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.保持自我更新的IMOP细胞<ol><li>准备IMOP培养基:DMEM / F12，1X B27添加剂，25微克/毫升carbenecillin和20毫微克/毫升的bFGF。请50毫升IMOP培养基用消毒试剂。热身49毫升DMEM / F12在50毫升的锥形在37℃水浴。 <ol><li>解冻50X B27添加剂和过滤除菌100毫克/毫升carbenecillin在37℃水浴等份5分钟。解冻100微克/ ml的bFGF等分在RT。加入1毫升50X B27，10微升100微克/ ml的bFGF和12.5微升100毫克/毫升carbenecillin入的DMEM / F12。 </li></ol></li><li>用3毫升媒体60毫米组织培养皿培养IMOP细胞。通过介质的体积加倍添加新鲜培养基来培养物每隔一天。确保的bFGF的浓度不低于5毫微克/毫升。 <ol><li>培养IMOP细胞在37℃，5％的CO 2，传代细胞后5 -如在下面的步骤中列出7天培养。传输文化15毫升的锥形使用10毫升的枪头。 </li></ol></li><li>使1毫摩尔EDTA于HBSS稀释0.1毫升的0.5M EDTA pH 8.0的入50ml的HBSS。预暖1mM EDTA中在37℃水浴中作出在HBSS。 </li><li>收获的细胞通过重力沉降或离心，在200×g离心5分钟。在RT。对于重力沉降，将15毫升锥形含有在37℃培养箱中培养5 - 10分钟。该时间后，观察otospheres收集在15毫升的锥形的底部。 注意:过大的离心力会导致细胞损伤。 </li><li>小心吸用2ml的吸移液管，而不会干扰细胞沉淀用过的培养基。添加预热的1mM EDTA的HBSS溶液0.5毫升至细胞沉淀。使用P1000的吸管，吸管轻轻向上和向下2 - 3倍。放置锥形，在37℃孵育&lt;5分钟，以促进解离成单细胞。 </li><li>轻轻转动电池解决方案，以确定是否otospheres被分解。如果没有OTO球体沉降到圆锥形的底部，将细胞解离。孵育时间可能会有所不同。 注:长期孵化与EDTA会导致过度的细胞死亡。 </li><li>从37℃取出细胞，加入2毫升的媒体中和，稀释EDTA。收集细胞，离心，在200×g离心5分钟，室温。吸出使用2 ml的抽吸吸移管加入5 ml的1X PBS中洗涤细胞稀释EDTA中。 </li><li>旋转细胞下来，在200×g离心5分钟，RT，并吸出的1X PBS用2ml的吸移液管。 3次 - 轻轻吹打上下2重悬在0.5ml IMOP培养基使用P1000吸管细胞。 </li><li>计数使用微流体粒子计数器与相应的盒28细胞 。 IMOP细胞融合板将包含〜6 X10 6个细胞 。在媒体的100，加入75微升的细胞溶液放入纸盒计数:稀释细胞1。板1×10 6个细胞在IMOP文化前进一个6cm皿重介质（〜1:10稀释）。通道iMOPs每5 - 7天。 注:形成大otospheres或附着在组织培养皿将分化的底部细胞。如果文化是在融合细胞也会死去。 </li></ol> 2.冻融IMOP细胞<ol><li>通过融化和平衡冷冻细胞之前向溶液至4℃制备合成冷冻介质。 </li><li>从IMOP细胞融合6cm皿收集细胞。用10毫升吸管和转移的细胞（〜6 - 8×10 6个细胞）放入15毫升的锥形。 </li><li>收获的细胞通过重力沉降或离心，在200×g离心5分钟，室温。如果otospheres是小的，细胞不会因重力沉降沉降。可选:计数的应用步骤1.9确定总细胞数的细胞。 </li><li>抽吸用2毫升吸移吸移管，同时使松散细胞沉淀后面用过的培养基。 </li><li>添加0.25毫升合成冷冻介质悬浮行政长官LLS以约5×10 5至3×10 6个细胞 / ml的密度。轻轻吸取细胞P1000的吸管。细胞悬液转移到使用P1000吸管用1ml过滤尖低温小瓶中。 </li><li>将小瓶在醇游离冷冻容器和放置冷冻容器在-80℃，以减少每分钟的温度1℃，直至温度达到-80℃。 </li><li>对于长期存储单元，将小瓶转移到液氮贮罐的蒸汽相。以解冻细胞培养，平衡含有冷冻的细胞在-80℃下低温小瓶中。 </li><li>前期预热IMOP培养基在37℃水浴。 </li><li>由漩小瓶的底部在37℃水浴中快速解冻冷冻瓶。加入1毫升预热IMOP培养基来解冻细胞一旦最后冰晶消失。传递细胞入15ml锥形并增加一个额外的4 ml的IMOP培养基</li><li>旋转的15毫升合作nical 5分钟。在200 XG，吸用过的介质。悬浮在一个直径6cm培养皿的细胞在2毫升IMOP培养基和板细胞。孵育培养物在37℃，5％CO 2的膨胀。 </li></ol> 3.区分IMOP细胞分化成感觉上皮细胞<ol><li>准备50毫升IMOP感觉上皮分化培养基中:DMEM / F12，1X B27添加剂，25微克/毫升carbenecillin。请50毫升IMOP感觉上皮分化培养基中。热身49毫升DMEM / F12在50毫升的锥形在37℃水浴。解冻50X B27添加剂和100毫克/毫升carbenecillin等份5分钟。在37℃水浴。加入1毫升50X B27和12.5微升100毫克/毫升carbenecillin入的DMEM / F12。 </li><li>为了收获，分离，悬浮和计数细胞重复步骤1.4-1.9 </li><li>板1×10 6个细胞在一直径6cm培养皿在第-3天使用IMOP培养基。在第0天，使用转印培养10毫升吸管入15ml圆锥形。收集OTO球靠重力沉降如在步骤1.6所述。吸出使用2ml的吸移液管用过的培养基并离开otospheres在锥形的底部。 </li><li>轻轻地加入2毫升感觉上皮分化培养基中。转移otospheres使用大孔10毫升吸管一个直径6cm培养皿。 