Name: in vitro differentiation of human pluripotent stem cells into trophoblastic cells
Uploaded: 2017-03-16T12:30:00.000+00:00
Duration: 8 min 21 s
Description: Watch this Scientific Journal Video about In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells at JoVE.com

This work was supported by a Pennsylvania Health Research Formula Fund.

注:对于任何一个人类胚胎干细胞或iPS细胞分化成滋养层祖细胞，生长在小鼠胚胎成纤维细胞（MEF）hPSCs的转变开始分化与BMP4 / A / p之前无饲养了两个通道的条件。这个过程消除了分化细胞的MEF污染。这里，我们提出了人类胚胎干细胞分化的协议，和相同的协议可以被应用到人iPS细胞。 1.文化和辐照小鼠胚胎成纤维细胞的人类胚胎干细胞恢复细胞（MEF）（制剂） <ol><li> 的MEF的γ-辐射 <ol><li>制成500毫升培养的MEF培养基:DMEM含10％FBS，2mM的L-谷氨酰胺，1×青霉素/链霉素（青霉素/链霉素），和0.1mM非必需氨基酸（NEAA）。 </li><li>解冻初级MEFs中的一小瓶，并使用MEF培养基用于培养细胞。展开主MEF中使用MEF培养基30板。 注意:氧浓度没有此步骤至关重要，因此任一培养箱内生理（4％）或环境（20％）的条件下都可以使用。 </li><li>收获MEFS。使用0.25％胰蛋白酶，以从板取下的MEF并将细胞转移到锥形管中。放置的MEF到照射仪器并暴露细胞3500灰色。 注:的时间量将取决于照射单元内的源的活性。 </li><li>悬浮用MEF培养基照射的MEF和计数使用血球细胞的数目。 </li><li>沉淀使用离心机该MEF并添加50％的MEF培养基和50％的细胞冷冻培养基（80％FBS和20％的MEF培养基）。等分试样每瓶1×10 6个细胞。 </li><li>放置在-80℃的冰箱中过夜的辐照MEF小瓶，然后将它们转移到液氮长期贮存。 </li></ol></li><li> 为解冻冷冻文化MEF中与人类胚胎干细胞 <ol><li>使500毫升人类胚胎干细胞培养基:DMEM / F-12，用20％血清Replac键相，0.1毫NEAA，2mM的L-谷氨酰胺，10纳克/ ml的bFGF，0.1毫β-巯基乙醇，和1x青霉素/链霉素。 </li><li>解冻胚胎干细胞解冻前MEF中一天一小瓶。涂覆6孔板有0.1％明胶，用1毫升每孔中。孵育在室温下至少20分钟，然后取出明胶。 </li><li>从存储在液氮中删除的MEF的小瓶中并浸入小瓶在37℃水浴中。观察间歇直到只剩下小冰晶保持小瓶。迅速将小瓶中的内容传送9mL的MEF培养基的15毫升锥形管内。 </li><li>通过在200 xg离心离心5分钟沉淀细胞。吸出上清液并重新悬浮在MEF培养基的细胞沉淀。均匀分装的MEF到6孔板中。放板放入37℃的低氧（4％）培养箱中过夜。 </li></ol></li><li> 在MEF中人类胚胎干细胞的常规培养 <ol><li>从液氮中取出的胚胎干细胞小瓶用3迅速解冻小瓶7℃水浴。轻轻吸取人类胚胎干细胞到含有9毫升人类胚胎干细胞培养基的15毫升锥形管和自旋向下在200 XG 5分钟。除去培养基并用1ml人类胚胎干细胞培养基中重悬细胞。 </li><li>吸从步骤1.2.4准备好的平板的MEF中，加入1 ml人类胚胎干细胞培养基。 ROCK抑制剂Y-27632（10μM）加入到人类胚胎干细胞培养基。转移到人类胚胎干细胞MEF中。 </li><li>放置细胞进入37℃的低氧（4％）培养箱中过夜。每天更换人类胚胎干细胞中。刮去分化的细胞用巴斯德移液管，在4X直立显微镜下观察。 </li><li>通道的人类胚胎干细胞每4-6天，取决于细胞汇合和人类胚胎干细胞集落的大小。一天前传代MEF中准备一盘。当细胞准备通道，取出胚胎干细胞培养基，并用1毫升的PBS洗孔。 </li><li>加入1毫克/毫升胶原酶（预热至37℃），孵育在37℃下5分钟，然后取出胶原酶。 W¯¯灰的细胞用PBS，每孔加入1 ml人类胚胎干细胞培养基中，并手动刮去细胞进入使用5毫升吸管的尖端小团块。转移悬浮细胞团块到一个新的MEF涂覆的孔（多个）。 </li></ol></li></ol> 2.由MEF人类胚胎干细胞的过渡到无饲养的细胞外基质，涂层板条件<ol><li> 准备MEF条件培养基（CM） <ol><li>板在T75烧瓶中的照射的MEF在90-100％汇合;重要的是，该MEF密集电镀。观察用显微镜，以确定它们是否已连接于烧瓶底部的细胞（MEF中附上约6小时的电镀或第二天之后）。 注意:使用显微镜观察时未连接的细胞漂浮在介质中。第二天，除去MEF培养基，并添加25毫升人类胚胎干细胞培养基中缺乏的B-FGF。 </li><li>孵化后24小时收集条件培养基。用新鲜培养基每天最多12天进行更换。 </l我&gt; <li>使用0.22微米过滤器过滤所收集的CM。在使用之前，添加新鲜的B-FGF到CM。 注:CM能够在-80℃冷冻并贮存长达1年。可替代地，在4℃下储存在CM 2周。 </li></ol></li><li> 从转变对文化MEF中的人类胚胎干细胞的馈线自由外涂层矩阵板。 <ol><li>细胞外基质的制备用于涂覆组织培养板<ol><li>解冻的细胞外基质的在冰上的等分试样（约3-4小时）。使用冰冷的提示，一冷，50ml锥形管中，并DMEM / F12培养基，传送2毫克的细胞外基质的成24毫升冰冷的DMEM / F-12（稀释取决于细胞外基质浓度从供应商）。 </li><li>立即锥形管转移50毫升冰和储存于4℃。用于涂覆组织培养板中，稀释细胞外基质的适量添加到孔（例如，将1ml每孔在6孔板）。漩涡涂覆该板，并在室温下孵育至少1小时。 </li></ol></li><li>使用含有B-FGF（10毫微克/毫升）的CM通道的hESCs从该MEF（如在步骤1.3）。 <ol><li>抽吸以从新印版移除细胞外基质并添加含B-FGF的CM。使用移液管从MEF板传送刮下细胞团块，并将其分装到细胞外涂覆基质板。孵育在37℃的低氧培养箱中过夜。 </li><li>更换每日B-FGF介质CM;介质的量将依赖于培养瓶的大小。用2毫升培养基用于一个6孔井。刮去分化的细胞用巴氏吸管。 </li><li>人类胚胎干细胞通过每6-7天，如果在显微镜下观察菌落明亮。 注:生长在涂外基质平板菌落人类胚胎干细胞生长速度比MEF培养的细胞大。 </li><li>当无饲养层细胞准备通道，取出介质，通过广告洗采用吸移管吸出，除去PBS丁1ml的PBS中，添加0.5毫克/毫升分散酶（预热至37℃）用移液管，并且在37℃下孵育5分钟。 </li><li>加入2毫升的PBS每孔并随后吸移用PBS洗细胞。加入1ml每孔的CM和手动刮去细胞进入使用5毫升吸管的尖端小团块。 </li><li>板悬浮细胞团块进入新的细胞外基质包被的平板; 24小时后的细胞要坚持。 </li></ol></li></ol></li></ol> 3.使用人类胚胎干细胞BMP4 / A / P值分化<ol><li>准备分化培养基。使用CM（缺乏B-FGF），并添加（鲜）BMP4（50毫微克/毫升）中，A83-01（1μM），和PD173074（0.1μM）。添加在使用前这些抑制剂到CM。 </li><li>使用无饲养层人类胚胎干细胞的细胞外基质包被的平板1通行增长，定在4％氧气的培养箱内培养。所述第一通道到细胞外涂覆基质的平板后，让细胞附着24小时。第二天，开始分化。除去（含B-FGF）的CM和用含有BMP4 / A / p（2毫升，每孔在6孔板）的CM代替它。继续培养用4％的氧含量的细胞。 </li><li>更换含BMP4 / A / P中的CM（2毫升/一个6孔板），每2天。吸删除旧的介质，并使用吸管添加新的CM。