Name: sirna transfection and emsa analyses on freshly isolated human villous cytotrophoblasts
Uploaded: 2016-09-20T08:00:00.000+00:00
Duration: 9 min 57 s
Description: Watch this Scientific Journal Video about siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts at JoVE.com

这项工作是由加拿大国家科学与工程研究理事会（NSERC）（＃298527）（BB）的资助。 CT是由一个机构FARE奖学金支持。 AV是由NSERC·格雷厄姆·贝尔博士支持奖学金。 BB召开了加拿大研究主席在人类反转录病毒学（第2层）。由于碧翠丝Beisner在修改文本的帮助。

该UQAM伦理委员会已经批准了这些协议，这是按照大学中心医院，蒙特利尔的圣 - 吕克医院（蒙特利尔，加拿大）的伦理委员会的指导方针。与会者签署了知情同意书。 1.介质制备工艺和主代绒毛滋养细胞的分离<ol><li>通过补充的Dulbecco氏改良的Eagle氏培养基（DMEM）用25mM HEPES，10％胎牛血清（FBS）和1％青霉素/链霉素制备用于人原绒毛细胞滋养细胞的培养基。过滤通过无菌0.22微米的过滤器，并存储所制备的介质在-20℃在50毫升分装℃冰箱。 </li><li>隔离从新鲜胎盘以下标准协议3个主要代绒毛滋养细胞。 <ol><li>用钳子，轻轻挠它关闭删除该组织胎膜，同时小心不要去除绒毛组织。切在使用手术刀和剪刀约3×3厘米的立方体剩余的胎盘绒毛。彻底含0.9％的NaCl除去母体血液清水洗净。 <ol><li>持用钳子每个立方体，小心翼翼地从剩下的血管和纤维组织用剪刀去除绒毛组织。 </li><li>为15至30毫克的绒毛组织，消化四倍的Hank氏平衡盐溶液（HBSS）包含胰蛋白酶（从9.6×10 5〜1.8×10 6 U;终浓度为1.2mg / ml）和脱氧核糖核酸酶I（DNA酶I）（最终浓度200微克/毫升），用于在连续摇动的水浴在37℃下30分钟。 注意:使用150毫升总体积为第一消化，将100ml用于第二消化和75ml对剩下的两个消化。 </li></ol></li><li>收集含有分散细胞，离心上清在1250 XG为不带刹车15分钟。在1毫升预热DMEM含有去除上清和悬浮细胞1％青霉素/链霉素。 </li><li>层上的不连续的5％的顶部的分散的细胞- 70％聚乙烯吡咯烷酮包覆二氧化硅梯度（见梯度制备表1）和离心机在507 xg离心23分钟，不制动。通过抽吸除去1.017之间的密度层以1.033和收集40％和50％之间的各层中使用新的吸管的梯度。用10ml预热的DMEM含有1％青霉素/链霉素洗涤细胞广泛。 注意:将收集的馏分对应密度1.048 1.062和，其中港口之间的滋养层。 </li><li>算使用血球细胞。简单地说，混合细胞，并在新的离心管中加入10微升。加10μl台盼蓝并轻轻地再次混合。绘制出细胞悬液，并填补了血细胞计数室。算下10X目标细胞。 </li><li>放置1×10 6和4.5×10 6细胞到用于分离的试管中的部分1.3和2中，各自LY。种子的剩余的细胞以1.5×10 6个细胞/孔在补充的DMEM培养基和培养物的密度在37℃和5％CO 2的最多4天的其它实验。 </li></ol></li><li>孤立的主要代绒毛滋养细胞的纯度评价<ol><li>自旋为1×10的离心管在300×克新鲜分离的细胞滋养细胞的6。弃上清，加入1ml预热的磷酸盐缓冲盐水（PBS）的。离心机的细胞在300 XG，并通过抽吸去除PBS。 </li><li> 1毫升冷甲醇添加到细胞中并在-20℃下孵育20分钟。离心细胞，在300×g离心5分钟，并通过抽吸除去甲醇。 注意:使用面膜罐，护目镜和橡胶手套在处理甲醇。 </li><li>加入1毫升的PBS在室温下再水合的细胞，离心分离，在300 xg离心并通过抽吸除去PBS中。 </li><li>孵育含有FBS（1:50 [体积/体积]稀释）和一个的FcR的细胞在PBS中在室温下阻断45分钟试剂（1:10），以消除非特异性结合。 </li><li>用500μl室温的PBS，离心细胞洗两次，在300×g离心5分钟，并通过抽吸除去PBS中。悬浮细胞在100μl室温PBS中。 </li><li>孵育细胞与小鼠单克隆异硫氰酸荧光素（FITC）缀合的抗人细胞角蛋白-7抗体克隆LP5K（1:200）或在PBS中的对照同种型匹配的非特异性抗体含有0.2％BSA的45分钟，在室温下黑暗。洗涤细胞在PBS中。 </li><li>通过流式细胞术3分析细胞。