注:从恶劣的移液机械剪切可以从otospheres分离细胞。 </li><li>加入2毫升新鲜感觉上皮细胞分化传媒文化隔日1次。如果需要的话，细胞可以通过重力沉降和培养基替换来收集。 </li><li>通过转移到15ml锥形并允许otospheres沉积物如步骤指出1.6.Aspirate使用2ml的吸移液管的用过的培养基和离开otospheres不受干扰收集otospheres在第10天。 </li><li>通过温育在4％甲醛中的1X PBS 15分钟在室温固定otospheres。除去甲醛溶液，冲洗otospheres用洗涤缓冲液（1X PBS含0.1％的TritonX-10（1×PBS含10％正常山羊血清和0.1％的Triton X-100），1小时0）之前孵育在封闭缓冲液。 <ol><li>更换缓冲液并培育样品在封闭缓冲液，用稀抗体。孵育在4℃下。洗液样品用含1×PBS 0.1％的Triton X-100的受试者otosphere到免疫染色27。转移otospheres到1X PBS和地点otospheres到使用P1000的吸管载玻片安装介质。放盖玻片在样品，并允许安装的介质干燥，在4℃。 </li></ol></li><li>获得使用倒置显微镜的设置配备了16位的CCD照相机以及一个20X 0.75空气或40X 1.3 NA油浸物镜落射荧光图像。蓝色（377±25毫微米/ 447±30纳米），绿色（475±25毫微米/ 540±25纳米），红色（562±20nm的/和625:从使用列出的（激发和发射）波长不同颜色通道收集荧光±20纳米）和红外（628±20纳米/ 692±20纳米）。 </li></ol> 4.试金EDU公司注册<ol><li>在-3天，板1×10 6 IMOP细胞在6 cm培养皿。三天后在第0天，收获，分离，悬浮和通过重复步骤1.4-1.9计数细胞。 </li><li>板2.5×10 5个细胞在IMOP培养基和5×10 5个细胞在感觉上皮分化培养基在6孔培养皿的不同孔中。 </li><li>通过使用EDU掺入法测定S期细胞由EDU掺入在第3天的百分比<ol><li>直接添加EDU库存到IMOP培养物，以获得在培养基中为1μMEDU的终浓度。 </li><li>在IMOP培养孵育EDU 2小时，在37℃，5％的CO 2。收获，分离并收集细胞重复步骤1.4-1.9。加在4％甲醛中的1X PBS 15分钟。在室温修复细胞。收获细胞，离心，在200×g离心5分钟，室温。除去甲醛溶液properlÿ处置。 </li><li>细胞用Hoechst的标签细胞核并结合EDU与绿色荧光染料叠氮化物根据制造商的协议。 注:所有洗涤均用1X PBS，3％BSA和0.1％吐温20洗涤细胞用1X PBS清洗两次完成。 </li><li>使用抗淬灭剂，并在幻灯片上安装电池放置1.5盖玻片上的样品。获得使用荧光显微镜标记细胞的荧光图像。 </li></ol></li></ol> 5.区分IMOP源性神经元<ol><li>使50个ml的神经元分化培养基:的Neurobasal介质，1X B27添加剂，2mM的L-谷氨酰胺。在37℃水浴5分钟解冻瓶的50X B27和200mM的L-谷氨酰胺。加入1ml 50X B27和0.5ml的200mM L-谷氨酰胺至48.5的Neurobasal介质毫升。 </li><li>涂层玻璃罩通过放置在无菌的10个厘米板12毫米的圆形1.5的玻璃盖玻片，并添加70％乙醇到板以灭菌和清洁的盖玻片。 </li><li>轻轻地搅动海湾rslips以确保它们被覆盖在乙醇。离开该板在室温10分钟。冲洗盖玻片3次，用无菌PBS洗涤，洗出剩余的乙醇。用无菌H 2 0冲洗盖玻片一次洗出剩余的1X PBS。 </li><li>用2ml的吸吸管吸出H 2 O，让盖玻片干。暴露的盖玻片于UV光在组织培养橱中15分钟。商店盖玻片在无菌环境中，如果不立即使用。 </li><li>将一个12毫米一轮盖玻片的24孔板的每一个孔。摇板轻轻以确保该盖玻片平躺在井的底部。 </li><li>解冻2毫克/毫升聚D-赖氨酸原液在RT，并稀释至在1X PBS中的10微克/毫升多聚-D-赖氨酸的浓度。加入0.25毫升10微克/毫升多聚-D-赖氨酸到孔中。离开该板在37℃培养箱中培养1小时。 </li><li>用无菌的1X PBS洗涤各孔3次。解冻10毫克/毫升的层粘连蛋白原液在RT，并稀释至10微克/米升在1X PBS中。加入0.25毫升10微克/毫升的层粘连蛋白工作溶液到24多孔盘的单个孔。孵育板在37℃孵化器。吸出用2ml的吸吸管层粘连蛋白的解决方案。 </li><li>通过加入在1ml 1X PBS中与P1000吸管和用2ml的吸移液管吸移去除1X PBS洗涤盖玻片3次，用1×PBS。离开的1X PBS从在井最后一次洗涤直到细胞准备要电镀。发起的神经元分化通过收获，解离和在步骤在6cm皿在第-3天计数细胞1.4-1.9.Plate 1×10 6个细胞。 </li><li>关于第0天，收获，分离并通过重复1.4-1.9计数细胞。种子1×10 5 - 1.5×10 5 IMOP细胞变成0.5 ml的每孔中24多井培养皿预热神经元分化介质。吸和预热的神经元分化的媒体加入到文化隔日1次。 </li><li>固定在4％甲醛IMOP衍生神经元15分钟。一个吨RT上日7.取下甲醛溶液，洗IMOP衍生的神经元用洗涤缓冲液（1×PBS含有0.1％的TritonX-100）培养在封闭缓冲液前（1×PBS含10％正常山羊血清和0.1％的Triton X-100） 1小时。 <ol><li>更换缓冲液并培育样品在封闭缓冲液，用稀抗体。孵育在4℃下。洗涤样品用1X PBS含有0.1％的Triton X-100主体的细胞免疫染色27。放置到安装媒体前洗净用1X PBS的玻璃罩。允许安装媒体在4℃采集落射荧光图像，在步骤3.8上市之前，干燥。 </li></ol></li></ol>

