş ve dondurmadan önce hücrelere 4 ° C&#39;ye kadar çözüm dengeye sentetik dondurma ortamı hazırlayın. </li><li> IMOP hücrelerinin konfluent 6 cm çanak hücreleri toplamak. 15 ml konik içine - (8 x 10 6 hücre ~ 6) 10 ml pipet ve transfer hücreleri kullanın. </li><li> Yerçekimi çökelme veya oda sıcaklığında 5 dakika boyunca 200 x g&#39;de santrifüj ile hasat hücreleri. Otospheres küçükse, hücreler yerçekimi sedimantasyon razı olmaz. İsteğe bağlı: toplam hücre numaralarını belirlemek için adım 1.9 kullanarak hücreleri saymak. </li><li> Aspire arkasında gevşek hücre pelet bırakarak pipet aspirasyon bir 2ml kullanarak medya geçirdi. </li><li> Ce tekrar süspansiyon sentetik donma medya 0.25 ml ekleyin~ 5 x 10 5 6 10 x 3 hücre / ml bir yoğunlukta lls. Yavaşça P1000 pipet ile hücreleri pipetle. Ucu süzülmüş 1 ml P1000 pipet kullanarak kriyojenik flakon hücre süspansiyonu aktarın. </li><li> Bir alkol serbest dondurma kapta şişeleri yerleştirin ve sıcaklık ulaşıncaya kadar -80 ° C, dakikada 1 ° C düşürmek için -80 ° C&#39;de dondurma kap yerleştirin. </li><li> Hücrelerin uzun süreli depolama için bir sıvı azot depolama tankının gaz fazına şişeleri aktarın. Kültür hücreleri çözülme, -80 ° C donmuş hücreleri içeren kriyojenik flakon dengeye. </li><li> 37 ° C su banyosunda Öncesi sıcak iMOP kültür ortamı. </li><li> 37 ° C su banyosu içinde şişenin alt dönen tarafından hızlı bir şekilde dondurulmuş şişe çözülme. Son buz kristali ortadan kalktığı anda çözülmüş hücrelere önceden ısıtılmış iMOP kültür ortamı 1 ml ilave edilir. Transferi 15 ml konik içine hücreleri ve iMOP kültür ortamı ilave 4 ml ekleyin </li><li> 15 ml co Spin5 dakika boyunca nical. 200 xg harcanan medya aspire ve. 6 cm tabak iMOP kültür ortamı ve plaka hücreleri 2 ml tekrar süspansiyon hücreleri. Genişlemesi için% 5, 37 ° C&#39;de CO2 kültürleri inkübe edin. </li></ol> 3. Duyu Epiteli içine IMOP Hücreleri Farklılaşan <ol><li> DMEM / F12, 1X B27 takviyesi, 25 ug / ml carbenecillin: iMOP duyu epitelyum farklılaşma medya 50 ml hazırlayın. IMOP duyusal epitel farklılaşma medya 50 ml olun. 37 ° C bir su banyosu içinde 50 ml konik DMEM / F12 49 ml kadar ısıtın. Çözülme 50X B27 takviyesi ve 5 dakika süreyle 100 mg / ml carbenecillin alikotları. 37 ° C su banyosu içinde. 1 mi 50X B27 ve DMEM / F12 içinde 100 mg / ml carbenecillin 12.5 ul ekle. </li><li> Hasat, tekrar süspansiyon ayırmak ve saymak için hücreler 1.4-1.9 adımları tekrarlayın </li><li> Gün -3 kültür ortamı iMOP kullanarak bir 6 cm çanak Plaka 1 x 10 6 hücre. Gün 0 günü, transfer kültürleri 15 ml konik içine 10 ml pipet kullanarak. Otoimmun toplayınyerçekimi sedimantasyon küreler olarak adım 1.6 belirtti. Aspire dışarı pipet aspirasyon bir 2 ml kullanan medya geçirdi ve konik altındaki otospheres bırakın. </li><li> Yavaşça duyu epitelyum farklılaşma medya 2 ml ekleyin. Transferi büyük delik 10 ml pipet kullanarak 6 cm çanak otospheres. NOT: Sert pipetleme Makine kesme otospheres hücreleri ayırmak olabilir. </li><li> Her gün kültürlere taze duyusal epitel farklılaşma medya 2 ml ekleyin. Gerekirse, hücreler yerine yerçekimi sedimantasyon ve medya tarafından tahsil edilebilir. </li><li> 15 ml konik aktarma ve otospheres adımda belirtildiği gibi harcanan medya pipet aspirasyon bir 2 ml kullanılarak 1.6.Aspirate tortu ve rahatsız otospheres bırakmak için izin vererek Gün 10&#39;da otospheres toplayın. </li><li> Oda sıcaklığında 15 dakika boyunca 1X PBS% 4 formaldehit içinde inkübe edilerek otospheres sabitleyin. , Formaldehit solüsyonu çıkarın yıkama tamponu (PBS 1X% 0.1 TritonX-10 içeren otospheres yıkayın0) blokaj tamponunda inkübe edildi (1X PBS,% 10 normal keçi serumu ve 1 saat süre ile,% 0.1 Triton X-100) ihtiva etmektedir. <ol><li> Tampon değiştirin ve sulandırılmış antikor ile tampon engelleme örnekleri kuluçkaya yatmaktadır. 4 ° C&#39;de inkübe edin. 1X PBS 27 immün% 0.1 Triton X-100 ihtiva eden konuyu otosphere ile yıkayın örnekleri. P1000 pipet kullanarak bir bardak slayt medyayı montaj içine 1X PBS içine aktarın otospheres ve yer otospheres. Numune üzerinde lamel koyun ve montaj medya 4 ° C&#39;de kurumaya bırakın. </li></ol></li><li> 16 bit CCD kamera ve 20X 0.75 havaya ya da bir 40X 1.3 NA immersiyon yağı amacı ile donatılmış bir inverted mikroskop kurulumu kullanarak Epifloresans görüntüleri elde edin. Mavi (377 ± 25 nm / 447 ± 30 nm), yeşil (475 ± 25 nm / 540 ± 25 nm), kırmızı (562 ± 20nm / ve 625: Listelenen (uyarma ve emisyon) dalga boyları kullanarak farklı renk kanallarından floresan toplayın ± 20nm) ve