使用细胞制剂，其通过流式细胞术进一步的实验表明最低96％的CK-7阳性细胞。 </li></ol></li></ol> 2.人力小学代绒毛滋养细胞的转染<ol><li>使用微孔设备的人原代绒毛滋养细胞的转染<ol><li>制备含有6孔板2ml中微穿孔细胞的培养补充的DMEM培养基中。 </li><li>主细胞滋养细胞（见1.2节）的分离后，取细胞的等分试样中的悬浮液，并使用血细胞计数器确定细胞数。 </li><li>在300 XG转移1.5×10 6细胞到微量离心管中并离心沉淀细胞，在室温下5分钟。清洗细胞用1ml的Dulbecco PBS（DPBS）（Mg 2+离子 ，无Ca 2+）中并在300 xg离心离心沉淀细胞，在室温下5分钟。 </li><li>除去上清液并轻轻重悬细胞于100μl重悬浮缓冲液中。 注意:避免保持细胞悬浮液，在室温下超过15-30分钟，以最大限度地提高细胞生存力和转染效率。 </li><li>加合胞素-2的siRNA（或控制加扰的siRNA）的300毫微克至1.5 ml离心管，并用100微升转染吸管吸出细胞的siRNA混合物。插吸管入站（见材料清单）和邮件主题单元为30毫秒的1300 V单脉冲。 </li><li>立即将细胞转移到含有预热的37℃补充的培养基，以避免细胞损伤的6孔板（在步骤2.1.1的方法制备）。 </li><li>重复步骤其余样品2.1.5和2.1.6。 </li><li>轻轻摇动平板30秒，以确保即使在细胞的分布。孵育在37℃的板在加湿的 CO 2培养箱中培养24至48小时。刷新培养基在转染后24小时。 </li><li>通过评估Western blot分析转染效率（参见步骤3到6）。 </li></ol></li><li>在人类细胞滋养细胞主要的siRNA脂质体<ol><li>绒毛细胞滋养细胞的分离后，种子的细胞以在2ml培养基中每孔1.5×10 6个细胞在6孔板的密度，孵育18小时。 </li><li>稀释合胞-2特异性siRNA的300纳克（或控制加扰RNA干扰）和5μl基于脂质的转染试剂的单独1.5毫升管中的10微升抗生素和无血清培养基的总体积。孵育5分钟。 </li><li>混合经稀释的试剂，并在室温下孵育另外的20分钟，以形成转染复合物。添加90微升抗生素和无血清培养基的到混合至100μl的终体积。 </li><li>从6孔板（步骤2.2.1）除去培养基。加入2毫升的新鲜培养基中，并在100微升转染混合物到每个孔中。孵育在37℃下将细胞在CO 2培养箱中24至48小时。刷新培养基在转染后24小时。 </li><li>通过评估Western blot分析转染效率（参见步骤3到6）。 </li></ol></li></ol>从全细胞裂解液培养3.制备<ol><li>取出介质和冲洗每口井（从步骤2.1.8和2.2.4）采用预热DPBS（镁离子 ，钙离子免费）。吸出DPBS，加入500微升的0.25％胰蛋白酶/乙二胺四乙酸（EDTA）。在CO 2培养箱中孵育1-3分钟，在37℃。通过加入500微升含血清的生长培养基的停止胰蛋白酶处理。 注:按瓶/皿的类型使用适当的介质卷:1毫升，每107 个细胞/ 100mm皿/ 150 平方厘米瓶; 0.5毫升每5×10 6个细胞/ 60mm培养皿/ 75cm 2的烧瓶中。 </li><li>转移细胞到1.5ml微量离心管中，沉淀细胞，在300×g离心在室温下2分钟离心。离心300 xg离心清洗细胞用DPBS（镁离子 ，无Ca 2+）和沉淀的细胞在室温下2分钟。 </li><li>小心取出上清液，而不会干扰颗粒。放置在冰上的管中。重悬沉淀50-200微升冰冷无线电免疫沉淀测定（RIPA）缓冲液（ 见表2缓冲组合物）含有新鲜添加编蛋白酶和磷酸酶抑制剂在1倍终浓度。简单地说，涡管，在冰上留下30分钟。 </li><li>离心16,000 XG 20分钟，在4℃。小心地将管冰上。使用移液管，将上清液转移到新鲜的预冷却的离心管保持在冰上。弃沉淀。 </li></ol>从培养细胞的核提取物的制备4 <ol><li>每口井（步骤1.2.5），洗细胞滋养细胞两次用1毫升预热DPBS（镁离子 ，钙离子免费）的。吸DPBS，加入500微升0.25％胰蛋白酶/ EDTA的。在CO 2培养箱中孵育1-3分钟，在37℃，并通过加入500微升含血清的生长培养基的停止胰蛋白酶处理。 </li><li>转移细胞到1.5ml微量离心管中，沉淀细胞，在300×g离心在室温下2分钟离心。通过离心洗细胞用DPBS（镁离子 ，无Ca 2+）和颗粒细胞在300×g离心在室温下2分钟。仔细清除所有上清而不破坏颗粒，然后将管冰上。 </li><li>根据制造商的说明提取核蛋白。确定使用蛋白定量测定（布拉德福德或双金鸡宁酸测定法（BCA））各样品的蛋白质浓度。 </li></ol> 5.样品制备和电泳<ol><li>确定用于使用蛋白质定量测定各细胞提取物中的蛋白质浓度（例如，布拉德福德8或BCA 9）。 </li><li>根据制造商的说明（如果抗体可在还原和变性条件下使用），2×Laemmli样品缓冲液（ 表2）的等体积的增加总蛋白的20-30微克。 </li><li>煮沸细胞溶胞产物在100℃下5分钟。等分试样50-100微升裂解物，以避免重复的冷冻/解冻循环和剩余的管储存于-20℃以备将来使用的。 </LI&gt; <li>而将样品加热，制备1升1倍的运行缓冲液（ 表2）。 </li><li>旋转样品以16,000 xg离心几秒钟，将它们放置在冰上。 </li><li>加载蛋白质的等量成12％的SDS-PAGE凝胶上（ 见表2为凝胶组合物）的各孔中，用分子量标记一起。在100 V.运行1凝胶2小时</li></ol> 6.从凝胶转移的蛋白质的膜<ol><li>激活用甲醇聚偏二氟乙烯（PVDF）膜1分钟，并用1×转移缓冲液/ 10％甲醇溶液冲洗。根据制造商的说明，进行30分钟依赖于蛋白质大小转移至1小时。 注意:转印效率可使用丽春红染色的封闭步骤之前进行评估。 </li></ol> 7.抗体染色<ol><li>在室温下1小时或过夜，在4℃下，用5％封闭溶液阻断膜。 </li><li> 6或用抗甘油醛-3-磷酸脱氢酶（GAPDH; 0.4微克/毫升; 1:500），在5％封闭溶液过夜:用抗合胞素-2抗体（5000 1 4微克/毫升）孵育膜4℃（抗合胞-2）或在室温下45分钟。三次洗膜在Tris缓冲盐水吐温（TBST; 见表2缓冲组合物）5分钟。 </li><li>孵育适当辣根过氧化物酶（HRP）的膜缀合的抗兔或抗小鼠抗体（1:10,000），在5％在室温下1小时阻断在TBST缓冲液中。洗膜在TBST中3次5分钟，然后在TBS中漂洗。检测使用基于化学发光印迹底信号。 </li></ol> 8.单链寡核苷酸的放射性标记<ol><li>磷酸化反应<ol><li>在微量离心管中，加入50毫微克的正向寡核苷酸一起用1×T4多核苷酸激酶缓冲液，1UT4多核苷酸激酶和20微居里（γ-32 P）ATP和弥补无核酸酶的水反应体积为20微升。 </li><li>孵育在37℃下30分钟。停止加入5微升的0.5M EDTA的反应。补足体积至50μl无核酸酶的水。 </li></ol></li><li>由寡核苷酸未掺入的核苷酸的去除<ol><li>制备具有下列制造商的说明TE缓冲液（ 表2）平衡过的G-25柱。放置在一个新鲜的无DNase的1.5 ml离心管色谱柱。 </li><li>慢慢加入探针（从步骤8.1.2）的50微升过树脂，小心不要去打扰树脂床。自旋2分钟，在350 xg离心收集在1.5 ml离心管中纯化的样品。在350 xg离心洗用36微升不含核酸酶的水和自旋的G-25柱2分钟以收集它在离心管中。 </li></ol></li><li>杂交用互补链醇igonucleotide <ol><li>放射性标记的探针（86微升）转移到1.5ml微量离心管中，并添加反向寡核苷酸的200毫微克与退火缓冲液的1倍（ 见表2）。热，在95℃5分钟并在室温下留下过夜冷却。 </li><li>通过向前加入50毫微克的未标记的制备非放射性的双链寡核苷酸和反向寡核苷酸以及退火缓冲液1×在1.5ml微量离心管中。补足体积至100μl无核酸酶的水。热火并保持在室温下按步骤8.3.1所述。 </li></ol></li></ol> 9.准备一个非变性聚丙烯酰胺凝胶的EMSA <ol><li>用1.5毫米的空间梳（ 见表2为凝胶组合物）制备的4％非变性凝胶（10×12厘米）。清洁用蒸馏水所有玻璃板。 注意:这是至关重要的板完全不含离子洗涤剂，如SDS。 </li><li>马上p我们在板之间的丙烯酰胺溶液允许聚合进行（约30分钟）。 </li><li>组装电泳装置和填充0.5X TBE坦克。在150V样品装载之前预先进行30分钟的凝胶到1小时。 </li></ol> 10. DNA结合反应<ol><li>在1.5ml微量离心管中，在1×凝胶移位结合缓冲液（ 表2）中添加核提取（步骤4.3）的10微克和补足终体积为10微升用无核酸酶水。作为对照，准备一管有没有核提取物。对于竞争反应，加1μL冷非特异性或双链寡核苷酸（步骤8.3.2）的。 </li><li>从步骤8.3.1到每个反应加入1μl放射性探头。在室温下孵育20分钟进行反应。加入1μl的10倍的凝胶上样缓冲液的每反应和加载部分9中制备的凝胶上的每个样品。 </li><li>在15运行1 1/2凝胶至2小时0 V迁移后，包装在塑料包装和定位胶和玻璃下面荧光屏曝光磁带。在4℃离开盒24小时。 </li><li>从曝光纸盒中取出屏幕，并在扫描荧光成像装置插入。 </li></ol>