<ol><li>Petit, C., Richardson, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Linking+genes+underlying+deafness+to+hair-bundle+development+and+function%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Linking genes underlying deafness to hair-bundle development and function.</a> Nat Neurosci. 12 (6), 703-710 (2009).</li>
<li>Kujawa, S. G., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adding+insult+to+injury%3A+cochlear+nerve+degeneration+after+%27temporary%27+noise-induced+hearing+loss%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Adding insult to injury: cochlear nerve degeneration after 'temporary' noise-induced hearing loss.</a> J Neurosci. 29 (45), 14077-14085 (2009).</li>
<li>Kujawa, S. G., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acceleration+of+age-related+hearing+loss+by+early+noise+exposure%3A+evidence+of+a+misspent+youth%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Acceleration of age-related hearing loss by early noise exposure: evidence of a misspent youth.</a> J Neurosci. 26 (7), 2115-2123 (2006).</li>
<li>Huth, M. E., Ricci, A. J., Cheng, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+aminoglycoside+ototoxicity+and+targets+of+hair+cell+protection%5BTitle%5D)+AND+%22Int+J+Otolaryngol%22%5BJournal%5D)">Mechanisms of aminoglycoside ototoxicity and targets of hair cell protection.</a> Int J Otolaryngol. 2011, 937861-93 (2011).</li>
<li>Brigande, J. V., Heller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quo+vadis%2C+hair+cell+regeneration%3F%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Quo vadis, hair cell regeneration?</a> Nat Neurosci. 12 (6), 679-685 (2009).</li>
<li>Groves, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+challenge+of+hair+cell+regeneration%5BTitle%5D)+AND+%22Exp+Biol+Med+%28Maywood%29%22%5BJournal%5D)">The challenge of hair cell regeneration.</a> Exp Biol Med (Maywood). 235 (4), 434-446 (2010).</li>
<li>Okano, T., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+cell+therapy+for+the+inner+ear%3A+recent+advances+and+future+directions%5BTitle%5D)+AND+%22Trends+Amplif%22%5BJournal%5D)">Stem cell therapy for the inner ear: recent advances and future directions.</a> Trends Amplif. 16 (1), 4-18 (2012).</li>
<li>Ronaghi, M., Nasr, M., Heller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Concise+review%3A+Inner+ear+stem+cells--an+oxymoron%2C+but+why%3F%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Concise review: Inner ear stem cells--an oxymoron, but why?</a> Stem Cells. 30 (1), 69-74 (2012).</li>
<li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Restoration+of+auditory+evoked+responses+by+human+ES-cell-derived+otic+progenitors%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Restoration of auditory evoked responses by human ES-cell-derived otic progenitors.</a> Nature. 490 (7419), 278-282 (2012).</li>
<li>Koehler, K. R., Mikosz, A. M., Molosh, A. I., Patel, D., Hashino, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+inner+ear+sensory+epithelia+from+pluripotent+stem+cells+in+3D+culture%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture.</a> Nature. , (2013).</li>
<li>Oshima, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanosensitive+hair+cell-like+cells+from+embryonic+and+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells.</a> Cell. 141 (4), 704-716 (2010).</li>
<li>Diensthuber, M., Oshima, K., Heller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem%2Fprogenitor+cells+derived+from+the+cochlear+sensory+epithelium+give+rise+to+spheres+with+distinct+morphologies+and+features%5BTitle%5D)+AND+%22J+Assoc+Res+Otolaryngol%22%5BJournal%5D)">Stem/progenitor cells derived from the cochlear sensory epithelium give rise to spheres with distinct morphologies and features.</a> J Assoc Res Otolaryngol. 10 (2), 173-190 (2009).</li>
<li>Doetzlhofer, A., White, P., Lee, Y. S., Groves, A., Segil, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prospective+identification+and+purification+of+hair+cell+and+supporting+cell+progenitors+from+the+embryonic+cochlea%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Prospective identification and purification of hair cell and supporting cell progenitors from the embryonic cochlea.</a> Brain Res. 1091 (1), 282-288 (2006).</li>
<li>Doetzlhofer, A., White, P. M., Johnson, J. E., Segil, N., Groves, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+growth+and+differentiation+of+mammalian+sensory+hair+cell+progenitors%3A+a+requirement+for+EGF+and+periotic+mesenchyme%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">In vitro growth and differentiation of mammalian sensory hair cell progenitors: a requirement for EGF and periotic mesenchyme.</a> Dev Biol. 272 (2), 432-447 (2004).</li>
<li>Martinez-Monedero, R., Yi, E., Oshima, K., Glowatzki, E., Edge, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+inner+ear+stem+cells+to+functional+sensory+neurons%5BTitle%5D)+AND+%22Dev+Neurobiol%22%5BJournal%5D)">Differentiation of inner ear stem cells to functional sensory neurons.</a> Dev Neurobiol. 68 (5), 669-684 (2008).</li>
<li>Oshima, K., Senn, P., Heller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+sphere-forming+stem+cells+from+the+mouse+inner+ear%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Isolation of sphere-forming stem cells from the mouse inner ear.</a> Methods Mol Biol. 493, 141-162 (2009).</li>
<li>Nishimura, K., Nakagawa, T., Sakamoto, T., Ito, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fates+of+murine+pluripotent+stem+cell-derived+neural+progenitors+following+transplantation+into+mouse+cochleae%5BTitle%5D)+AND+%22Cell+Transplant%22%5BJournal%5D)">Fates of murine pluripotent stem cell-derived neural progenitors following transplantation into mouse cochleae.</a> Cell Transplant. 21 (4), 763-771 (2012).</li>
<li>Wu, D. K., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+mechanisms+of+inner+ear+development%5BTitle%5D)+AND+%22Cold+Spring+Harb+Perspect+Biol%22%5BJournal%5D)">Molecular mechanisms of inner ear development.</a> Cold Spring Harb Perspect Biol. 4 (8), a008409(2012).</li>
<li>Jiang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lineage+analysis+of+the+late+otocyst+stage+mouse+inner+ear+by+transuterine+microinjection+of+a+retroviral+vector+encoding+alkaline+phosphatase+and+an+oligonucleotide+library%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Lineage analysis of the late otocyst stage mouse inner ear by transuterine microinjection of a retroviral vector encoding alkaline phosphatase and an oligonucleotide library.</a> PLoS One. 8 (7), e69314(2013).</li>
<li>Sapede, D., Dyballa, S., Pujades, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+lineage+analysis+reveals+three+different+progenitor+pools+for+neurosensory+elements+in+the+otic+vesicle%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Cell lineage analysis reveals three different progenitor pools for neurosensory elements in the otic vesicle.</a> J Neurosci. 32 (46), 16424-16434 (2012).</li>
<li>Satoh, T., Fekete, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clonal+analysis+of+the+relationships+between+mechanosensory+cells+and+the+neurons+that+innervate+them+in+the+chicken+ear%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Clonal analysis of the relationships between mechanosensory cells and the neurons that innervate them in the chicken ear.</a> Development. 132 (7), 1687-1697 (2005).</li>
<li>Raft, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cross-regulation+of+Ngn1+and+Math1+coordinates+the+production+of+neurons+and+sensory+hair+cells+during+inner+ear+development%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Cross-regulation of Ngn1 and Math1 coordinates the production of neurons and sensory hair cells during inner ear development.</a> Development. 134 (24), 4405-4415 (2007).</li>
<li>Neves, J., Parada, C., Chamizo, M., Giraldez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Jagged+1+regulates+the+restriction+of+Sox2+expression+in+the+developing+chicken+inner+ear%3A+a+mechanism+for+sensory+organ+specification%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Jagged 1 regulates the restriction of Sox2 expression in the developing chicken inner ear: a mechanism for sensory organ specification.</a> Development. 138 (4), 735-744 (2011).</li>
