Name: developing hipsc derived serum free embryoid bodies for the interrogation of 3-d stem cell cultures using physiologically relevant assays
Uploaded: 2017-07-20T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays at JoVE.com

我们感谢伊丽莎白Benevides校对文章。感谢Drs。 John Hussman和Gene Blatt进行了有益的讨论和评论。

神经祖细胞的产生<ol><li> 在补充有小分子的人iPSC培养基中，在γ-照射的小鼠胚胎饲养细胞（MEF）细胞层上，将来自成纤维细胞和PBMC的hiPSC保持在6孔板中（参见材料表）。 注:日常保养是以前报道的程序1，16修改后的版本。 <ol><li>在6孔组织培养级板的每个孔上以600μL/孔推荐的培养基（以下制造商的方案）将6×10 5个 MEF板铺板。 </li><li> 48小时后，加入hiPSCs，用300μL/孔hiPSC培养基取代MEFs培养基1 h。 </li><li>在37℃水浴中迅速解冻hiPSC，并逐渐缓慢加入1mL hiPSC培养基。将hiPSC和培养基转移到15 mL锥形管中。加入培养基使总体积达到5 mL。以129 xg离心4分钟，吸出超新星在1:1,000浓度的ROCK抑制剂的1mL hiPSC培养基中轻轻重新悬浮沉淀。 </li><li>板中的细胞悬浮液（通常在30万个细胞/孔接种）到MEF细胞层，并在37℃，5％CO 2，95％湿度下孵育。 </li><li>使用光学显微镜密切监测iPSC培养。 48小时后，去除具有不均匀边缘的培养物，在光学显微镜下生长成为囊状或显示为黄棕色，以最小化自发分化。 注意:7 - 10天后，应形成hiPSC菌落。殖民地应该圆形，边界清晰，细胞密度均匀，没有松散包装的不均匀细胞。 </li><li>手动选择圆形hiPSC菌落，以清除自发分化细胞的培养。将它们放置在新鲜的MEF层上来扩展菌落。展开1:3，每7天，当接近融合的文化和冻结1，16。 </li></ol></li><li>在扩增和冷冻细胞（用于备用）后，在板上生长hiPSC菌落直至达到50-75％汇合。 </li><li>电镀后7天，使用温和的酶处理（用300μL酶溶液5-10分钟）和温和研磨（用1,000μL移液管2-4次）收获并制备hiPSC用于神经前体细胞分化。 </li><li>以129 xg离心细胞悬浮液4分钟，吸出上清液，并将沉淀重悬于5 mL人iPSC培养基中。将细胞悬浮液转移到0.1％明胶包被的5cm细胞培养皿中，在37℃下1小时以消除MEF并使hiPSC产率最大化。将培养基（非贴壁细胞）转移到另外5厘米明胶包被的培养皿中。 </li><li>使用转移吸管将非贴壁细胞转移到15 mL离心管中。用3 mL培养基轻轻冲洗平板，并加入15 mL管中。 </li><li>将细胞悬浮液以129 xg离心4分钟，吸出超级细胞将其沉淀在培养基中。使用自动细胞计数器确定细胞计数。在96孔低粘附V底板中板9,000个细胞/孔。 </li><li>离心板163 xg 3分钟。在37℃，95％湿度和5％CO 2下孵育该板。使用移液管，每隔一天更换50％的培养基，取出一半的培养基，并用新鲜的化学成分分化培养基1（DM1; 图1 ; 材料表）替换14天。 </li></ol> 2.无血清胚胎体（SFEB）诱导（ 图2 ） <ol><li>在加入聚集体之前，将40μm细胞培养插入物置于6孔板中，并加入1 mL DM1培养基至少1 h。 </li><li> 在第14天，使用200μL宽嘴尖将20μL培养基的聚集体转移到40μm细胞培养物插入物上，并将培养基改变为DM2。 <br/&gt;注意:细胞培养插入物是位于孔底部稍微偏离并由悬浮在塑料框架上的聚四氟乙烯膜组成的专用插入物。该膜是生物相容的，可以有效地维持营养和氧气输送到放置在顶部的SFEB。它们通常与来自小鼠或大鼠7，17，18取海马脑切片培养物使用。 <ol><li>将4-6个聚集体转移到一个细胞培养插入物，并允许聚集体之间的足够的空间。使用精细的吸头（ 例如 200μL提示）尽可能多地除去多余的溶液。 注意:SFEB可以短暂的干扰SFEB，因为它们将作为从SFEB周围移除的溶液移动到细胞培养插入物上。 </li><li>在37℃，95％湿度和5％CO 2的 1mL DM2中维持培养物。使用移液器，更换75％每隔一天中间除去四分之三的培养基，并用四分之三的新鲜培养基代替。例如，移除750μL培养基，加入750μL新鲜培养基。 </li></ol></li><li>培养14天后，每隔一天切换至含有75％培养基的DM3培养基，并持续16天。 </li><li>为了维持超过30天的细胞培养插入物的SFEB，每隔一天更换培养基60天，90天和120天。 注意:SFEBs的直径将增长至约1,000μm，通常为100-150μm厚1 。 </li></ol> 3.确定SFEB组成<ol><li>使用200μL宽口吸头，轻轻移液培养基，从插入物中分离SFEB。要转移SFEB，请使用宽口吸头将其轻轻吸入吸头。将SFEB装入移液管末端后，轻轻转移至12孔板中，用300μL磷酸盐缓冲盐水洗涤（PBS）。 注意:SFEB将悬浮在12孔板中的溶液中。这对于允许固定剂，封闭溶液和抗体的完全渗透是重要的。如果使用多个SFEB进行染色，则可以在12孔板中每孔加入10个SFEB。这将节省解决方案和抗体。 </li><li>在室温下将SFEB固定在300μL/孔4％多聚甲醛中30-45分钟。用300μLPBS洗涤固定的SFEB两次，每次洗涤3-5分钟。 注意:处理多聚甲醛时，穿戴适当的个人防护装备。 注:为探讨以免疫标记来确定成熟度，细胞类型等。在这项研究中鸡TUJ1在1使用标记的神经元:500，为其他标记，请见表2和参考文献1，16。 </li><li>渗透并用PBS中的0.1％Triton-X100和10％正常驴血清（封闭溶液;300μ; L /孔）在室温下1小时。 </li><li>去除阻断溶液，并在封闭溶液中用300μL一抗处理SFEB。在4℃下孵育过夜。在含有0.1％Triton-X的300μLPBS中洗涤SFEB三次（每次洗涤3-5分钟）。 </li><li>在阻断溶液中制备1:1000的二抗。在室温下用二次抗体孵育SFEB 1小时。用300μLPBS洗涤SFEB，3次，每次3-5分钟。 注意:在这个阶段，SFEB可以准备成像。 </li><li>为了成像，使用宽孔移液管将SFEB移液到载玻片上。使用温和的吸力去除SFEB周围的多余溶液。 注意:对于类似的染色条件，可以将多个SFEB放置在同一张载玻片上。 </li><li>在SFEB上添加一滴安装介质，放置玻璃盖玻片，轻轻按下以确保没有气泡。允许安装介质硬化并进行成像和定量（步骤3.8）。 <br/&gt;注意:安装介质硬化后，SFEB可以进行成像。 </li><li>通过利用穿过SFEB关注区域（ROI）的多个区域，并使用Z堆栈通过在一个标准共焦显微镜每个ROI具有最大投影图像组合，如参考文献1中 ，16中所述进行细胞定量。 注:全SFEBs可以使用基于图像的拼接（详见参考文献1，16）进行量化。由于SFEB薄至约100μm，组织中光的散射最小，因此不需要双光子显微镜。这个协议与成纤维细胞衍生iPSC的较早的用途制备的SFEB命运映射揭示复杂多样的结构与相似CGE和前端脑命运1,16转录身份的interneurons的亚群。 </sup&gt; </li></ol>记录SFEB中的神经元活动<ol><li>根据制造商的说明准备12孔MEA板。 </li><li>在无菌条件下用无菌H 2 O洗涤MEA板3次5分钟以进行清洁。用75％乙醇洗涤5分钟，然后用100％乙醇洗涤，对板进行灭菌。 </li><li>烘烤板在烘箱中在50℃下倒置4-5小时以完成灭菌过程。 </li><li>向每个孔中加入500μL的0.2％聚乙烯亚胺溶液（PEI），并在室温下孵育1小时。吸PEI和洗涤各孔用无菌蒸馏水H 2 O.空气干燥4倍在引擎盖过夜。 </li><li>在L15培养基中制备20μL/ mL层粘连蛋白溶液，并将10μL层粘连蛋白加入每个孔的中心。 注意:请勿涂覆周围的参考电极和接地电极。 </li><li>向周围的水库中加入无菌dH 2 O以防止介质或层粘连蛋白蒸发。在37℃孵育板1小时。 </li><li>将SFEB（见第1部分和第2部分）通过轻轻地将其吸入广泛的吸液管尖端并转移到层粘连蛋白包被的MEA板上。向每个孔中加入200μLDM2培养基，并在37℃，95％湿度和5％CO 2下孵育过夜。 </li><li>在24小时后再加入200μL培养基，并在37℃，95％湿度，5％CO 2下回收30分钟，然后再读板。 </li><li>将MEA板置于读板器中（预热至37℃）以记录神经元活动。使用相关的MEA录制软件记录活动10分钟。详见参考资料19 。 </li><li>使用统计分析软件生成原始数据 - 连续流和神经元活动的栅格图19 。 注意:MEA录音是在SFEB电镀7天后进行的。对于这项研究，记录采取10分钟以检测网络爆发活动，con连续痕迹和光栅图。 SFEB可以在MEA板材上长期保持，并且可以使用环境条件在更长的时间内记录（ 图6 ）。 </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=Functional+cortical+neurons+and+astrocytes+from+human+pluripotent+stem+cells+in+3D+culture.">Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture.</a> Nat Methods. 12 (7), 671-678 (2015).</li><li>Yan, X., 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=iPS+cells+reprogrammed+from+human+mesenchymal-like+stem/progenitor+cells+of+dental+tissue+origin.">iPS cells reprogrammed from human mesenchymal-like stem/progenitor cells of dental tissue origin.</a> Stem Cells Dev. 19 (4), 469-480 (2010).</li><li>Wang, 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=Generation+of+clinical-grade+human+induced+pluripotent+stem+cells+in+Xeno-free+conditions.">Generation of clinical-grade human induced pluripotent stem cells in Xeno-free conditions.</a> Stem Cell Res Ther. 6, 223 (2015).</li><li>Kwon, 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=Neuronal+Differentiation+of+a+Human+Induced+Pluripotent+Stem+Cell+Line+(FS-1)+Derived+from+Newborn+Foreskin+Fibroblasts.">Neuronal Differentiation of a Human Induced Pluripotent Stem Cell Line (FS-1) Derived from Newborn Foreskin Fibroblasts.</a> Int J Stem Cells. 5 (2), 140-145 (2012).</li><li>Li, X. 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=Coordination+of+sonic+hedgehog+and+Wnt+signaling+determines+ventral+and+dorsal+telencephalic+neuron+types+from+human+embryonic+stem+cells.">Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells.</a> Development. 136 (23), 4055-4063 (2009).</li><li>Sheridan, S. D., Surampudi, V., Rao, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+embryoid+bodies+derived+from+human+induced+pluripotent+stem+cells+as+a+means+to+assess+pluripotency.">Analysis of embryoid bodies derived from human induced pluripotent stem cells as a means to assess pluripotency.</a> Stem Cells Int. , 738910 (2012).</li><li>Smith, Q., Stukalin, E., Kusuma, S., Gerecht, S., Sun, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochasticity+and+Spatial+Interaction+Govern+Stem+Cell+Differentiation+Dynamics.">Stochasticity and Spatial Interaction Govern Stem Cell Differentiation Dynamics.</a> Sci Rep. 5, 12617 (2015).</li></ol>