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G., Vargas, A., Rassart, E., Barbeau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CRE/AP-1-Like+Motif+Is+Essential+for+Induced+Syncytin-2+Expression+and+Fusion+in+Human+Trophoblast-Like+Model.">A CRE/AP-1-Like Motif Is Essential for Induced Syncytin-2 Expression and Fusion in Human Trophoblast-Like Model.</a> PLoS One. 10, e0121468 (2015).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal. Biochem. 150, 76-85 (1985).</li><li>Blaschitz, A., Weiss, U., Dohr, G., Desoye, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+reaction+patterns+in+first+trimester+placenta:+implications+for+trophoblast+isolation+and+purity+screening.">Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening.</a> Placenta. 21, 733-741 (2000).</li><li>Desforges, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SNAT4+isoform+of+the+system+A+amino+acid+transporter+is+functional+in+human+placental+microvillous+plasma+membrane.">The SNAT4 isoform of the system A amino acid transporter is functional in human placental microvillous plasma membrane.</a> J. Physiol. 587, 61-72 (2009).</li><li>Felgner, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipofection:+a+highly+efficient,+lipid-mediated+DNA-transfection+procedure.">Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.</a> Proc Natl Acad Sci U S A. 84, 7413-7417 (1987).</li><li>Guilbert, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+functional+characterization+of+villous+cytotrophoblasts+free+of+syncytial+fragments.">Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments.</a> Placenta. 23, 175-183 (2002).</li><li>Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+term+human+trophoblast+cells.">Isolation and culture of term human trophoblast cells.</a> Methods Mol. Med. 121, 203-217 (2006).</li><li>Ma, B., Zhang, S., Jiang, H., Zhao, B., Lv, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoplex+morphologies+and+their+influences+on+transfection+efficiency+in+gene+delivery.">Lipoplex morphologies and their influences on transfection efficiency in gene delivery.</a> J. Control. Release. 123, 184-194 (2007).</li><li>Freeley, M., Long, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+siRNA+delivery+to+T-cells:+potential+clinical+applications+for+inflammatory+disease,+cancer+and+infection.">Advances in siRNA delivery to T-cells: potential clinical applications for inflammatory disease, cancer and infection.</a> Biochem. J. 455, 133-147 (2013).</li></ol>

