Name: generation of esc-derived mouse airway epithelial cells using decellularized lung scaffolds
Uploaded: 2016-05-05T15:00:00.000+00:00
Duration: 12 min 31 s
Description: Watch this Scientific Journal Video about Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds at JoVE.com

我们要感谢Rossant博士和碧萝博士在图1-3中描述的实验中使用的Nkx2-1 mcherry ESC。 FACS是在病童医院，UHN流式细胞仪进行融资。这项工作是由操作从加拿大卫生研究院的研究，并从创新的加拿大基金会的资助基础设施（CSCCD）资助。

动物实验按照医院的病童研究院动物护理委员会准则进行。 1.脚手架准备<ol><li>肺脱细胞<ol><li>使用二氧化碳安乐死室成年Wistar大鼠。放置动物在腔室，并以每分钟室体积10-30％的填充率开始100％的CO 2的曝光。 <ol><li>观察动物的意识障碍;在此之后大约2-3分钟会发生。如果神志不清不这段时间内发生，检查填充率和腔密封。以下失去知觉，对于褪眼睛颜色和缺乏呼吸的观察动物。如果两者都观察到，保持二氧化碳灌装1-2分钟，然后从室取出的动物的过程。 </li></ol></li><li>动物安全通过固定前爪和后腿解剖面，向下喷射的胸部和腹部地区用70％乙醇。进入心脏和鲁通过沿胸骨纵切口打开胸腔NGS。 </li><li>通过隔膜使小切口首先以引起肺缩回，减少穿刺肺的机会。结扎与缝合下腔静脉，并将在左心房小切口小解剖剪刀。 </li><li>使用准备好的10毫升注射器填充有肝素Hank氏平衡盐溶液（HBSS）（10U / ml肝素在HBSS）将25g针，通过将针插入右心室通过肺循环推缓冲器开始肺灌注（速率为2毫升/分钟）。直到肺部变为白色，并从左心房流动的流体运行明确继续此过程。 </li><li>灌注之后，暴露气管导管插入接近甲状软骨的塑料导管和到位用缝线固定。 </li><li>安装设定10毫升注射器固定在铁架台和夹子重力灌注系统。删除和Discard注射器推杆。安全注射器针筒的最大充装点在20厘米以上的肺。附加一个双向活塞在注射器的端部，和一个长塑料管向活栓递送脱细胞溶液到插管气管的另一端。倾溶液注入注射器中，并允许溶液以填充所连的塑料管和导管。 <ol><li>通过填充到肺总容量（约12毫升），持续1分钟灌洗肺和从气管去除塑料导管，以允许流体流出肺的。灌洗肺部时保持低于20厘米H 2 O.压力不填注射器超过10毫升</li></ol></li><li>肺部八次与脱细胞溶液的重复灌洗，然后用10漂洗用磷酸盐缓冲盐水（PBS）。 </li><li>解剖气管和肺部从颈部和胸腔游离并从动物取出。保持组织在冷PBS在4℃下直到用于vibratome本身制备ctioning。 </li></ol></li><li>厚款代<ol><li>制备约15毫升的2％和4％（重量/体积）琼脂糖，和足够6％（重量/体积）琼脂糖嵌入所有裂片成小的矩形块。通过微波溶解在PBS低熔点琼脂糖粉末。转移琼脂糖到50毫升管的热块上，并维持在40℃以上的温度，以避免凝胶。 </li><li>在每个叶支气管分离用小剪刀每叶（颅，中间，附件，和尾部右边叶和左叶）的端部解剖脱细胞肺癌。专利使用吸收性片材以除去过量的PBS中，并放置内2％干每个叶（重量/体积）琼脂糖而加热块上。 </li><li>在琼脂糖5分钟涂布后除去每个叶，放置在培养皿中并允许表面胶凝1分钟上的冷板。 </li><li>轻轻地放在裂片放回4％（重量/体积）琼脂糖，凉5分钟后，用6％（重量/体积）琼脂糖再次重复涂布。 </li><li>经过连续合作每个叶的阿婷，使用金属基体材料的模具周围从边缘的组织的琼脂糖至少3mm分别嵌入各叶6％（重量/体积）琼脂糖。东方每个叶使用镊子通过在面对实验者金属模具的表面定位瓣最大平坦的边缘。