作者没有竞争的经济利益。

这里描述的方案提供了将hiPSC源区分为重现前额叶皮质早期发育阶段的3-D结构的条件。该过程产生可以询问电生理学的结构，同时也适用于显微镜。 SFEB的最终形态类似于器官型脑切片培养物的形态，并允许高质量的详细共聚焦成像。该方案可以成功地从成纤维细胞和外周血单核细胞衍生的hiPSC中产生SFEB。由外周血单个核细胞来源的hiPSCs制成的SFEB表现出与从成纤维细胞衍生的hiPSC产生的形态特征相...

我们以前报告了由患者衍生的人类多能干细胞（hiPSC）产生的3-D模型系统，其概括了早期皮层网络发展的某些方面1 。此3-D模型，无血清胚状体（SFEB），改善了以前的简单聚合的hiPSC模型2,3。工作的一个增长的身体被显露像我们SFEBs 3-D结构，神经发育的近似方面在体内和在比2维（2-D）观察到更早的时间点通常观察到的/单层的hiPSC模型4,5。初步研究集中在3-D体的自组织复杂性，而不表现出其生理复杂性2 。 本文描述的方案已用于源自成纤维细胞的未分化的hiPSC<html>外周血单核细胞（PBMCs）。这些细胞维持在γ照射的小鼠胚胎喂养器（MEFs）上。将这些hiPSC菌落手工清洗自发分化的细胞，酶促收获，并重新悬浮于含Rho-Kinase抑制剂Y-27632（ROCKi）的培养基中。将未分化的hiPSC在转移到96孔低粘附性V底板之前进行离解和离心。电镀后，使用双SMAD抑制（SB431542和LDN193189以及dickkopf 1（DKK-1））启动神经诱导，以驱动前额叶前脑神经元命运谱系6 。 14天后，将SFEB转移到6孔板中的细胞培养插入物中。一旦转移，圆形SFEB开始扩散和稀释，同时维持局部网络连接，如使用类似的细胞培养插入物1的海马器官切片培养物制备中常见的那样，ss ="xref"&gt; 7。 以这种格式使用基于SFEB的3-D平台适用于可以使用基于细胞的生理测定例如钙成像或电生理测定（例如单细胞记录或多电极阵列（MEA））来询问的皮层网络的有效生产） 1 。虽然3-D系统具有早期皮层发育的标志，但其他研究已经表明，这些3-D体可能需要更长的孵育时间，以允许人体组织发育的本质上较慢的速度8 。该SFEB协议成功地从未分化的hiPSC产生3-D SFEB，其捕获皮质早期发育的方面。 SFEBs在神经系统疾病中模拟网络畸变的潜力是该系统的优势。源自患者组织的hiPSC可以生长成受屁股的神经系统的细胞与细胞生物学有关的ays以及伴随的基因表达。人类iPSC被用来确定大群体的个体的遗传谱与不同神经障碍具有复杂的病因，如孤独症谱系障碍（ASD），精神分裂症9，Rett综合症10，和阿尔茨海默氏病11，12。直到最近，iPSC模型通常是单层制剂，虽然精通评估分子相互作用，但在破译体内发现的复杂细胞相互作用方面是不够的。动物模型已经成为重建整个器官平台的默认替代品。这些动物模型受到研究结果差的翻译困扰，复制由大型遗传筛选研究确定的人类遗传图谱的能力有限。因此，iPSC开发的3-D系统增加了人类所需的复杂层次疾病建模13，14。用于3D的hiPSC平台下一个步骤是，以适应使用基于细胞的测定筛选15高通量的大规模要求。