The authors have nothing to disclose.

研究人类胎盘功能和发展已通过旨在优化各种胎盘细胞群的分离方案有了很大的提高。其中最好的研究的胎盘细胞群仍是代绒毛滋养细胞，其研究有很大的优化，从许可协议的高效，可靠的隔离受益。这进一步允许了若干实验，如转染和促进剂的研究。使用先前描述的方案3中 ，可以得到主绒毛细胞滋养细胞的纯群体。 CK-7是一种中间丝蛋白，其在滋养细胞中表达。针对此蛋白的单克隆抗?...

Human placental dysfunction is associated with the development of several pregnancy-associated diseases like preeclampsia and intrauterine growth restriction 1. An important cell constituent of the placenta is the trophoblasts, which can be classified as either extravillous or villous cytotrophoblasts. Upon fusion, villous cytotrophoblasts further differentiate into the syncytiotrophoblast layer, a multinuclear cell structure with an important role in feto-maternal exchange and hormone production 2. Human primary villous cytotrophoblasts and their differentiated counterparts represent important biological samples and allow researchers to study a number of placenta-related processes, such as cell fusion, in culture. Furthermore, substantial efforts are ongoing to identify markers that will facilitate appropriate management and improve preventive therapies specific to pregnancy-related diseases. Laboratories routinely isolate primary human villous cytotrophoblasts from fresh placentas, using a standard isolation procedure based on trypsin digestion of placenta villi 3. As cultured cytotrophoblasts lose their capacity to proliferate and quickly differentiate in a syncytiotrophoblast-like layer upon culture 4, very efficient transfection methods and optimized analysis approaches are needed. Previous studies have determined optimal conditions of transfection of these primary cytotrophoblasts 5. Herein, a different method of siRNA transfection, which has been previously tested in this cell type 6, is presented. In comparison to a lipofection-based approach, this microporation method improves transfection, as assessed by the extent of silencing of specific genes.
Promoter and gene expression studies also provide a better understanding of placental function. Although more difficult to use owing to the short time frame for which primary villous cytotrophoblasts can be cultured, promoter analyses using standard protocols can nonetheless be addressed, as previously published 7. Electrophoretic Mobility Shift Assay (EMSA) is one of these commonly used in vitro methods, allowing for fast and easy monitoring of DNA-protein interactions. Nuclear extracts from these primary trophoblasts were used to test a region of the Syncytin-2 promoter for specific interactions. Results revealed that bound factors could be detected at different time points of culture and in a specific and reproducible manner.
Data presented in this protocol confirm that our transfection approach and the EMSA protocol can be used for isolated primary villous cytotrophoblasts and will be of great use to study the diverse functions of villous cytotrophoblasts in normal or pathological conditions.