该边缘将是固定在试样板上vibratome切片的一侧。 </li><li>允许块在冷板与vibratome切片之前胶凝至少30分钟。在4℃下在高达12小时的加湿室商店块vibratome切片之前。 </li><li>设置的vibratome用冷PBS 12填充切片室。保持整个与周围的冰浴切片冷温度。从金属模具中取出的块，并使用刀片修剪下来过量琼脂糖周边波瓣，而从组织的边缘保持约3毫米。 </li><li>使用黏合剂固定组织，以试样板中心，淹没板入T他PBS填充切片室。设置通过选择分别以下速度，幅度和厚度值切片上vibratome边界0.2毫米/秒，1.85毫米，350微米。 注意:叶取向的两个纵向和横向部分是可以接受的。 </li><li>每节完全叶。手动切割部免费使用小剪刀，如果一个部分没有完全由叶片在段序列的末端分离。轻轻收集支架部分，并保持在PBS在冰上，直到下一个步骤。 注意:产生部分将包括在近端和远端肺领域和可用于recellularization两个来源;然而，大部分的表面的将包括远端肺。 </li></ol></li><li>支架部分去污<ol><li>从转移的PBS 350微米厚的支架部分，以离心管（最多30段/管）和核酸治疗（PBS中90 U / ml）的（见材料清单）12 - 在室温24小时，上一个旋转器。 </li><li>以下核酸酶处理，用钳子向新的离心管中，并在无菌条件下的抗微生物溶液（200U / ml青霉素链霉素和25微克/毫升两性霉素B的PBS）6小时在RT，在旋转器上对待转印部。 注意:支架可以在使用前被储存在抗微生物溶液在4℃最多一周℃。 </li><li>在与抗菌液去污一步，冲洗支架用PBS两次无菌条件下并转移到无血清分化培养基（SFDM）与细胞接种前。 </li></ol></li></ol> 2，内胚细胞的制备<ol><li>定型内胚层诱导<ol><li>保持使用条件2I 13无饲养层，无血清培养下鼠标ESC线。通过胰蛋白酶消化去除附着的多能干细胞培养开始内胚层诱导。 </li><li>重悬SFDM和种子细胞以20,000密度细胞三天低附着板/ ml，未介质改为允许EB形成。 </li><li>三天后轻轻用10毫升吸管的胚转移到50ml锥形管，让他们在底部收集在室温下3分钟。 </li><li>小心吸媒体和补充新鲜SFDM媒体辅以50毫微克/毫升激活素A. </li><li>种子细胞返回到在1，低附着板:2密度和文化三天，才能达到最终的内胚层分化。 </li></ol></li><li>定型内胚层的富集<ol><li>收集日6 EBS，用胰蛋白酶分解，并结合使用荧光抗体标记14对c-KIT和CXCR4的表达。 </li><li>使用这两种标志物的表达荧光激活细胞分选术（FACS）分类标记的细胞，以获得富集定形内胚层的人口15。 </li></ol></li></ol> 3. R​​ecellularization设置<ol><li>气 - 液界面文化设置<ol><li>转自SFDM每个支架脱细胞部分（步骤1.2.9）上使用无菌镊子疏水膜浮动（8微米孔径）。确保支架部分是对膜均匀地分布。 </li><li>由分别与1或0.5毫升SFDM，填充井制备6-或12孔板中。轻轻地放在膜进入井，允许浮在介质顶部的膜，形成空气 - 液体培养设置。 </li></ol></li><li> 3D支架的播种<ol><li>对于定型内胚层标志物（步骤2.2.2）以下FACS计数使用血球排序细胞，降速为400 XG在SFDM 5分钟，重悬。重悬的细胞以获得含有约100,000个细胞/ 10微升/支架的体积。 </li><li>要recellularize支架，吸管10微升的细胞，直接到步骤3.1.2各准备一节。 </li><li>在文化更换SFDM媒体每48小时。通过在稍微倾斜端着盘子吸出旧媒体为了避免破坏文化。慢慢的新鲜SFDM沿井的侧添加到文化，以防止浮膜下沉。 </li><li>保持长达21天的气 - 液培养，实现种子细胞分化到成熟的气道上皮细胞。 注意:Recellularized部分可以用于组织染色和免疫荧光（IF）显微镜在任何时间点的细胞培养16中进行处理。 </li></ol></li></ol>