上添加BMP4和除去的B-FGF（考虑分化第2天）后的第二天，细胞形态发生变化，用显微镜观察时出现大。 注:未分化细胞，表现出明亮的圆边，将不存在。 </li><li>收集的细胞以期望的时间点。分化的细胞会停止约2周后分裂。在这段时间内转染细胞（参见下一部分）。 </li></ol> 4.转染滋养层细胞与siRNA的或质粒DNA <ol><li> 准备转染滋养层细胞 </STRONG&gt; <ol><li>对细胞外基质的两个通道从该MEF它们转移后的文化人类胚胎干细胞（见步骤2.2）。使用含有B-FGF人类胚胎干细胞中。 </li><li>分裂的胚胎干细胞分化上之前，新的细胞外基质板一天。高细胞融合是非常重要的，所以1分裂细胞:1到一个新的板块/孔，这将确保至少50％汇合第二天。孵育细胞过夜在低氧气培养箱。 注:胚胎干细胞不需要在这个步骤中被计数。 </li><li>第二天，替换用分化培养基的培养基（含有BMP4 / A / P和缺乏B-FGF，使用2-毫升/孔的6孔板的CM）。通过抽吸除去旧培养基并转移使用移液管的新培养基（2ml）中。放置细胞在低氧培养器（4％氧气）中过夜。 </li></ol></li><li> 使用的siRNA用于基因破坏或使用质粒DNA转染 <ol><li>分化细胞，直到所需的时间点（之间的天1和14）。转染前一天，trypsinize使用0.05％胰蛋白酶进行5分钟的细胞。他们板至所需汇合（取决于转染试剂协议）到明胶包被的平板（0.1％明胶添加到组织培养板20分钟，并吸出除去）。添加含有BMP4 / A / P（缺乏B-FGF）CM和孵育过夜。 </li><li>第二天，新鲜分化培养基添加到细胞中。的siRNA转染使用siRNA转染试剂。对于质粒DNA，使用适当的脂质体试剂。 </li><li>按照产品协议为每个转染试剂描述。转移的siRNA脂质体复合物的细胞的每个孔/板，轻轻混合，培养细胞过夜以低氧气培养箱。 注意:有些转染试剂是由抗生素，需要CM缺乏笔/链球菌抑制。 </li><li>第二天，替换以新鲜分化培养基。 </li><li>收获细胞的欲望ð时间点（ 例如，24小时，48小时和72小时），利用定量RT-PCR检查基因破坏效率。收获细胞，使用0.05％胰蛋白酶，加入每孔1 ml，在37℃温育5分钟。加入5毫升，每孔MEF培养基的以中和胰蛋白酶。降速细胞在200×g离心5分钟，并使用用于RNA分离细胞沉淀。 </li></ol></li></ol>滋养层细胞5.病原感染:仙台病毒感染<ol><li> 准备分化的胚胎干细胞 <ol><li>为从该MEF转让后的细胞外基质两段文化人类胚胎干细胞（见步骤2.2）。使用含有B-FGF人类胚胎干细胞中。 </li><li>分裂的胚胎干细胞分化上之前，新的细胞外基质板一天。高蜂窝汇合是重要的，因此分裂细胞1:1到一个新的板/孔，这将确保至少50％汇合的第二天。孵育细胞过夜以低氧气培养箱（4％氧气）。&lt;BR /&gt;注:不需要在这一步要算人类胚胎干细胞。 </li><li>第二天，替换用分化培养基（含CM BMP4 / A / P和缺乏B-FGF）和培养过夜以低氧气培养箱（4％氧气）的介质。 </li></ol></li><li> 滋养层细胞的仙台病毒感染 <ol><li>所需的分化前一天一天，trypsinize使用0.05％胰蛋白酶的分化细胞（单细胞）5分钟，并将其转移到明胶包被的板。添加分化培养基和在低氧气培养箱孵育过夜。 </li><li>第二天，添加培养基感染（这将取决于病毒而变化）。使用CM缺乏含有BMP4 / A / P，以0.5毫升一个6孔板的一个孔青霉素/链霉素。添加仙台病毒对于MOI = 1孵育在低氧气孵化8小时。 </li><li>如果更多的时间点是必要的，删除含有病毒的培养基后4小时，并用BMP4 / A / p CM替换它。收集细胞用于RNA分离使用细胞刮。 </li><li>通过对所涉及的病毒反应18个基因进行定量RT-PCR确定病毒感染的效率。 </li></ol></li></ol>