<table><tbody><tr><td>&lt;strong&gt;SFEB Neuronal Differentiation Cell culture Media. Reagents. Components&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>STEMdiff Neural Induction Medium (hiPSC Media)</td><td>STEMCELL Technologies</td><td>0-5835</td><td>250 mL</td></tr><tr><td>PluriQ ES-DMEM Medium (MEF Media)</td><td>GlobalStem</td><td>GSM-2001</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;DM1 Media Components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>D-MEM/F-12 (1x), Glutamax liquid, 1:1</td><td>Invitrogen</td><td>10565018</td><td>385 mL</td></tr><tr><td>Knockout Serum Replacement</td><td>Invitrogen</td><td>10828028</td><td>20% 100 mL</td></tr><tr><td>Pen/Strep</td><td>Invitrogen</td><td>15140122</td><td>5 mL</td></tr><tr><td>Glutamax 200 mM</td><td>Invitrogen</td><td>35050061</td><td>5 mL</td></tr><tr><td>MEM Non-Essential Amino Acids Solution 10 mM (100x), liquid</td><td>Invitrogen</td><td>11140050</td><td>5 mL</td></tr><tr><td>2-Mercaptoethanol (1,000x), liquid</td><td>Invitrogen</td><td>21985023</td><td>900 &amp;mu;L</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;DM2 Media Components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>D-MEM/F-12 (1x), Glutamax liquid, 1:1</td><td>Invitrogen</td><td>10565018</td><td>500 mL</td></tr><tr><td>Glutamax 200 mM</td><td>Invitrogen</td><td>35050061</td><td>5 mL</td></tr><tr><td>Pen/Strep</td><td>Invitrogen</td><td>15140122</td><td>5 mL</td></tr><tr><td>N-2 Supplement (100x), liquid</td><td>Invitrogen</td><td>17502048</td><td>10 mL</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;DM3 Media Components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NEUROBASAL Medium (1x), liquid</td><td>Invitrogen</td><td>21103049</td><td>500 mL</td></tr><tr><td>B-27 Supplement Minus Vitamin A (50x), liquid</td><td>Invitrogen</td><td>12587010</td><td>10 mL</td></tr><tr><td>Glutamax 200 mM</td><td>Invitrogen</td><td>35050061</td><td>5 mL</td></tr><tr><td>Pen/Strep</td><td>Invitrogen</td><td>15140122</td><td>5 mL</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Small Molecules&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Thiazovivin</td><td>Stemgent</td><td>04-0017</td><td>2 &amp;mu;M</td></tr><tr><td>SB431542</td><td>Stemgent</td><td>04-0010-10</td><td>1:1,000 (10 &amp;mu;M)</td></tr><tr><td>Dorsomorphin</td><td>Stemgent</td><td>04-0024</td><td>1 &amp;mu;M</td></tr><tr><td>LDN-193189</td><td>Stemgent</td><td>04-0074-10</td><td>250 nM</td></tr><tr><td>Y27632 (ROCKi)</td><td>Stemgent</td><td>04-0012-10</td><td>10 &amp;mu;M</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Recombinant Protiens&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DKK-1</td><td>Peprotech</td><td>120-30</td><td>200 ng/mL</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Components/Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell Culture inserts 0.4 &amp;mu;M, 30mm Diameter</td><td>Millicell</td><td>PICM0RG50</td><td></td></tr><tr><td>Mouse Embryonic Fibroblasts</td><td>GlobalStem</td><td>GSC-6301G</td><td></td></tr><tr><td>96 well V bottom w/Lids</td><td>Evergreen</td><td>222-8031-01V</td><td></td></tr><tr><td>StemPro Accutase Cell Dissociation Reagent</td><td>ThermoFisher</td><td>A1110501</td><td></td></tr><tr><td>TritonX-100</td><td>ThermoFisher</td><td>85111</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>ThermoFisher</td><td>10010023</td><td>500 mL</td></tr><tr><td>Normal Donkey Serum</td><td>Jackson Labs</td><td>017-000-121</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium</td><td>ThermoFisher</td><td>11415114</td><td>500 mL</td></tr><tr><td>DRAQ5 (Nuclear Marker)</td><td>ThermoFisher</td><td>65-0880-96</td><td></td></tr><tr><td>MEA Plates</td><td>Axion Biosystems</td><td>M768-GL1-30Pt200</td><td></td></tr><tr><td>6 well flat bottom</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibody&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nestin</td><td>Millipore</td><td>MAB5326</td><td></td></tr><tr><td>Brn-2</td><td>Protein tech</td><td>14596-1-AP</td><td></td></tr><tr><td>VGLUT1</td><td>Synaptic Systems</td><td>135 303</td><td></td></tr><tr><td>Pax6</td><td>abcam</td><td>ab5790</td><td></td></tr><tr><td>Calretinin</td><td>abcam</td><td>ab702</td><td></td></tr><tr><td>Calbindin</td><td>abcam</td><td>ab11426</td><td></td></tr><tr><td>CoupTFII</td><td>R&amp;amp;D Systems</td><td>PPH714700</td><td></td></tr><tr><td>Nkx 2.1</td><td>abcam</td><td>ab12650</td><td></td></tr><tr><td>Tuj1</td><td>abcam</td><td>ab41489</td><td></td></tr><tr><td>Reelin</td><td>Millipore</td><td>MAB5364</td><td></td></tr><tr><td>Tbr1</td><td>Millipore</td><td>MAB2261</td><td></td></tr><tr><td>NFH</td><td>Dako</td><td>M0762</td><td></td></tr></tbody></table>

developing hipsc derived serum free embryoid bodies for the interrogation of 3-d stem cell cultures using physiologically relevant assays