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这里描述的协议产生只使用天然肺支架直接，没有其他补充了分化成熟的ESC源性上皮气道。这种文化建立的定义，无血清，价格低廉，重现性好。没有基础分化培养基中生长因子的补充是必要的。用于产生干细胞衍生的肺上皮细胞中以前发表的方法已使用2维的策略与生长因子补充，以促进谱系限制3,4,8,18,19。此处所描述的技术是在这些方法中有几个原因，包括有利:定义培养条件下，分化效率高功能性气道细胞，从其它内胚层谱系（甲状腺，肝，胰腺）有限污染，和一个三维分化的微环境。这种技术产生了类似的结果与小鼠源ECM，因此可以进行修改，以生成用小鼠肺脱细胞支架。大鼠的胚胎干细胞分化成定型内胚层，并使用此协议也分化成上皮细胞气管接种于支架材料。 脱细胞的支架可被存储在去污溶液在4℃recellularization之前最多7天。支架保持更长的持续时间可能是功能性的，但是我们没有测试证实了这一点。为了实现高效的组织分化来说，需要FACS以下诱导激活素A.非内胚层细胞中6天胚常表达多能性标志物Oct4和如果支架播种将继续没有组织和规范的增殖，以丰富的定型内胚层肺血统。接种密度也可根据细胞系特异性实现支架完整recellularization调整。 该协议的一个限制是它无力促进分化肺泡上皮细胞。虽然NKX七天后在支架培养的检测2-1 + / SOX9 +远侧祖细胞，这种人口不存在在第14天，以及第21天成熟型的标记我肺泡上皮细胞（AQP5）和II型肺泡上皮细胞（SFTPB，SFTPC）不会在支架培养过程中的任何阶段检测到。这可能是由于额外的生长因子的补充和浸没培养条件，以促进牙槽骨谱系规范的要求，而如这里描述的，基质蛋白和脱细胞支架的残余基质结合生长因子是足以促进呼吸道谱系分化。 促进定型内胚层分化为用气道支架肺上皮细胞的关键步骤是脱细胞过程的优化。细胞成分必须同时基质组分被保留完全除去。利用DAPI对核材料组织染色，超结构用扫描和透射电子显微镜，和DNA检测支架乌拉尔分析可以用来确认除去所有的细胞成分。支架的细胞外基质蛋白成分可以用IF染色和进行评估基质蛋白免疫印迹分析:胶原蛋白I，IV型胶原，弹性蛋白，纤连蛋白，硫酸肝素蛋白聚糖，和层粘连蛋白16。 这些干细胞衍生的肺上皮细胞可在气道相关的病理的疾病建模和药物发现平台，例如囊性纤维化被使用。在再生应用，例如组织修复和细胞治疗这些细胞-基质构建体的潜力可通过在不同的小鼠模型改造构建体在体内移植进行检查。此外，这款3D 在气道分化体外方法可以很容易地适应通过操纵生长因子的可用性和细胞基质信号传导研究各种参数，影响肺的血统规范的过程中对支架分化的不同阶段培养物。