<table><tbody><tr><td>HBSS without&amp;nbsp; Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp;</td><td>Sigma</td><td>#H2387</td><td></td></tr><tr><td>HBSS (10x)</td><td>Sigma</td><td>#14060-057</td><td></td></tr><tr><td>DMEM High Glucose without Hepes</td><td>Gibco</td><td>#12100-061</td><td></td></tr><tr><td>Hepes (1 M)</td><td>Gibco</td><td>#15630-080</td><td></td></tr><tr><td>Penicillin-Streptomycin-Neomycin (100x)</td><td>Gibco</td><td>#15640-055</td><td></td></tr><tr><td>Amphotericin B&amp;nbsp;</td><td>Sigma</td><td>#A2411</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>#C4901</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;.&lt;/sup&gt;7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Sigma</td><td>#M</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>#16170-078&amp;nbsp;</td><td></td></tr><tr><td>Percoll&amp;nbsp;</td><td>Sigma</td><td>#P-1644</td><td>For density gradient</td></tr><tr><td>Syncytin-2 siRNA</td><td>Ambion Life technologies</td><td>#AM16708</td><td></td></tr><tr><td>Scrambled siRNA</td><td>Qiagen</td><td># SI03650318</td><td></td></tr><tr><td>DNase I</td><td>Sigma-Aldrich</td><td>#D5025</td><td></td></tr><tr><td>Trypsine, type I&amp;nbsp;</td><td>Sigma</td><td>#T8003</td><td></td></tr><tr><td>DharmaFECT Lipotransfection&amp;nbsp; reagents&amp;nbsp;</td><td>GEhealthcare</td><td># T-2001-01</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Life technologies</td><td>#25300-062</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Roche Diagnostic</td><td>#11873580001</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>#23225</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>#A7906</td><td></td></tr><tr><td>TWEEN 20</td><td>Sigma</td><td>#P9416&amp;nbsp;</td><td></td></tr><tr><td>Anti-rabbit IgG, HRP-linked antibody&amp;nbsp;</td><td>Cell Signaling</td><td>#7074</td><td></td></tr><tr><td>BM Chemiluminescence Western Blotting Substrate (POD)</td><td>Roche Diagnostic</td><td>#11500708001</td><td></td></tr><tr><td>DPBS&amp;nbsp;</td><td>Life technologies</td><td>#14287-080</td><td></td></tr><tr><td>T4 Polynucleotide Kinase</td><td>NEB</td><td>#M0201S</td><td></td></tr><tr><td>ATP, [&amp;gamma;-&lt;sup&gt;32&lt;/sup&gt;P]</td><td>Perkin Elmer</td><td>#BLU002A100UC</td><td></td></tr><tr><td>Acrylmide</td><td>Sigma&amp;nbsp;</td><td>#A9099</td><td></td></tr><tr><td>TEMED</td><td>Life technologies</td><td>#17919</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Sigma&amp;nbsp;</td><td>#A3678</td><td></td></tr><tr><td>Anti-human cytokeratin-7 antibody clone LP5K, FITC conjugated</td><td>Millipore</td><td>CBL194F</td><td>Dilution 1:200</td></tr><tr><td>FcR blocking reagent&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130- 059-901</td><td>Dilution 1:10</td></tr><tr><td>Flow Cytometer BD Acuri system</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Microporator MP-100 apparatus&amp;nbsp;</td><td>Digital Bio</td><td></td><td></td></tr><tr><td>Resuspension Buffer R (Neon Transfection System 100&amp;nbsp;&amp;micro;l Kit)</td><td>Life technologies</td><td>MPK10096</td><td></td></tr><tr><td>PVDF membrane&amp;nbsp;</td><td>Millipore</td><td>IPVH00010&amp;nbsp;</td><td>Activate with methanol</td></tr><tr><td>Anti-human GAPDH antibody</td><td>Santa Cruz Biotechnology</td><td>sc-137179</td><td>1:500</td></tr><tr><td>HorseRadish Peroxidase (HRP)-conjugated goat anti-rabbit antibody or anti-mouse antibody</td><td>Cell Signalling</td><td>#7074</td><td>1:10,000</td></tr><tr><td>HorseRadish Peroxidase (HRP)-conjugated goat anti-mouse antibody</td><td>Cell Signalling</td><td>#7076&amp;nbsp;</td><td>1:10,000</td></tr><tr><td>NE-PER Nuclear and Cytoplasmic Extraction Reagent</td><td>Thermo Scientific</td><td>#78833</td><td></td></tr><tr><td>G-25 column&amp;nbsp;</td><td>GE Healthcare</td><td>#27-5325-01</td><td></td></tr><tr><td>Chemiluminsescence and fluorescence imaging device</td><td>Montr&amp;eacute;al Biotech</td><td>Fusion FX5</td><td></td></tr><tr><td>4% native gel</td><td>Home made</td><td></td><td></td></tr><tr><td>PBS</td><td>Home made</td><td></td><td>1x</td></tr><tr><td>Personal Molecular Imager (PMI) System</td><td>BioRad</td><td></td><td></td></tr></tbody></table>

sirna transfection and emsa analyses on freshly isolated human villous cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

从足月妊娠胎盘新鲜用于分离人主要代绒毛滋养细胞进行的一组中的协议部分提供实验。继他们的孤立，我们首先分析细胞滋养细胞的纯度通过使用角蛋白7标记（ 图1）。使用单克隆抗细胞角蛋白7的抗体的细胞制备物从而染色。 图1表示从以下主要绒毛细胞滋养细胞的纯化典型的实验中，通过标准的流式细胞仪分析的结果。以下的密度梯度步骤，...

