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>

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?...

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...

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

Research

JoVE Journal

Developmental Biology

8.4K 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/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/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/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/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/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 ...