多能细胞对肺谱系定向分化是依赖于微环境1,2-精确信号事件。由于此过程的动态本质，已经挑战模仿肺器官的准确事件体外 。最近的报道已经用于与二维培养物的可溶性生长因子补充分步进行谱系限制策略，实现肺分化3-8。在步分化方案，多能细胞，是否胚胎干细胞（ESC）或诱导的多能干细胞，首先分化为定形内胚层胚层。内胚层细胞随后推到前部内胚层的命运和其后肺祖细胞，所确定包含同源结构域转录因子NKX2-1的表达。这些肺祖细胞进一步分化近端（气）或远端（肺泡）肺上皮细胞的Wi日继续生长因子的补充。这种2维策略曾在产生肺上皮细胞取得了一些成功，但也有一些限制，包括不明效率，从其它内胚层谱系可能的污染，缺少一个3维（3D）结构，并且在某些情况下使用未定义培养物血清补充。上脱细胞肺支架多能或分化的细胞的培养日益用作测定，以评估在形成肺上皮结构3,5,6,8,9接种的细胞的再生潜能。这些报告接种文化与持续生长因子或血清补充支架的细胞。 龙开发涉及的划分，迁移，基因响应环境线索的表达和个体细胞的分化。细胞外基质（ECM）是糖蛋白的一个格子，除了提供结构支撑，引导Tissu酒店通过整合和调节这些过程10,11ê形态。通过使用肺ECM支架作为天然平台内胚层培养，以更好地模仿体内肺发育环境，我们已经产生干以限定三维培养细胞来源的呼吸道上皮细胞具有高效率和再现性设置。 由脱细胞以及小鼠ESC衍生的内胚层细胞产生的大鼠肺的ECM支架生成并随后接种到这些支架。 CXCR4和C-KIT蛋白的表达双重指示定型内胚层细胞的身份和阳性细胞都SOX2和NKX2-1的表达被确定为气道（近端肺）祖细胞。定形内胚层细胞在空气液体界面（ALI）长达三周生成官能呼吸道上皮细胞在体外进行培养。 该协议促进defini肺系分化略去内胚层早7天，用NKX2-1 + / + SOX2早期肺癌近祖的出现观察。第14天，文化成熟的气道上皮细胞群的21冒出包括纤毛（TUBB4A +），俱乐部（SCGB1A1 +），和基底（TRP63 +，KRT5 +）细胞的形态和功能相似本地鼠标气道。这个协议说明了3D-基质微环境的实现健壮分化到呼吸道上皮细胞的重要性。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Perfusion solution</td><td>Sigma</td><td>H0777</td><td>10 U/ml heparin</td></tr><tr><td>Perfusion solution</td><td>Gibco</td><td>14170112</td><td>dissolved in Hank&#039;s balanced salt solution (HBSS-)</td></tr><tr><td>Decellularization solution</td><td>BioShop</td><td>CHA003</td><td>8&amp;nbsp;mM CHAPS</td></tr><tr><td>Decellularization solution</td><td>Sigma</td><td>E9884</td><td>25&amp;nbsp;mM EDTA</td></tr><tr><td>Decellularization solution</td><td>BioShop</td><td>SOD002</td><td>1&amp;nbsp;M NaCl</td></tr><tr><td>Decellularization solution</td><td>Gibco</td><td>14190-144</td><td>dissolved in PBS</td></tr><tr><td>Benzonase nuclease</td><td>Novagen</td><td>70664-3</td><td>90&amp;nbsp;U/ml Benzonase nuclease&amp;nbsp;</td></tr><tr><td>Benzonase nuclease</td><td>Gibco</td><td>14190-144</td><td>diluted in PBS</td></tr><tr><td>Antimicrobial solution&amp;nbsp;</td><td>Gibco</td><td>15140</td><td>200&amp;nbsp;U/ml penicillin streptomycin</td></tr><tr><td>Antimicrobial solution&amp;nbsp;</td><td>Gibco</td><td>15290</td><td>25&amp;nbsp;&amp;mu;g/ml amphotericin B</td></tr><tr><td>Antimicrobial solution&amp;nbsp;</td><td>Gibco</td><td>14190-144</td><td>diluted in PBS</td></tr><tr><td>Trypsinization&amp;nbsp;</td><td>Gibco</td><td>12605-028</td><td>TrypLE</td></tr><tr><td>Serum free differentiation media (SFDM)</td><td>Gibco</td><td>IMDM 2440-053, F12 11765-054</td><td>3:1 ratio of IMDM and Ham&amp;rsquo;s modified F12 medium</td></tr><tr><td>Serum free differentiation media (SFDM)</td><td>Gibco</td><td>12587-010</td><td>B27 supplement (50x dilution)</td></tr><tr><td>Serum free differentiation media (SFDM)</td><td>Gibco</td><td>17502-048</td><td>N2 supplement (100x dilution)</td></tr><tr><td>Serum free differentiation media (SFDM)</td><td>Gibco</td><td>15260-037</td><td>0.05% (Fraction V) bovine serum albumin</td></tr><tr><td>Serum free differentiation media (SFDM)</td><td>Gibco</td><td>35050-061</td><td>200&amp;nbsp;mM Glutamax</td></tr><tr><td>Serum free differentiation media (SFDM)</td><td>Sigma</td><td>M6145</td><td>4&amp;nbsp;&amp;mu;M monothioglycerol</td></tr><tr><td>Serum free differentiation media (SFDM)</td><td>Sigma</td><td>A4403&amp;nbsp;</td><td>0.05&amp;nbsp;mg/ml ascobic acid</td></tr><tr><td>Endoderm induction</td><td>R&amp;amp;D</td><td>338-AC/CF</td><td>Activin A</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CDH1</td><td>BD Biosciences</td><td>610181</td><td>Mouse, non-conjugated, 1:100</td></tr><tr><td>C-KIT</td><td>BD Biosciences</td><td>558163</td><td>Rat, PE-Cy7, 1:100</td></tr><tr><td>CXCR4</td><td>BD Biosciences</td><td>558644</td><td>Rat, APC, 1:100</td></tr><tr><td>KRT5</td><td>Abcam</td><td>ab24647</td><td>Rabbit, non-conjugated, 1:1,000</td></tr><tr><td>NKX2-1</td><td>Abcam</td><td>ab76013</td><td>Rabbit, non-conjugated, 1:200</td></tr><tr><td>Laminin</td><td>Novus Biologicals</td><td>NB300-144</td><td>Rabbit, non-conjugated, 1:200</td></tr><tr><td>SCGB1A1</td><td>Santa Cruz</td><td>sc-9772&amp;nbsp;</td><td>Goat, non-conjugated, 1:1,000</td></tr><tr><td>SOX2</td><td>R&amp;amp;D Systems</td><td>AF2018&amp;nbsp;</td><td>Goat, non-conjugated, 1:400</td></tr><tr><td>TRP63</td><td>Santa Cruz</td><td>sc-8431</td><td>Mouse, non-conjugated, 1:200</td></tr><tr><td>TUBB4A</td><td>BioGenex</td><td>MU178-UC</td><td>Mouse, non-conjugated, 1:500</td></tr><tr><td>Goat IgG&amp;nbsp;</td><td>Invitrogen</td><td>A-11055</td><td>Donkey, Alexa Fluor 488, 1:200</td></tr><tr><td>Mouse IgG&amp;nbsp;</td><td>Invitrogen</td><td>A-21202</td><td>Donkey, Alexa Fluor 488, 1:200</td></tr><tr><td>Mouse IgG&amp;nbsp;</td><td>Invitrogen</td><td>A-31571</td><td>Donkey, Alexa Fluor 647, 1:200</td></tr><tr><td>Rabbit IgG</td><td>Invitrogen</td><td>A-21206</td><td>Donkey, Alexa Fluor 488, 1:200</td></tr><tr><td>Rabbit IgG&amp;nbsp;</td><td>Invitrogen</td><td>A-31573</td><td>Donkey, Alexa Fluor 647, 1:200</td></tr><tr><td>&lt;strong&gt;Other Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Low adherent plates</td><td>Nunc</td><td>Z721050&amp;nbsp;</td><td>Low cell binding plates,&amp;nbsp; 6 wells&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Air-liquid interface membranes&amp;nbsp;</td><td>Whatman</td><td>110614</td><td>Hydrophobic Nucleopore membrane, 8&amp;nbsp;&amp;mu;m pore size</td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1200S&amp;nbsp;</td><td>Leica Vibratome</td></tr><tr><td>Tissue Adhesive</td><td>Ted Pella</td><td>10033</td><td>Pelco tissue adhesive</td></tr></tbody></table>