<li>Mak, A. C., Szeto, I. Y., Fritzsch, B., Cheah, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+and+overlapping+expression+pattern+of+SOX2+and+SOX9+in+inner+ear+development%5BTitle%5D)+AND+%22Gene+Expr+Patterns%22%5BJournal%5D)">Differential and overlapping expression pattern of SOX2 and SOX9 in inner ear development.</a> Gene Expr Patterns. 9 (6), 444-453 (2009).</li>
<li>Kiernan, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sox2+is+required+for+sensory+organ+development+in+the+mammalian+inner+ear%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Sox2 is required for sensory organ development in the mammalian inner ear.</a> Nature. 434 (7036), 1031-1035 (2005).</li>
<li>Puligilla, C., Dabdoub, A., Brenowitz, S. D., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sox2+induces+neuronal+formation+in+the+developing+mammalian+cochlea%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Sox2 induces neuronal formation in the developing mammalian cochlea.</a> J Neurosci. 30 (2), 714-722 (2010).</li>
<li>Kwan, K. Y., Shen, J., Corey, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((C-MYC+transcriptionally+amplifies+SOX2+target+genes+to+regulate+self-renewal+in+multipotent+otic+progenitor+cells%5BTitle%5D)+AND+%22Stem+Cell+Reports%22%5BJournal%5D)">C-MYC transcriptionally amplifies SOX2 target genes to regulate self-renewal in multipotent otic progenitor cells.</a> Stem Cell Reports. 4 (1), 47-60 (2015).</li>
<li>Dittami, G. M., Sethi, M., Rabbitt, R. D., Ayliffe, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+mammalian+cell+counts%2C+cell+size+and+cell+health+using+the+Moxi+Z+mini+automated+cell+counter%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Determination of mammalian cell counts, cell size and cell health using the Moxi Z mini automated cell counter.</a> J Vis Exp. (64), (2012).</li>
<li>Levenstein, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Basic+fibroblast+growth+factor+support+of+human+embryonic+stem+cell+self-renewal%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Basic fibroblast growth factor support of human embryonic stem cell self-renewal.</a> Stem Cells. 24 (3), 568-574 (2006).</li>
<li>Furue, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heparin+promotes+the+growth+of+human+embryonic+stem+cells+in+a+defined+serum-free+medium%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium.</a> Proc Natl Acad Sci U S A. 105 (36), 13409-13414 (2008).</li>
<li>Lotz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sustained+levels+of+FGF2+maintain+undifferentiated+stem+cell+cultures+with+biweekly+feeding%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Sustained levels of FGF2 maintain undifferentiated stem cell cultures with biweekly feeding.</a> PLoS One. 8 (2), e56289(2013).</li>
<li>Brewer, G. J., Torricelli, J. R., Evege, E. K., Price, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+survival+of+hippocampal+neurons+in+B27-supplemented+Neurobasal%2C+a+new+serum-free+medium+combination%5BTitle%5D)+AND+%22J+Neurosci+Res%22%5BJournal%5D)">Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.</a> J Neurosci Res. 35 (5), 567-576 (1993).</li>
<li>Watanabe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat Biotechnol. 25 (6), 681-686 (2007).</li>
<li>Ruben, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+the+inner+ear+of+the+mouse%3A+a+radioautographic+study+of+terminal+mitoses%5BTitle%5D)+AND+%22Acta+Otolaryngol%22%5BJournal%5D)">Development of the inner ear of the mouse: a radioautographic study of terminal mitoses.</a> Acta Otolaryngol. , Suppl 220. 1-44 (1967).</li>
<li>Matei, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Smaller+inner+ear+sensory+epithelia+in+Neurog+1+null+mice+are+related+to+earlier+hair+cell+cycle+exit%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">Smaller inner ear sensory epithelia in Neurog 1 null mice are related to earlier hair cell cycle exit.</a> Dev Dyn. 234 (3), 633-650 (2005).</li>
<li>Lee, Y. S., Liu, F., Segil, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+morphogenetic+wave+of+p27Kip1+transcription+directs+cell+cycle+exit+during+organ+of+Corti+development%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">A morphogenetic wave of p27Kip1 transcription directs cell cycle exit during organ of Corti development.</a> Development. 133 (15), 2817-2826 (2006).</li>
<li>Chen, P., Segil, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((p27%28Kip1%29+links+cell+proliferation+to+morphogenesis+in+the+developing+organ+of+Corti%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">p27(Kip1) links cell proliferation to morphogenesis in the developing organ of Corti.</a> Development. 126 (8), 1581-1590 (1999).</li>
<li>Hasson, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unconventional+myosins+in+inner-ear+sensory+epithelia%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Unconventional myosins in inner-ear sensory epithelia.</a> J Cell Biol. 137 (6), 1287-1307 (1997).</li>
<li>Barclay, M., Ryan, A. F., Housley, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Type+I+vs+type+II+spiral+ganglion+neurons+exhibit+differential+survival+and+neuritogenesis+during+cochlear+development%5BTitle%5D)+AND+%22Neural+Dev%22%5BJournal%5D)">Type I vs type II spiral ganglion neurons exhibit differential survival and neuritogenesis during cochlear development.</a> Neural Dev. 6, 33(2011).</li>
<li>Bermingham, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Math1%3A+an+essential+gene+for+the+generation+of+inner+ear+hair+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Math1: an essential gene for the generation of inner ear hair cells.</a> Science. 284 (5421), 1837-1841 (1999).</li>
<li>Izumikawa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Auditory+hair+cell+replacement+and+hearing+improvement+by+Atoh1+gene+therapy+in+deaf+mammals%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals.</a> Nat Med. 11 (3), 271-276 (2005).</li>
<li>Kim, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NeuroD-null+mice+are+deaf+due+to+a+severe+loss+of+the+inner+ear+sensory+neurons+during+development%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">NeuroD-null mice are deaf due to a severe loss of the inner ear sensory neurons during development.</a> Development. 128 (3), 417-426 (2001).</li>
<li>Liu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Essential+role+of+BETA2%2FNeuroD1+in+development+of+the+vestibular+and+auditory+systems%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">Essential role of BETA2/NeuroD1 in development of the vestibular and auditory systems.</a> Genes Dev. 14 (22), 2839-2854 (2000).</li>
<li>Ma, Q., Anderson, D. J., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurogenin+1+null+mutant+ears+develop+fewer%2C+morphologically+normal+hair+cells+in+smaller+sensory+epithelia+devoid+of+innervation%5BTitle%5D)+AND+%22J+Assoc+Res+Otolaryngol%22%5BJournal%5D)">Neurogenin 1 null mutant ears develop fewer, morphologically normal hair cells in smaller sensory epithelia devoid of innervation.</a> J Assoc Res Otolaryngol. 1 (2), 129-143 (2000).</li>
<li>Pang, Z. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+human+neuronal+cells+by+defined+transcription+factors%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Induction of human neuronal cells by defined transcription factors.</a> Nature. 476 (7359), 220-223 (2011).</li>
<li>Vierbuchen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+conversion+of+fibroblasts+to+functional+neurons+by+defined+factors%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Direct conversion of fibroblasts to functional neurons by defined factors.</a> Nature. 463 (7284), 1035-1041 (2010).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+single-step+induction+of+functional+neurons+from+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Rapid single-step induction of functional neurons from human pluripotent stem cells.</a> Neuron. 78 (5), 785-798 (2013).</li>
<li>Blanchard, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+conversion+of+fibroblasts+into+peripheral+sensory+neurons%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Selective conversion of fibroblasts into peripheral sensory neurons.</a> Nat Neurosci. 18 (1), 25-35 (2015).</li>
</ol>