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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=Differentiation+of+trophoblast+cells+from+human+embryonic+stem+cells:+to+be+or+not+to+be?.">Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be?.</a> Reproduction. 147 (5), D1-D12 (2014).</li><li>Sarkar, P., 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=Activin/nodal+signaling+switches+the+terminal+fate+of+human+embryonic+stem+cell-derived+trophoblasts.">Activin/nodal signaling switches the terminal fate of human embryonic stem cell-derived trophoblasts.</a> The Journal of biological chemistry. 290 (14), 8834-8848 (2015).</li><li>Penkala, I., 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=lncRHOXF1,+a+Long+Noncoding+RNA+from+the+X+Chromosome+That+Suppresses+Viral+Response+Genes+during+Development+of+the+Early+Human+Placenta.">lncRHOXF1, a Long Noncoding RNA from the X Chromosome That Suppresses Viral Response Genes during Development of the Early Human Placenta.</a> Mol Cell Biol. 36 (12), 1764-1775 (2016).</li><li>Penkala, I., 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=lncRHOXF1,+a+Long+Noncoding+RNA+from+the+X+Chromosome+That+Suppresses+Viral+Response+Genes+during+Development+of+the+Early+Human+Placenta.">lncRHOXF1, a Long Noncoding RNA from the X Chromosome That Suppresses Viral Response Genes during Development of the Early Human Placenta.</a> Molecular and cellular biology. 36 (12), 1764-1775 (2016).</li><li>Hockemeyer, D., 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=Genetic+engineering+of+human+pluripotent+cells+using+TALE+nucleases.">Genetic engineering of human pluripotent cells using TALE nucleases.</a> Nature biotechnology. 29 (8), 731-734 (2011).</li><li>Bernardo, A. S., 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=BRACHYURY+and+CDX2+mediate+BMP-induced+differentiation+of+human+and+mouse+pluripotent+stem+cells+into+embryonic+and+extraembryonic+lineages.">BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages.</a> Cell stem cell. 9 (2), 144-155 (2011).</li><li>Zhang, P., 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=Short-term+BMP-4+treatment+initiates+mesoderm+induction+in+human+embryonic+stem+cells.">Short-term BMP-4 treatment initiates mesoderm induction in human embryonic stem cells.</a> Blood. 111 (4), 1933-1941 (2008).</li><li>Vallier, 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=Early+cell+fate+decisions+of+human+embryonic+stem+cells+and+mouse+epiblast+stem+cells+are+controlled+by+the+same+signalling+pathways.">Early cell fate decisions of human embryonic stem cells and mouse epiblast stem cells are controlled by the same signalling pathways.</a> PloS one. 4 (6), e6082 (2009).</li><li>Arman, E., Haffner-Krausz, R., Chen, Y., Heath, J. K., Lonai, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+fibroblast+growth+factor+(FGF)+receptor+2+suggests+a+role+for+FGF+signaling+in+pregastrulation+mammalian+development.">Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (9), 5082-5087 (1998).</li><li>Yu, P., Pan, G., Yu, J., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF2+sustains+NANOG+and+switches+the+outcome+of+BMP4-induced+human+embryonic+stem+cell+differentiation.">FGF2 sustains NANOG and switches the outcome of BMP4-induced human embryonic stem cell differentiation.</a> Cell stem cell. 8 (3), 326-334 (2011).</li><li>Sudheer, S., Bhushan, R., Fauler, B., Lehrach, H., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+inhibition+directs+BMP4-mediated+differentiation+of+human+embryonic+stem+cells+to+syncytiotrophoblast.">FGF inhibition directs BMP4-mediated differentiation of human embryonic stem cells to syncytiotrophoblast.</a> Stem cells and development. 21 (16), 2987-3000 (2012).</li><li>Bischof, P., Irminger-Finger, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+cytotrophoblastic+cell,+a+mononuclear+chameleon.">The human cytotrophoblastic cell, a mononuclear chameleon.</a> The international journal of biochemistry &amp; cell biology. 37 (1), 1-16 (2005).</li><li>Cole, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycosylated+hCG,+a+review.">Hyperglycosylated hCG, a review.</a> Placenta. 31 (8), 653-664 (2010).</li><li>Apps, R., 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=Human+leucocyte+antigen+(HLA)+expression+of+primary+trophoblast+cells+and+placental+cell+lines,+determined+using+single+antigen+beads+to+characterize+allotype+specificities+of+anti-HLA+antibodies.">Human leucocyte antigen (HLA) expression of primary trophoblast cells and placental cell lines, determined using single antigen beads to characterize allotype specificities of anti-HLA antibodies.</a> Immunology. 127 (1), 26-39 (2009).</li></ol>