generation of esc-derived mouse airway epithelial cells using decellularized lung scaffolds

肺谱系分化需要复杂的环境线索，包括生长因子信号传导，细胞 - 细胞相互作用和细胞 - 基质相互作用的集成。由于这种复杂性， 在体外肺发育的重演，促进干细胞肺上皮细胞的分化已经挑战。在这个协议中，脱细胞的肺支架被用来模仿肺的3维环境，并产生干细胞衍生的呼吸道上皮细胞。小鼠胚胎干细胞首先分化为使用具有激活素A.内胚层细胞的胚状体（EB）培养方法的内胚层谱系，然后接种于脱细胞的支架，并在长达21天的空气 - 液体界面中培养。这种技术促进无需额外生长因子的补充接种细胞功能的气道上皮细胞（纤毛细胞，俱乐部细胞和基底细胞）分化。这种文化建立的定义，塞鲁M-免费，价格低廉，重现性好。虽然有有限污染来自非肺胚层谱系中培养，该协议仅产生呼吸道上皮人群和不引起肺泡上皮细胞。与此协议产生的气道上皮细胞可用于在肺器官发生和疾病建模或气道相关的疾病，如囊性纤维化的药物发现平台来研究细胞 - 基质相互作用。

在本协议所述，定型内胚层分化稳健成熟的气道可以用脱细胞上的脚手架肺切片种子细胞的扩展培养来实现上皮细胞。建议将脱细胞支架的特征在于，以确保（1）的宿主细胞被完全除去，和（2）的细胞外基质蛋白在使用前支架分化保留。脱细胞可以通过组织染色用苏木精和曙红（H＆E）和4&#39;，6-二脒基-2-苯基吲哚（DAPI）进行评估，如在图1A中所示。高倍率扫描电子显微镜（SEM）的支架的分析证实了不存在的宿主细胞和完整矩阵的体系结构，如在图1B中示出。通过拉伸试验和免疫染色对基质蛋白质的其余支架的附加特征可以被进行，以确保preservat以下16脱细胞ECM的离子。 在SFDM媒体胚胎干细胞六天结果， 如图2A所示的细胞聚集体（EBS）的形成的非贴壁培养。三天激活素的定型内胚层诱导的处理结果。 ESC的谱系限制定形内胚层可通过上调和内胚层谱系相关基因和蛋白17的表达的确认，并且不能形态断言。内胚层诱导效率从50-70％，取决于使用的ESC线的范围内。实验是在小鼠ESC线进行:R 1（Nkx2-1 mCherry），G4（DsRed的 -MST），和129 /奥拉（ 百瑞GFP / Foxa2的-hCD4），而在该原稿示出的所有数据使用R 1单元线。 如图2B所示的双阳性CXCR4 + / C-KIT +定形内胚层人口进行排序，接种到三维肺支架，以及用于在SFDM空气 - 液体界面21天培养。它进行排序和富集定形内胚层为限于组织和分化与未分选的细胞的培养从异质日达到6的EB， 如图2C所示是很重要的。排序细胞支架上形成的结构让人联想开发， 如图2C-D所示肺（13.5胚胎天）。根据我们的经验，10分选的细胞/支架的接种密度已经取得了最好的支架复育。 分化为肺宗族观察如下脚手架文化早7天。使用如图3A所示的IF共聚焦分析被检测气道正用于NKX2-1和SOX2上皮的祖细胞。 SOX2阳性，NKX2-1阴性细胞是已不分化至肺里可能多能内胚层细胞neage。对于较长的培养这些细胞可能开始表达NKX2-1或凋亡，而不在微足够的支持。中频第21天培养物的分析显示， 如图3B所示包含TUBB4A +纤毛细胞，SCGB1A1 +俱乐部细胞和TRP63 + / KRT5 +基底细胞成熟呼吸道上皮人口。第21天培养物的表面的SEM分析表明干细胞衍生的培养物，以小鼠气道的形态相似。 当与定形内胚层接种并在相同条件下培养上面16所描述的两个分离的基质蛋白（胶原蛋白I，胶原蛋白IV，纤连蛋白，层粘连蛋白）和脱细胞的大鼠肾支架无法促进肺谱系分化。这表明，肺源性支架创建的3D微环境在其推动seede肺系分化能力，从肾的支架不同ð内胚层细胞。 <img alt="图1" src="/files/ftp_upload/54019/54019fig1.jpg" /> 图1.脱细胞技术除去细胞成分和保留的ECM。（A）的H＆E（顶部图）和DAPI（下图）的自然和脱细胞的肺组织染色示出以下脱细胞没有核的。右图显示，而不去细胞最优从肺组织切片的例子。箭头显示在支架由于不完整的脱细胞的细胞核残留，突出recellularization前优化技术的重要性。比例尺= 25微米。天然的和脱细胞肺的（B）的SEM图像，示出了不存在的细胞和对脱细胞支架矩阵结构的保存。比例尺= 10微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54019/54019fig1large.jpg" target="_blank">请点击此处</a>查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54019/54019fig2.jpg" /> 图2.排序ESC衍生定型内胚层导致最佳recellularization。下面激活素A对促进定型内胚层分化治疗三天（A）日6胚状体（EBS）。 （B）中的胚是通过FACS对表面标记的CXCR4双重表达和c-KIT排序以分离定形内胚层的人口。这个人口将脱细胞支架接种。 （C）H＆E recellularized构架中的7天和培养21天时染色。左图代表组织，气管状结构在与排序细胞播种，右面板代表了支架突出recellularization之前细胞分选的重要性排列紊乱，高度增殖的细胞无序。规模巴= 30微米。 （D）H＆E胚胎13.5天小鼠肺的染色。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54019/54019fig2large.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/54019/54019fig3.jpg" /> 以下对脱细胞支架7天培养的定形内胚层的天然肺支架图3.扩展培养定向分化为呼吸道上皮细胞。（A）的近端肺祖细胞（NKX2-1 + / SOX2 +）进行检测。比例尺= 15微米。 （B）左图显示成熟的假复层基底膜层粘连蛋白的蛋白质结构上的基底侧内衬上皮细胞（CDH1 +）的结构。中心和右侧图像显示气道成熟上皮完整的纤毛细胞（TUBB4A +），CLUB细胞（SCGB1A1 +）和基底细胞（KRT5 + / TRP63 +）。左边的图像比例尺= 30微米;中间和右侧图像比例尺= 15微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54019/54019fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds at JoVE.com