The authors have nothing to disclose.

监测IMOP文化 一种协议，用于维持自我更新和促进新颖IMOP细胞系的分化的描述和附加的电镀格式包括（ 表1）。这有助于常规扩张和IMOP细胞分化的几个关键步骤指出。类似的多能干细胞培养，细胞IMOP理论上具有不确定的使用寿命。以确保细胞系适当的维护，IMOP培养物进行常规的细胞活力，自我更新和分化潜能监测。要分解后确定细胞活力，我们采用台盼蓝染色。在一般情况下，细胞的70％以上是离解后存活。为了监测自我更新的IMOP细胞，用c-Myc和Sox2的抗体免疫染色完成。在增殖培养条件，IMOP细胞具有〜18小时的倍增时间。在自我更新标记或做表情的改变ubling时间被改变自我更新的潜在指标。并监控IMOP细胞，早期标记物感觉上皮或神经元细胞周期出口和表达的分化潜能被用来评估细胞的分化潜能。如果IMOP细胞不通过上述判据中的任何一个，这将是可取终止培养。 这影响IMOP文化变量 在IMOP培养细胞的一个关键步骤是维持的bFGF的活性浓度的培养物，以促进自我更新。 bFGF的是据报道，在37℃，高度不稳定的培养细胞29,30时。文化的成长可显著由于bFGF的不稳定性受到自发分化。在当前的协议，新鲜培养基不断地供给到培养物中以维持bFGF的浓度高于5毫微克/毫升。使用受控的bFGF水平的稳定释放乙醇酸和乳酸聚酯微球能降低维护媒体31 bFGF的稳定水平需要媒体的频率发生变化。此外，不断释放的bFGF的会减少需要经常添加媒体保持bFG​​F的水平IMOP文化。 在维持IMOP细胞的生存力的另一个关键步骤是限制暴露于解离试剂和机械剪切。在当前的协议，解离与1m​​M EDTA中完成孵育。遏制过度的培养时间用EDTA，减少因剧烈吹打过大的机械剪切力和传代细胞IMOP时，将有助于提高细胞活力限制多个离心步骤。注意给予这些步骤扩增培养期间可以有效地保持大量的细胞生存能力。 期间IMOP细胞的常规培养，几种方法用于离解的细胞进行了测试。 Mechan从移液的iCal剪切可用于离解otospheres，但并不均匀地或一致地产生的单细胞。使用用于IMOP细胞的细胞解离市售试剂可以有效地产生单细胞otospheres，但这些试剂影响细胞的粘合性能改革otospheres和粘附到处理过的表面。为了使用商用试剂中，需要多次洗涤用人分化方法前去除解离试剂。 在当前的分化方法，既感觉上皮和神经元分化的条件导致观察到在单细胞或死细胞的团块的形式，细胞死亡。凋亡性细胞死亡可能是由于长期体外培养期间缺乏适当的分化或存活线索。含B27的补充这两种分化条件，以促进细胞的存活。虽然B27 supplem耳鼻喉科已经显示出促进初级海马神经元32的存活，这可能不足以促进IMOP细胞的存活。此外，如ROCK抑制剂可能有助于维持，并在常规培养和体外分化加大IMOP细胞的存活化合物。 ROCK抑制剂已经被广泛地用于多能干细胞培养物，以促进细胞在无血清培养物的生存，而不会影响分化33。除了ROCK抑制剂的培养基可以帮助增加在长期分化协议细胞存活，而不影响IMOP细胞的分化潜能。增加细胞的生存将允许协议的发展，有效地生成大量的成熟毛细胞或SGNS的。 增加CDKN1B表达为IMOP分化的早期标志物 在早期耳蜗发育，细胞的初始事件循环退出先分化为毛细胞，支持细胞和SGNS 34。神经祖细胞的有丝分裂航站楼利差从基地出发，逐步迈向耳蜗35的顶点一浪。在相对 ​​梯度，prosensory祖，为毛细胞和支持细胞产生的终端有丝分裂的进展，从顶点到耳蜗34,36的基地。虽然CDKN1B的时空表达以及相关的有丝分裂后的状态，这是最有可能不是促进在发展耳蜗细胞周期退出的唯一因素。为了支持这一点，CDKN1B突变的动物仍然可以开发有丝分裂后的毛细胞37。相反，CDKN1B可以用作早期指标标记分化的发作。在非制导IMOP分化培养，早期标志物的分化将协助开发协议，以促进分化的早期阶段明显的形态特征可以被观察到了。&lt;/ P&gt; 类似的耳部发展，循环出口启动分化和导致增加CDKN1B水平IMOP细胞。在增殖IMOP细胞，CDKN1B只有低水平表达可以观察到。其他细胞周期蛋白依赖性激酶抑制剂如CDKN1A（p21的CIP）可以负责在IMOP细胞正常细胞周期进展。期间感觉上皮和IMOP细胞的神经元分化，单细胞定量分析显示细胞CDKN1B的增加表达的一个子集在鉴别IMOP培养物（ 图6A，C）。 CDKN1B在这些细胞中的表达是IMOP细胞经历终末分化的指示。 IMOP细胞作为蜂窝式平台为研究细胞命运决定 因为IMOP细胞可以从一个祖状态到体外不同耳谱系过渡，它们可以被用来研究耳部细胞命运决心。目前，所描述的协议中使用无制导分化程序，以产生不同的耳部细胞类型。该IMOP系统允许增加生物活性分子，化合物和定义的基因，引导IMOP细胞向成熟的毛细胞，支持细胞和SGN谱系。一个用途IMOP蜂窝式平台的是测试候选转录因子（TF），其促进分化。的关键因子表达可以用来驱动毛细胞或神经元的分化。 CDKN1B沿既定标记或毛细胞和SGNS的形态特征的表达可用于确定候选转录因子到不同的耳部谱系38,39的贡献。 候选的转录因子如ATOH1是毛细胞发展的必要条件，并已改变用途，以促进头发细胞分化受损耳蜗40,41。简介ATOH1到IMOP细胞可能有助于促进头发细胞的分化。在developi纳克耳蜗，既Ngn1和NEUROD1 在体内42-44需要适当SGN分化。 Ngn1或NEUROD1在IMOP细胞中的表达可促进SGN分化。这些实验是类似于产生诱导神经元细胞（IN），其中，单独或组合的转录因子的表达可以转换成纤维细胞或多能干细胞为神经元45-48。这是毛细胞或SGNS的发育过程中正常表达的转录因子的引入是指导IMOP细胞向毛细胞或SGN谱系一个很好的策略。 促进大量IMOP来源的毛细胞和SGNS分化条件将提供疾病建模，小分子筛选和毒理学试验一个强大的移动平台，可以快速完成，并经济高效地之前在啮齿类动物的劳动密集型测试启动。描述的协议提供可用于个简单和通用的系统tudying耳祖分化，是适合于大规模的实验。