The authors declare that they have no competing financial interests.

我们提出了分化的hESC入滋养层祖细胞的基本步骤。这个协议最近被优化以迅速分化的hESC与除了激活素/ Nodal信号抑制剂，增加分化为滋养层细胞和避免胚层祖细胞，其通常单独用BMP4治疗观察到的产生。该模型BMP4系统允许滋养层沿袭规格和扩展的最早阶段的审查。此外，这种BMP4模型系统也调查滋养层细胞向特定子谱系，使用绒毛膜细胞系和绒毛外滋养细胞系，这是不可能的分化是有用的。 体?...

怀孕期间胎盘需要胎儿的生长和存活，并促进气体，营养物，废物和激素的母体和胎儿循环之间的交换。哺乳动物胚胎发育过程中形成的第一器官是胎盘，它开始显影6-7天在人类受孕后和在小鼠中1，2，3，4 3.5-4.5天。滋养层细胞是胎盘的最重要的细胞，这些细胞所代表的哺乳动物胚胎的最早谱系分化的事件之一。他们从植入前胚泡的外胚外滋养层细胞产生。我们的胎盘发育的早期阶段的知识是通过模拟早期人类发展的伦理和后勤限制的限制。 在胚胎移植，滋养细胞侵入母体上皮细胞和分化成专门的祖细胞5。细胞滋养细胞（CTB介）的单核，即融合和分化成合体（SYN包）和绒毛外滋养层细胞侵入（EVTs）未分化的祖细胞，它的锚胎盘在子宫。 SYN包的多核，即合成所需的持续受孕激素影响终末分化细胞。产生EVTs和SYN包的早期分化事件对于胎盘形成所必需的，如在滋养层细胞的损伤导致流产，先兆子痫，宫内生长受限1。已经开发人类滋养细胞系的类型包括永生个CTB和绒毛膜癌，其产生的胎盘激素和显示侵入属性6。从人类早期妊娠胎盘的主要滋养层细胞可以分离，但很快细胞DIFferentiate并停止在体外增殖。重要的是，转化和原代细胞系具有不同的基因表达图谱，表明致瘤性和永生滋养层细胞系可能不准确地表示主滋养层7。此外，这些线是不可能像胎盘滋养层干细胞祖细胞，因为它们是从后级通过第三孕期导出的第一。 有为了研究胎盘的形成和功能的早期事件需要在早期人类滋养细胞体外培养体系稳健。人类胚胎干细胞（胚胎干细胞），它们共享与早期胚胎的内细胞团性质，常用于早期人类的发展模式，包括早期胎盘的形成。人类诱导多能干细胞（iPS细胞）和人类胚胎干细胞可以分化成骨用在铁道部滋养细胞体外phogenic蛋白4（BMP4）8，9，10，11，12，13，14，15。多能细胞用BMP4的滋养层细胞这种转换是特异于人细胞，并广泛用于研究早期人类胎盘的发展，因为它不要求访问人类早期胚胎9,16。最近，人们发现，在加入抑制剂A83-01（A）和PD173074（P），其阻断SMAD2 / 3和MEK1 / 2信号途径，增加HPSC分化的效率为滋养外胚层样祖细胞，主要的SYN和EVTs，没有广泛代中胚层，内胚层或外胚层细胞9，17的</suP&gt;。使用这些介质的条件下，人类胚胎干细胞分化12天具有相似的基因表达谱从人胚泡期胚胎中分离滋养层细胞和分泌多种胎盘特异性生长激素，支撑该体外模型系统9，11的有效性。在这里，我们提出了hPSCs 体外分化为使用BMP4 / A / p培养基中的人类祖先滋养了详细的方案。这些条件产生细胞的数量丰富为各种各样的应用，包括RNA测序，基因破坏用的siRNAs，病原体感染，遗传修饰使用脂转染介导的转染。

<table><tbody><tr><td>DMEM/F12</td><td>Invitrogen</td><td>11330-057</td><td></td></tr><tr><td>Knock Out Serum Replacement</td><td>Invitrogen</td><td>10828-028</td><td>This is referred to as &quot;serum replacement&quot; in this protocol.