kızılötesi (628 20nm ± / ve 692 ± 20nm). </li></ol> 4. Assaying Edu Ortaklığın <ol><li> Gün -3 günü, 6 cm tabağına 1 x 10 6 iMOP hücreleri plaka. Üç gün sonra 0. günde, hasat, ayrışır, tekrar süspansiyon ve adımları 1,4-1,9 tekrarlayarak hücreleri saymak. </li><li> Plaka iMOP kültür ortamı 2.5 x 10 5 hücre ve 6 kuyu yemeğin farklı kuyularda duyusal epitelyum farklılaşma medyada 5 x10 5 hücre. </li><li> Bir edu birleştirme yöntemi kullanılarak 3. günde edu katılmasıyla S fazında hücrelerin yüzdesi belirlemek <ol><li> Kültür ortamında 1 uM edu bir nihai konsantrasyon elde etmek üzere iMOP kültürlere edu stokları ekleyin. </li><li> 2 saat boyunca iMOP kültürlerinde edu inkübe ve% 5 CO2 ile 37 ° C &#39;de inkübe edilir. Hasat, ayırmak ve toplamak hücreler adımları 1,4-1,9 tekrarlayın. 15 dakika boyunca 1X PBS içinde% 4 formaldehit içinde ekleyin. RT&#39;de hücreleri gidermek için. Oda sıcaklığında 5 dakika boyunca 200 x g&#39;de santrifüj ile hasat hücreleri. Formaldehit çözeltisi ve properl kaldıry atmayın. </li><li> Etiket Hoechst hücrelerin çekirdekleri ve üreticinin protokolüne uygun olarak yeşil floresan boya azid ile edu dahil. Not: tüm yıkamalar 1X PBS, iki kez 1X PBS ile% 3 BSA ve% 0.1 Tween 20 ile yıkayın hücreleri ile yapılır. </li><li> Ve antifade reaktif kullanılan bir slayt Mount hücreleri numune üzerinde 1.5 cam kapak yerleştirin. Bir Epifloresans mikroskopi kullanılarak işaretli hücrelerin floresan görüntüler elde. </li></ol></li></ol> 5. IMOP Türevli Nöronlar Farklılaşan <ol><li> Neurobasal ortam, 1X B27 takviyesi, 2 mM L-Glutamin: nöronal farklılaşma medya 50 ml sağlayın. 5 dakika için 37 ° C su banyosu içinde 50 kat B27 ve 200 mM L-Glutamin çözülme şişe. Neurobasal ortam 48.5 mi 1 ml 50X B27 ve 0.5 ml 200 mM L-Glutamin ekleyin. </li><li> Steril 10 cm plaka yuvarlak 12 mm 1.5 cam lamelleri yerleştirilmesi ve sterilize lamelleri temizlemek için plakaya% 70 EtOH eklemek tarafından Coat coverglass. </li><li> Yavaşça koya ajiterslips onlar etanol kaplıdır sağlamak. Oda sıcaklığında 10 dakika boyunca plaka bırakın. Kalan etanol yıkamak için steril 1X PBS ile lamelleri 3 kere yıkayın. 1X PBS kalan yıkamak için steril H 2 0 kez lamel durulayın. </li><li> Pipet aspirasyon bir 2 ml kullanılarak H 2 0 aspire ve lamelleri kurumaya bırakın. 15 dakika boyunca doku kültürü kaputu UV ışığına lamelleri Açığa. Hemen kullanılmadığı takdirde mağazası steril bir ortamda lamelleri. </li><li> 24 iyi bir çanak her bir kuyunun bir 12 mm yuvarlak lamel yerleştirin. Lamelleri kuyunun dibinde düz yalan emin olmak için hafifçe sallayın plaka. </li><li> Çözülme 2 mg / 1X PBS içinde 10 ug / ml poli-D-lisin konsantrasyonuna oda sıcaklığında poli-D-lizin stok çözeltisi ml seyreltin. 10 ug / ml poli-D-lisin 0.25 ml çukurlara ekleyin. 1 saat boyunca 37 ° C inkübatör plaka bırakın. </li><li> Steril 1X PBS ile 3 kez yıkayın. Oda sıcaklığında 10 mg / ml stok çözeltisi, laminin ve Çözme 10 ug / m seyreltin1X PBS l. Ekle 10 ug 24 çoklu kuyu çanak tek kuyuya çözüm çalışma / ml laminin 0.25 ml. 37 ° C inkübatör de plaka inkübe edin. 2 ml aspire pipet kullanarak laminin çözüm aspire. </li><li> P1000 pipet ile 1X PBS 1 ml ilave edilmesi ve pipet aspire 2 ml aspire 1X PBS kaldırarak lamel 1X PBS ile 3 kez yıkayın. Hücreler kaplama için hazır olana kadar kuyuda son yıkama 1X PBS bırakın. Dissociating ve Gün -3 de 6 cm tabak adımlarla 1.4-1.9.Plate 1 x 10 6 hücre hücre sayımı, hasat nöronal farklılaşma başlatın. </li><li> Gün 0, hasat günü, ayırmak ve 1.4-1.9 tekrar ederek hücreleri saymak. Tohum 1 x 10 5 - kuyuda 24-kuyu tabak çoklu başına önceden ısıtılmış nöronal farklılaşma medya 0.5 ml x 10 5 iMOP hücreleri 1.5. Aspire ve her gün kültürlere önceden ısıtılmış nöronal farklılaşma medya ekleyebilirsiniz. </li><li> 15 dakika boyunca% 4 formaldehit içinde iMOP türetilmiş nöronlar sabitleyin. birt RT Gün 7&#39;de çıkarın formaldehit çözeltisi, yıkama tamponu ile iMOP türetilmiş nöronlar yıkama blokaj tamponunda inkübe edildi (1X PBS,% 0.1 Triton X-100 ihtiva eder) (1X PBS,% 10 normal keçi serumu ve% 0.1 Triton X-ihtiva eden 100) 1 saat karıştırıldı. <ol><li> Tampon değiştirin ve sulandırılmış antikor ile tampon engelleme örnekleri kuluçkaya yatmaktadır. 4 ° C&#39;de inkübe edin. 1X PBS% 0.1 Triton X-100 denek hücreleri içeren ile yıkayın örnekleri 27 immün için. Montaj medya üzerine yerleştirmeden önce 1X PBS ile coverglass yıkayın. Montaj medya adım 3.8 listelenen Epifloresans görüntüleri almadan önce 4 ° C&#39;de kurumaya bırakın. </li></ol></li></ol>