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的提款跌幅增殖IMOP细胞 为了减少IMOP细胞的增殖能力，并启动IMOP细胞分化和bFGF从培养物取出。要确认生长因子撤出降低扩散，埃杜掺入用作增殖试验。细胞的掺入EDU从otospheres与IMOP培养基（含有bFGF）的和otospheres在感觉上皮培养基中培养培养（不含bFGF）的百分比进行比较。 Otosphere培养物脉冲与核苷酸类似物EDU，收获并固定。团EDU核苷酸进行了荧光标记使用的点击化学Alexafluor 488叠氮化物。从在不存在bFGF对生长otospheres细胞显示EDU相对掺入降低到细胞的bFGF（图1A-F）中培养。由于大小Of显示otosphere增加，这是越来越难，从可视化用表面荧光显微镜otosphere所有单元格。为了解决这个问题，将细胞解离，并安装成使它们可明确地显现连接质量 。甚至解离和固定后，将细胞重新聚集。以保持细胞在离解状态，洗涤剂，阻止从重新聚集的细胞进行了测试。吐温20是防止细胞再聚集的洗涤剂中的一个。将解离的细胞用0.1％吐温20和安装到载玻片上。 EDU标记细胞，然后用荧光显微镜（ 图1G，H）可视化。从单个实验的代表性结果（N = 5）表明后的bFGF戒断（P &lt;0.005）后3天在EDU的百分比显著下降标记的细胞从48％至19％。这些结果证实在S期细胞的百分比和IMOP细胞后增殖潜力急剧下降bFGF的撤出。 IMOP衍生感觉上皮细胞快递CDKN1B和显示的形态变化 的IMOP感觉上皮分化协议时间线显示（ 图2A）。从增殖培养Otospheres解离成单细胞，并允许恢复在IMOP培养基3天。此方法有助于富集增殖细胞和选择抗有丝分裂后的细胞在这些起始培养物。在3天之后，新形成的otospheres接种在感觉上皮分化培养基，并使其经受无制导分化10天。含otospheres在接种后不同时间点的典型文化的亮场图像显示的大小先降otospheres启动大小（ 图2B-E）增加了。 27的形态学和分子特征。以确定是否CDKN1B（p27蛋白KIP）的表达可被用作标记用于分化，增殖从或感觉上皮分化IMOP培养otospheres进行比较。荧光标记物进行观察，并用荧光显微镜拍摄的。从增殖培养otospheres的代表图像显示，几乎没有毒伞素染色（ 图3 AD）CDKN1B的低表达细胞核之外。 Otospheres在感觉上皮分化培养基中培养显示的增加的核CDKN1B伴随的表达与鬼笔环肽标签中的细胞 （图3 EH）的周缘的外观。在增殖IMOP otospheres，CDH1（E-cadherin）和鬼笔环肽是弱标记（图3 IL）。在分化IMOP otospheres，显鬼笔环肽和CDH1标记突出显示在肌动蛋白丝和细胞-细胞粘附（图3 MP），类似于先前结果27个地区的形态变化。在感觉上皮细胞的分化，在肌动蛋白丝的形态变化平行CDH1表达的细胞间粘附位点的出现。在这个协议中，随着变化鬼笔环肽和CDH1增加CDKN1B表达服务的感觉上皮分化早期指标中IMOP细胞。 IMOP源性神经元快递CDKN1B和TUBB3 的IMOP神经元分化协议的示意图所示（ 图4A）。类似于上述协议，IMOP细胞解离和人lowed恢复为IMOP培养3天。 Otospheres收获，解离成单细胞并接种到聚-D-赖氨酸和层粘连蛋白包被的盖玻片。使细胞分化为7天。代表亮场图像的时间点显示的渐进式形态的变化，因为它们分化 ​​成IMOP源性神经元（ 图4B-E）。 7日，长突起可以看出，从细胞体（ 图4E箭头）延伸。的变化来判断在CDKN1B的表达水平在神经​​元分化，增殖IMOP细胞和IMOP衍生的神经元进行了比较。 Otospheres都被标记了赫斯特和CDKN1B抗体。从增殖otospheres IMOP细胞有少数细胞与低核CDKN1B 表达 （图4 FH）。此外，IMOP衍生的神经元显示增加细胞核CDKN1B表达的数目的神经元分化（图4 7天后IK）。以确定是否CDKN1B细胞采用神经元谱系，IMOP衍生的神经元与神经元标记TUBB3贴标签已完成。 IMOP源性神经细胞的代表性图像显示CDKN1B和TUBB3（ 图5 AD）的共同标签。放大并从这些细胞中神经突的定量表明与每个TUBB3相关联的平均1.5突起标记的细胞（60）（图5 EG）。在我们的神经元分化的协议，与CDKN1B和TUBB3免疫染色可以用作分化的指标进双极或伪单极IMOP衍生的神经元。 启动分化后核CDKN1B表达水平的提高 在otospheres形态变化显示IMOP细胞质的变化，因为它们发生分化。为了实现从IMMU定量结果nofluorescence图像来表示早期分化事件，CDKN1B从单个增殖IMOP细胞（N = 239），并IMOP细胞经历感觉上皮分化的细胞核荧光强度（N = 246）进行测定。正常化从独立实验CDKN1B荧光信号，CDKN1B，以从单个细胞的Hoechst荧光的比值来确定。 CDKN1B的标准化荧光强度从增殖IMOP细胞（黑色）和感觉上皮分化IMOP细胞（红色）直方图作图（图6A）。在高CDKN1B表达细胞的数目增加，观察到直方图的右移为感觉上皮分化（红色）相对于所述直方图增殖IMOP细胞（黑色）。为了确定增加的细胞表达CDKN1B，对归一化CDKN1B荧光强度单位的阈值（0.65）的比例被设定（箭头）和细胞的百分比ABO<html>已经阈值确定。后感觉上皮分化，IMOP细胞显示增加的25.3％的细胞表达CDKN1B（图6B）的67.1％。相同的定量分析应用到IMOP衍生的神经元。当比较增殖IMOP细胞（N = 525）和7天IMOP衍生的神经元（N = 583），其与IMOP衍生神经元相关联的直方图右移，观察相对于直方图增殖IMOP 细胞 （图6C）。确定细胞高于阈值的百分比显示增加CDKN1B表达细胞从23.5％至85.5％（图6D）。使用所描述的协议，CDKN1B表达的定量分析证实了增加的细胞，在感觉上皮和神经元分化上调CDKN1B的数目。 CDKN1B表达细胞的数目增加可以用来确认IMOP细胞在这些协议的分化。 jove_content"FO:保持together.within页="1"&gt; <img alt="图1" src="/files/ftp_upload/53692/53692fig1.jpg" /> 图1. EDU团作为检测，以确定IMOP文化的增殖状态。IMOP衍生otospheres中bFGF的存在下培养。细胞的细胞核标记（A），赫斯特和 （B）EDU Alexafluor 488（C）的赫司特和埃杜荧光合并后的图像。 Otospheres培养3天缺乏bFGF的媒体。从培养细胞用的赫斯特和EDU标记（D）的赫斯特和 （E）EDU Alexafluor 488.（F）的合并的图像。 Hoechst公司（品红色）和EDU（ ​​绿色）标记的细胞解离otospheres和洗涤用含有1×PBS 0.1％吐温20细胞后合并荧光图像分别来自otospheres中bFGF的存在或（H）的情况下的bFGF培养（G）。标尺长条是10微米，除非EDU的指示。（I）的百分比从标记IMOP细胞bFGF的存在或不存在的培养细胞。分析柱状图及误差棒内被表示为单个细胞的数量被描述为平均值（SEM）的标准误差。细胞计数从每组5个独立实验。学生的t检验是为了确定统计显着性。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53692/53692fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53692/53692fig2.jpg" /> 图2.一般 原理感觉上皮细胞分化的。（A）时间轴分化IMOP细胞分化成感觉上皮。线图表示细胞解离和介质变化的时间。 （二）典型相衬otospheres期间无制导感觉上皮分化第0天的图像，（C）3，（D）7 和 （E）10。