</td></tr><tr><td>NEAA</td><td>Invitrogen</td><td>11140-050</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>16000-044</td><td></td></tr><tr><td>L-Glutamine</td><td>Invitrogen</td><td>10828-028</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-155</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M-7522</td><td></td></tr><tr><td>B-FGF</td><td>Millipore</td><td>GF-003</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>11965-118</td><td></td></tr><tr><td>Dispase</td><td>Invitrogen</td><td>17105-041</td><td></td></tr><tr><td>Collagenase Type IV</td><td>Invitrogen</td><td>17104-019</td><td></td></tr><tr><td>Rock inhibitor Y27632</td><td>Calbiochem</td><td>688000</td><td></td></tr><tr><td>Irradiated CF1 MEFs</td><td>GlobalStem</td><td>6001G</td><td>MEFs can be generated from embryonic day 13.5 embyos and irradiated.</td></tr><tr><td>0.22 &amp;micro;m syringe filter</td><td>Millipore</td><td>SLGS033SS</td><td></td></tr><tr><td>Heracell 150i low oxygen incubator</td><td>Heracell/VWR</td><td>89187-192</td><td>Any tissue culture incubator with capacity to regulate oxygen concentrations is sufficient.</td></tr><tr><td>BMP4</td><td>R&amp;amp;D Systems</td><td>314-BP-01M</td><td></td></tr><tr><td>A 83-01</td><td>R&amp;amp;D Systems</td><td>2939/10</td><td></td></tr><tr><td>PD173074</td><td>R&amp;amp;D Systems</td><td>3044/10</td><td></td></tr><tr><td>RNAiMax</td><td>Invitrogen</td><td>13778150</td><td></td></tr><tr><td>Trizol</td><td>ThermoFisher</td><td>15596026</td><td>Trizol is used to isolate total RNA.</td></tr><tr><td>X-tremeGENE 9</td><td>Roche</td><td>6365779001</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>356231</td><td>This is referred to as &quot;extracellular matrix&quot; in this protocol.</td></tr></tbody></table>

in vitro differentiation of human pluripotent stem cells into trophoblastic cells