The authors have nothing to disclose.

IMOP Kültürleri İzleme Yeni bir iMOP hücre çizgisinin farklılaşmasını kendini yenileme korunması ve geliştirilmesi için bir protokol açıklanan ve ek kaplama biçimleri (Tablo 1) yer almaktadır. Rutin genişleme ve iMOP hücrelerin farklılaşması yardımcı Birkaç kritik adımlar belirtilmiştir. Pluripotent kök hücre kültürleri benzer, iMOP hücreleri teorik belirsiz ömrünü var. Hücre hatları düzgün korunur emin olmak için, iMOP...

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.

IMOP Hücreleri bFGF Çekilme Azaltmaktadır Çoğalma IMOP hücrelerin proliferatif kapasitesinin azaltılması ve iMOP hücrelerinin farklılaşmasını başlatmak için, bFGF kültürlerinden geri çekildi. Büyüme faktörü çekilmesi çoğalmasını azaltır olduğunu doğrulamak için, edu dahil bir çoğalma tahlilinde olarak kullanılmıştır. (BFGF) olmadan iMOP kültür ortamı (bFGF ihtiva eden) v...

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.

Ölümsüzleştirdi Multipotent Otic Progenitor Hücrelerde Farklılaşma başlatılıyor

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 de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.4K Görüntüleme.  Rutgers University. The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

Video: Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

İnsan Fetal Beyin gelen Progenitör-türetilmiş Oligodendrosit Kültür Sistemi

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

Nörobilim

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.

Embriyonik Kök Hücreler farklılaşma oligodendrosit Öncüleri

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

Biyoloji

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.

İnsan Kaynaklı Pluripotent Kök Hücrelerden Osteoklastların Farklılaşması ve Karakterizasyonu

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

Gelişim Biyolojisi

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.

Germ benzeri hücreler içine Yenidoğan Fare Cilt Türetilmiş Kök Hücre Farklılaşma<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.

Nöron-Oligodendrosit Etkileşimlerini Modellemek için Pluripotent Kök Hücrelerden İnsan Nöronlarının ve Oligodendrositlerin Üretilmesi

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

Differentiating Human Embryonic Stem Cells into Neural Progenitors

Source: Jeon, K. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/60571">Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells.</a> J. Vis. Exp.&#160;(2020)
This video demonstrates the differentiation of human embryonic stem cells, or hESCs, directly into neural progenitor cells without using feeder cells or any intermediate steps. The selective inhibition of bone morphogenetic protein or BMP and transforming growth factor-beta, or TGF-&#946;, signaling pathways leads to hESCs differentiation into neural progenitor cells, or NPCs.

İnsan Embriyonik Kök Hücrelerinin Nöral Progenitörlere Farklılaşması

1. Preparation of human embryonic stem cells (hESC)-qualified Basement Membrane Matrix-coated Plates

	Slowly thaw the hESC-qualified basement membrane ...

JoVE Encyclopedia of Experiments

Nöronal Kültür Teknikleri

Gelişmiş Nöron Modelleri

A Co-culture Method to Model Neuron-Oligodendrocyte Interactions

Source: Assetta, B.,et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/61778">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions.</a> J. Vis. Exp. (2020).
This video demonstrates the establishment of a co-culture of human induced neurons (iNs) and induced oligodendrocyte precursor cells (iOPCs). iNs are mixed with iOPCs in a co-culture and then incubated to support the maturation of both iNs into neurons and iOPCs into oligodendrocytes. Neurons form synaptic connections with other neurons and oligodendrocytes, while oligodendrocytes develop myelin sheaths around the neurons&#39; axons.

Nöron-Oligodendrosit Etkileşimlerini Modellemek için Bir Ko-Kültür Yöntemi

1. Human neuron induction from human pluripotent stem cells

	Lentivirus preparation (~5 days)
	
		Plate ~1 million Human embryonic kidney 293T (HEK293T) ...

Çoklu kültür ve Etkileşim Çalışmaları

Differentiation of Neural Progenitor Cells into Neurons

Source: Mao, X., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
The video showcased a cell culture method for differentiating neural progenitor cells into neurons. By incubating cells in a nutrient-rich medium supplemented with growth factors, the progenitor cells matured into neurons with developed axons and dendrites. A stabilizing agent ensured cell viability and protected against oxidative damage throughout the differentiation process.

Nöral Progenitör Hücrelerin Nöronlara Farklılaşması

1. Differentiation from NPCs to neurons (Phase II)

	Seed about 5 x 105 mouse embryonic stem cells or mESC derivatives within 2 mL of basal ...

Differentiating Otic Progenitor Cells Into Sensory Epithelial Cells Followed by Immunostaining

Source: Azadeh, J. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/53692">Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells.</a>&#160;J. Vis. Exp. (2016).
This video demonstrates the differentiation of immortalized multipotent otic progenitor (iMOP) cells into sensory epithelial cells. It outlines the steps involved in culturing iMOP cells, forming otospheres, inducing differentiation, and visualizing cellular components and differentiation markers through fluorescence microscopy to confirm successful cell differentiation.

Otik progenitör hücrelerin duyusal epitel hücrelerine farklılaşması ve ardından immün boyama

1. Maintaining Self-renewal in IMOP Cells

	Prepare iMOP culture media: DMEM/F12, 1X B27 supplement, 25 &#181;g/ml carbenicillin and 20 ng/ml basic ...

Differentiating Immortalized Multipotent Otic Progenitors into Spiral Ganglion Neurons and Evaluating the Differentiation

Source: Azadeh, J., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53692">Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells.</a> J. Vis. Exp. (2016).
The video demonstrates the differentiation of immortalized multipotent otic progenitors (iMOPs) into spiral ganglion neurons (SGNs) and immunofluorescence-based confirmation of the differentiation. The iMOPs are plated onto culture substrate-coated coverslips in a neuronal differentiation medium for differentiation into SGNs. The differentiated cells are labeled with antibodies specific for neuronal differentiation markers and analyzed under a microscope to confirm differentiation.

Ölümsüzleşmiş Multipotent Otik Progenitörlerin Spiral Ganglion Nöronlarına Ayrılması ve Farklılaşmanın Değerlendirilmesi

1. Differentiating iMOP-Derived Neurons 

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