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.

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			<td height="20" style="height:20px;width:455px;">Moxi Z Cassette Type S</td>
			<td style="width:196px;">Orflo&#160;</td>
			<td style="width:145px;">MXC002</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:455px;">Recombinant Murine Fibroblast Growth Factor, basic (bFGF)</td>
			<td style="width:196px;">Peprotech</td>
			<td style="width:145px;">450-33</td>
			<td style="width:280px;">Resuspended in 0.1% BSA in H20 and stored as 20 mg/ml aliquots</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:455px;">Poly-D-Lysine</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:145px;">P7886</td>
			<td style="width:280px;">Resuspended in 1X PBS and stored as 10 mg/ml 100 &#181;l aliquots</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:455px;">Carbenicillin, Disodium Salt</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">BP2648-1</td>
			<td style="width:280px;">Resuspended in 10mM Hepes pH 7.4 and stored as 100 mg/ml aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">5 ml pipet individually wrapped paperback (200/case)</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td>1367811D</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">10 ml pipet individually wrapped paperback (200/case)</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td>1367811E</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Tissue Culture Treated Biolite 24 -Well Plate</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">130188</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Tissue Culture Treated Biolite 6 -Well Plate</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">130184&#160;</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Tissue Culture Treated 6 cm Dish</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">130181&#160;</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EMD Millipore Millex Sterile Syringe PVDF Filter Pore size: 0.22&#956;m</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 0.1-10 ul elongated filter tip</td>
			<td>USA Scientific</td>
			<td>1120-3810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 1-20 ul filter tip</td>
			<td>USA Scientific</td>
			<td>1120-1810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 1-200 ul&#160; filter tip</td>
			<td>USA Scientific</td>
			<td>1120-8810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 101-1000 ul filter tip</td>
			<td>USA Scientific</td>
			<td>1126-7810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">15 ml conical tubes sterile 20 bags of 25 tubes (500 tubes)</td>
			<td>USA Scientific</td>
			<td>1475-0511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">50 ml conical tubes sterile 20 bags of 25 tubes (500 tubes)</td>
			<td>USA Scientific</td>
			<td>1500-1211</td>
			<td></td>
		</tr>
	</tbody>
</table>

initiating differentiation in immortalized multipotent otic progenitor cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

bFGF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

Universidad de Cartagena UDEC

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 ...