胎盘是胚胎发育过程中，开发的第一个器官，并需要对发育中的胚胎的生存。胎盘包括从植入前胚泡的胚外滋养外胚层细胞分化的各种滋养细胞。因此，我们的人胎盘的早期分化事件的认识，是因为对人胚胎的分离和操作的伦理和法律限制的限制。人多能干细胞（hPSCs）是用于调查人类发展的稳健的模型系统，也可以在体外分化成表达各滋养层细胞类型的标志物滋养层细胞。在这里，我们提出了一个详细的协议，用于区分hPSCs到使用骨形态发生蛋白4和激活素/ Nodal信号通路抑制剂的滋养层细胞。该协议生成可以用的siRNA转染各种滋养层细胞类型调查失功能表型或可感染病原体。此外，hPSCs可以进行遗传修饰，然后分化成滋养层祖换的功能增益分析。用于产生人类滋养细胞从hPSCs开始本体外分化的方法克服了与早期的人类胚胎工作的伦理和法律的限制，并且该系统可用于多种应用，包括药物发现和干细胞研究。

hPSCs的体外分化概述 该体外分化方案开始生长在被转换到无饲养为一个通路（ 图1A）的条件的MEF未分化的人类胚胎干细胞。虽然我们在本协议中所述人类胚胎干细胞的分化，我们使用这个协议成功地分化成iPS细胞滋养层细胞。细胞外基质的过渡去除大部分照射的MEF，这是不可取的，需要人的滋养层细胞...

Watch this Scientific Journal Video about In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells at JoVE.com

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 ...

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In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

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8.5K 次观看.  University of Pennsylvania. 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.

视频: In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

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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 ...

发育生物学

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

协议小鼠胚胎成纤维细胞直接转化为滋养层干细胞

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

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 ...

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 ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

从人多能干细胞定向多巴胺能神经元分化

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

神经科学

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

鼠标滋养层干细胞从胚泡的推导

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

生物学

Protocol for Human Blastoids Modeling Blastocyst Development and Implantation

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend on its ability to efficiently, timely, and faithfully recapitulate the sequences of blastocyst development (morphogenesis, specification, patterning), and to form cells reflecting the blastocyst stage. Here we show that na&#239;ve human pluripotent stem cells cultured in PXGL conditions and then triply inhibited for the Hippo, transforming growth factor- &#946;, and extracellular signal-regulated kinase pathways efficiently undergo morphogenesis to form blastoids (&gt;70%). Matching with developmental timing (~4 days), blastoids unroll the blastocyst sequence of specification by producing analogs of the trophoblast and epiblast, followed by the formation of analogs of the primitive endoderm and the polar trophoblasts. This results in the formation of cells transcriptionally similar to the blastocyst (&gt;96%) and a minority of post-implantation analogs. Blastoids efficiently pattern by forming the embryonic-abembryonic axis marked by the maturation of the polar region (NR2F2+), which acquires the specific potential to directionally attach to hormonally stimulated endometrial cells, as in utero. Such a human blastoid is a scalable, versatile, and ethical model to study human development and implantation in vitro.

A protocol outlining the formation of human blastoids that efficiently, timely, and sequentially generate blastocyst-like cells.

人爆破模拟胚发育和植入的协议

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend ...

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 ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

分离小鼠原代滋养细胞及滋养细胞侵袭实验

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...