中比例尺长度为100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53692/53692fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53692/53692fig3.jpg" /> 标记 IMOP细胞IMOP培养基中培养的指示细胞周期阻滞和分化IMOP衍生感觉上皮细胞。Otosphere图3中的表达。细胞的细胞核标记与 （A）的Hoechst 和 （B）CDKN1B抗体。丝状肌动蛋白被标记（C），毒伞。 （D）的含有增殖IMOP细胞典型otospheres的合并图像。从IMOP细胞Otospheres培养于感官epithElia的培养基10天。从otopsheres细胞标记（E）赫司特，（F）CDKN1B和（G），毒伞。 otospheres的（H）合并图像显示增加CDKN1B和鬼笔标记。增殖otospheres进行标记与 （Ⅰ）的Hoechst，（J）CDH1，（K）的鬼笔环肽。 （L），合并后的图像示出了从这些标记弱荧光。 Otospheres分化成感觉上皮细胞也被标记（M）赫司特，（N）CDH1，（O），毒伞。 （P）合并后的图像显示了典型的强CDH1和鬼笔标记。的比例尺的长度是10微米除非另有说明。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53692/53692fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <im<html>摹ALT ="图4"SRC ="/文件/ ftp_upload / 53692 / 53692fig4.jpg"/&gt; 图4.一般原理神经分化的。（A）时间轴分化IMOP细胞分化为神经元。 IMOP细胞进行神经分化的（B）第1天，（C）3（D）5 和 （E）7.箭头指向轴突的相衬图像。 IMOP细胞作为标记（F），赫司特和 （G）CDKN1B IMOP培养基otospheres培养的荧光图像。 （H）标记的细胞用Hoechst和CDKN1B的合并图像。 IMOP细胞后在神经分化培养基培养7天的荧光图像。细胞的细胞核标记（Ⅰ）的Hoechst和（J）的 CDKN1B。 （K）合并的单元格标记赫斯特和CDKN1B的形象。的比例尺的长度是10微米，除非另有说明。<html> OM /文件/ ftp_upload / 53692 / 53692fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/53692/53692fig5.jpg" /> 图分子标记指示性的细胞周期退出和神经元分化5.表达。IMOP细胞神经分化后第7天。细胞核标记与 （A）的Hoechst，（B）的TUBB3（神经β微管蛋白）的抗体以突出细胞形态和 （C）CDKN1B抗体。 （D）与CDKN1B和TUBB3的单个细胞标记合并图像。的比例尺的长度为20微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53692/53692fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> OAD / 53692 / 53692fig6.jpg"/&gt; 图6.单细胞定量荧光强度CDKN1B分析。（A）的标准化荧光CDKN1B表达的强度通过计算从各个细胞CDKN1B和Hoechst的荧光强度的比率来确定。归一化的荧光强度作图相对于细胞数为直方图。示CDKN1B从增殖IMOP细胞因子bFGF（黑色）和IMOP otospheres的存在下分化成感觉上皮（SE）（红）中培养标准化荧光强度。甲阈设定在0.65标准化荧光强度单位（箭头）（B）中表达CDKN1B高于阈值IMOP细胞的百分比，从增殖（黑色）和感觉上皮分化培养物（红色）。 （C）的合并标记用Hoechst和Myo6抗体感觉上皮分化IMOP细胞荧光图像。 （D </str翁&gt; CDKN1B表达从增殖IMOP细胞（黑色）和神经元分化的细胞IMOP（红色）归一化的荧光强度）作图和阈值设定（箭头），如上所述。 （E） - IMOP细胞增殖（黑色）和神经元分化培养物（红色）表达CDKN1B的百分比。用于分析个体细胞表示在每个条形图。结果是从不同的实验编译（N = 3）和误差棒描绘为SEM表示。学生t检验用来确定统计显着性。 （六）合并双极或标记赫斯特和TUBB3伪单极IMOP源性神经元的荧光图像。的比例尺的长度是20微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53692/53692fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 细胞培养容器 </td><td> 苏rface面积（cm 2）的</td><td> 种子队的日常维护细胞的数量 </td><td> 种子细胞的感觉上皮细胞分化的数量 </td><td colspan="2"> 种子队的神经元分化细胞的数量 </td><td> 缩放因子相对于60mm培养皿 </td><td> 媒体的使用量（毫升） </td></tr><tr><td> Multwell板 </td><td></td><td></td><td></td><td colspan="2"></td><td></td><td></td></tr><tr><td> 96 </td><td> 0.3 </td><td>万</td><td> 20000 </td><td colspan="2"> 10,000-50,000 </td><td> 0.015 </td><td> 0.1 </td></tr><tr><td> 24 </td><td> 2 </td><td> 90000 </td><td> 180000 </td><td colspan="2"> 100,000-150,000 </td><td> 0.100 </td><td> 0.5 </td></tr><tr><td> 12 </td><td> 4 </td><td> 180000 </td><td> 360000 </td><td colspan="2"> 300,000 </td><td> 0.200 </td><td> 1 </td></tr><tr><td> 6 </td><td> 10 </td><td> 500000 </td><td>百万</td><td colspan="2"> 500,000-750,000 </td><td> 0.500 </td><td> 2 </td></tr><tr><td> 餐具 </td><td></td><td></td><td></td><td colspan="2"></td><td></td><td></td></tr><tr><td> 35毫米</td><td> 10 </td><td> 500000 </td><td>百万</td><td colspan="2"> 500000 </td><td> 0.500 </td><td> 2 </td></tr><tr><td> 60毫米</td><td> 20 </td><td>百万</td><td> 2000000 </td><td colspan="2"> 1,000,000-1,500,000 </td><td> 1.000 </td><td> 3 </td></tr><tr><td>百毫米</td><td> 60 </td><td> 2500000 </td><td> 5000000 </td><td colspan="2"> 2,500,000- 3000000 </td><td> 3.000 </td><td> 8 </td></tr></tbody></table> 表1.单元号为IMOP细胞的分化在瓦尔维护白条组织培养板形式。