Watch this Scientific Journal Video about siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts at JoVE.com

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

这个协议描述了使用微孔化有效转染的siRNA在新鲜分离的人绒毛细胞滋养细胞和鉴定在这些细胞中DNA的蛋白质复合物的方法。转染的细胞可以通过Western印迹进行监测，并在为期4天的培养时间，EMSA分析。

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

sirna-transfection-emsa-analyses-on-freshly-isolated-human-villous

Research

JoVE Journal

Genetics

10.5K 次观看.  University of Quebec in Montreal. 这个协议描述了使用微孔化有效转染的siRNA在新鲜分离的人绒毛细胞滋养细胞和鉴定在这些细胞中DNA的蛋白质复合物的方法。转染的细胞可以通过Western印迹进行监测，并在为期4天的培养时间，EMSA分析。 

视频: siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

人胎盘滋养层细胞培养模型，研究褪黑素对缺氧的保护作用/复氧引起的中断

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density ...

科研

发育生物学

Transcriptional Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells differentiate into primary trophoblast giant cells (TGCs) while the polar trophectoderm overlying the inner cell mass continues to proliferate later differentiating into secondary TGCs. TGCs play a key role in developing placenta and are essential for a successful pregnancy. Investigation of transcriptional regulation of specific genes during post-implantation development can give insights into TGCs development. Cells of the ectoplacental cone (EPC) from embryos at 7-7.5 days of gestation (E7-7.5), derived from the polar trophectoderm, differentiate into secondary TGCs1. TGCs can be studied in situ, on cryostat sections of embryos at E7 although the number of TGCs is very low at this stage. An alternative means of analyzing secondary TGCs is to use short-term cultures of individual EPCs from E7 embryos. We propose a technique to investigate the transcriptional status of genes of interest both in vivo and in vitro at the single-cell level using fluorescent in situ hybridization (RNA FISH) to visualize nascent transcripts. This technique provides a direct readout of gene expression and enables assessment of the chromosomal status of TGCs, which are large endoreplicating cells. Indeed, a key feature of terminal differentiation of TGCs is that they exit the cell cycle and undergo multiple rounds of endoreplication.This approach can be applied to detect expression of any gene expressed from autosomes and/or sex chromosomes and can provide important information into developmental mechanisms as well as placental diseases.

Transcriptional  Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

Trophoblast giant cells (TGCs) play a key role in the placenta to ensure a healthy pregnancy. We present a protocol for assessing the transcriptional status of genes in TGCs by nascent fluorescent in situ hybridization on cryostat sections of post-implantation embryos or short-term cultures of embryonic day 7 ectoplacental cones.

转录分析的新生RNA FISH<em&gt;在体内</em&gt;滋养巨细胞或<em&gt;体外</em&gt; Ectoplacental锥外植体的短期文化

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

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 ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

体外检测评估神仙体头三个月滋养细胞细胞系的迁移、入侵和增殖

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

使用基于流式的方法改善离体胎盘外植体培养

Ex Vivo Placental Explant Flow Culture - Mimicking the Dynamic Conditions In Utero

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models inadequately reflect the dynamic in utero setting, where the placenta encounters constant slight shear stress due to plasma or blood flow. To address this limitation, a flow culture system has been devised to bring ex vivo placental explant cultivation closer to the in utero flow conditions experienced within the maternal body. Within this approach, placental explants are cultivated in a sequence of five interconnected flow chambers. This setting&#160;maintains physiological oxygen concentrations and a consistent flow rate. The collected data reveals that under flow conditions, the preservation of tissue morphology exhibits notable enhancement compared to conventional static methods. This innovative technique introduces a straightforward means of ex vivo placental explant culture, offering a more faithful representation of the dynamic in vivo environment. Moreover, this study introduces new&#160;possibilities for investigating the functional dynamics of the feto-maternal interface. By embracing feasible dynamic methodologies, a deeper comprehension of placental biology is facilitated, underscoring its relevance for maternal-fetal health.

作者聚焦:通过胎盘组织中的单细胞测序和空间组织学推进研究 

Here is a protocol for culturing placental explants under constant flow conditions. This approach enhances traditional static villous culture systems by enabling the replication of dynamic physiological environments.

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models ...

Analyzing Angiogenesis: In Vitro Assay for Tube Formation on BM Matrix

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of ...

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

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人胎盘滋养层细胞培养模型，研究褪黑素对缺氧的保护作用/复氧引起的中断

转录分析的新生RNA FISH

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

在人多能的体外干细胞向滋养层细胞

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

体外检测评估神仙体头三个月滋养细胞细胞系的迁移、入侵和增殖

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis