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.

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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

8.4K 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

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

从胚胎干细胞衍生心脏祖细胞

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

科研

发育生物学

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

在人多能的体外干细胞向滋养层细胞

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

在体外人骨髓间充质干细胞分化为心肌样细胞功能

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

低血清条件下心脏祖细胞内皮分化的诱导作用

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

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