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.

在永生多能耳科内皮祖细胞分化启动

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.5K 次观看.  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.

视频: 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.

来自祖，人脑少突胶质细胞培养系统

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

科研

神经科学

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.

胚胎干细胞分化成少突胶质前体

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

生物学

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.

破骨细胞与人诱导多能干细胞的分化和表征

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

发育生物学

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.

新生小鼠皮肤干细胞分化成生殖细胞<em在体外</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.

从多能干细胞生成人类神经元和少突胶质细胞，用于模拟神经元-少突胶质细胞相互作用

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

Differentiating Human Embryonic Stem Cells into Neural Progenitors

Source: Jeon, K. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/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.

将人类胚胎干细胞分化为神经祖细胞

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

	Slowly thaw the hESC-qualified basement membrane ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

A Co-culture Method to Model Neuron-Oligodendrocyte Interactions

Source: Assetta, B.,et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/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.

一种模拟神经元-少突胶质细胞相互作用的共培养方法

1. Human neuron induction from human pluripotent stem cells

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

Co-culture and Interaction Studies

Differentiation of Neural Progenitor Cells into Neurons

Source: Mao, X., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/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.

神经祖细胞分化为神经元

1. Differentiation from NPCs to neurons (Phase II)

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

Differentiating Otic Progenitor Cells Into Sensory Epithelial Cells Followed by Immunostaining

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

将耳祖细胞分化为感觉上皮细胞，然后进行免疫染色

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.remotexs.ntu.edu.sg/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.

将永生化多能耳祖细胞分化为螺旋神经节神经元并评估分化

1. Differentiating iMOP-Derived Neurons 

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