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成

该协议有效地引导小鼠胚胎干细胞衍生定形内胚层成熟呼吸道上皮细胞。这种分化技术使用的3维脱细胞肺支架直接肺谱系说明书中，在一个确定的，无血清培养的设置。

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

generation-esc-derived-mouse-airway-epithelial-cells-using

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.6K 次观看.  Hospital for Sick Children. 该协议有效地引导小鼠胚胎干细胞衍生定形内胚层成熟呼吸道上皮细胞。这种分化技术使用的3维脱细胞肺支架直接肺谱系说明书中，在一个确定的，无血清培养的设置。 

视频: Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

龙工程的程序

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

科研

生物工程

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

非人灵长类龙脱细胞和Recellularization使用专门的大型器官生物反应器

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Decellularization of the Murine Cardiopulmonary Complex

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and composition. It is based on a microsurgical procedure designed to catheterize the trachea and aorta of a euthanized mouse to perfuse the heart and lungs with decellularizing agents. The decellularized cardiopulmonary complex can subsequently be immunostained to reveal the location of structural ECM proteins. The whole procedure can be completed in 4 days.
The ECM scaffolds resulting from this protocol are free of dimensional distortions. The absence of cells enables structural examination of ECM structures down to submicron resolution in 3D. This protocol can be applied to healthy and diseased tissue from mice as young as 4-weeks old, including mouse models of fibrosis and cancer, opening the way to determine ECM remodeling associated with cardiopulmonary disease.

This protocol aims to decellularize the heart and lungs of mice. The resulting extracellular matrix (ECM) scaffolds can be immunostained and imaged to map the location and topology of their components.

鼠心肺复合物的脱细胞化

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and ...

癌症研究

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

小鼠胚胎的龙文化，一个系统来评价分枝的分子机制

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

生物学

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research.
Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells.
Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-&#223; and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects.
Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures.

从人多能干细胞中产生气道上皮细胞气液界面培养物

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major ...

发育生物学

<meta charset="utf8"></head><body>用于研究肺屏障完整性、疾病和药物发现的气道模型

iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the nasopharynx to the smallest alveolar sac. Interactions between these cells are crucial in lung development and disease, acting as a barrier against harmful chemicals and pathogens. Current in vitro co-culture models utilize immortalized cell lines with different biological backgrounds, which may not accurately represent the cellular milieu or interactions of the lung. We differentiated human iPSCs into 3D lung organoids (containing both epithelium and mesenchyme), endothelial cells, and macrophages. These were co-cultured in an air-liquid interface (ALI) format to form an epithelial/mesenchymal apical barrier invested with macrophages and a basolateral endothelial barrier (iAirway). iPSC-derived iAirways showed a reduction in barrier integrity in response to infection with respiratory viruses and cigarette toxins. This multi-lineage lung co-culture system provides a platform for studying cellular interactions, signaling pathways, and molecular mechanisms underlying lung development, homeostasis, and disease progression. iAirways closely mimic human physiology and cellular interactions, can be generated from patient-derived iPSC&#39;s, and can be customized to include different cell types of the airway. Overall, iPSC-derived iAirway models offer a versatile and powerful tool for studying barrier integrity to better understand genetic drivers for disease, pathogen response, immune regulation, and drug discovery or repurposing in vitro, with the potential to advance our understanding and treatment of airway diseases.

<meta charset="utf8"></head><body>作者聚焦:使用同基因上皮细胞、内皮细胞和免疫细胞对人类气道重塑和病毒反应进行建模

This article describes the generation of a complex, multi-cellular airway barrier model composed of induced pluripotent stem cell (iPSC)-derived lung epithelium, mesenchyme, endothelial cells, and macrophages in an air-liquid interface culture.

iPSC 衍生的上皮细胞、间充质细胞、内皮细胞和免疫细胞共培养，以模拟肺部健康和疾病中的气道屏障完整性

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

由去细胞化肺切片制备的工程肺组织

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in regenerative medicine and bioreactor technologies offer opportunities to produce lab-grown functional tissue and organ constructs that can be used to screen drugs, model disease, and engineer tissue replacements. Here, a miniaturized bioreactor coupled with an imaging modality that allows in situ visualization of the inner lumen of explanted rat trachea during in vitro tissue manipulation and&#160;culture is described. Using this bioreactor, the protocol demonstrates imaging-guided selective removal of endogenous cellular components while preserving the intrinsic biochemical features and ultrastructure of the airway tissue matrix. Furthermore, the delivery, uniform distribution, and subsequent prolonged culture of exogenous cells on the decellularized airway lumen with optical monitoring in situ are shown. The results highlight that the imaging-guided bioreactor can potentially be used to facilitate the generation of functional in vitro airway tissues.

The protocol describes an imaging-enabled bioreactor that allows the selective removal of the endogenous epithelium from the rat trachea and homogenous distribution of exogenous cells on the lumen surface, followed by long-term in vitro culture of the cell-tissue construct.

成像引导的生物反应器，用于生成生物工程气道组织

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in ...

用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成

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此视频中的章节

龙工程的程序

非人灵长类龙脱细胞和Recellularization使用专门的大型器官生物反应器

鼠心肺复合物的脱细胞化

小鼠胚胎的龙文化，一个系统来评价分枝的分子机制

从人多能干细胞中产生气道上皮细胞气液界面培养物

iPSC 衍生的上皮细胞、间充质细胞、内皮细胞和免疫细胞共培养，以模拟肺部健康和疾病中的气道屏障完整性

iPSC 衍生的上皮细胞、间充质细胞、内皮细胞和免疫细胞共培养，以模拟肺部健康和疾病中的气道屏障完整性

iPSC 衍生的上皮细胞、间充质细胞、内皮细胞和免疫细胞共培养，以模拟肺部健康和疾病中的气道屏障完整性

由去细胞化肺切片制备的工程肺组织

成像引导的生物反应器，用于生成生物工程气道组织