虽然已经使用hiPSC开发了许多体外疾病模型，但一个限制是这些二维（2-D）系统可能不代表携带疑似疾病变异的受影响个体的基础细胞结构和功能复杂性。传统的二维模型仍然是体内结构的不完整表现，并没有充分地捕捉大脑的复杂性。因此，现在需要更多的基于3-D hiPSC的模型，其可以更好地概括在体内系统中看到的细胞相互作用和功能。 在这里，我们报告了一种基于无血清胚状体（SFEB）从未分化的hiPSC开发3-D系统的方案。这种3-D模型反映了开发的腹侧新皮层的各个方面，并且允许研究活体神经细胞和完整组织的整合功能，例如迁移，连通性，通讯和垫uration。具体来说，我们证明使用我们的方案的SFEB可以使用生理相关和高含量的基于细胞的测定法进行询问，如钙成像和多电极阵列（MEA）记录，而不进行冷冻切片。在MEA记录的情况下，我们证明SFEB在长期培养期间增加了穗活动和网络爆发活动。该SFEB协议提供了一个强大和可扩展的系统，用于研究三维模型中网络形成的研究，捕捉早期皮质发育的方面。

使用我们的技术生长的SFEB产生具有形态学特征的组织，其类似于具有广泛的Tuj1阳性神经元以及神经祖细胞的早期发展的皮质脑室下区域（ 图3A ）。在SFEB的外层和内层观察到许多发育中的皮质玫瑰花结（ 图3B ）。 SFEB的外缘类似于含有神经元神经元的显影皮层。 Brn2的表达支持这一表达，Brn2是最外侧皮质层中神经祖细胞的标志...

Watch this Scientific Journal Video about Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays at JoVE.com

Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

在这里，我们报告了一种由称为无血清胚状体（SFEB）的人诱导多能干细胞（hiPSCs）开发三维（3-D）系统的方案。这种3-D模型可以像器官切片培养物一样用于模拟人类皮质发育和生理学询问发展中的神经回路。

开发HiPSC衍生的无血清胚胎体用于使用生理相关测定的3-D干细胞培养物的询问

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not ...

developing-hipsc-derived-serum-free-embryoid-bodies-for-interrogation

Research

JoVE Journal

Neuroscience

8.8K 次观看.  The Hussman Institute for Autism. 在这里，我们报告了一种由称为无血清胚状体（SFEB）的人诱导多能干细胞（hiPSCs）开发三维（3-D）系统的方案。这种3-D模型可以像器官切片培养物一样用于模拟人类皮质发育和生理学询问发展中的神经回路。 

视频: Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

科研

生物工程

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

从多能干细胞中无血清自由生产三维人类肝球

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

发育生物学

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

基于2D和3D人诱导多能干细胞的模型，用于剖析新皮质发育过程中原发性纤毛受累

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.

We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics.

功能性 3D 球状体细胞培养物的半自动表型分析

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell ...

生物化学

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

人诱导多能干细胞来源的心脏球体的生成、高通量筛选和生物样本库

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

鼠胚体外发育无血清全胚胎培养系统

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

生物学

从 iPSC 高效生成人肠道类器官的基质比较

Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

作者聚焦：使用 iPSC 推进用于药物开发的类器官生成

Organoids have become valuable tools for disease modeling. The extracellular matrix (ECM)&#160;guides cell fate during organoid generation, and using a system that resembles the native tissue can improve model accuracy. This study compares the generation of induced pluripotent stem cells-derived human intestinal organoids in animal-derived ECM and xeno-free hydrogels.

基底膜基质用于人类干细胞维持和肠道类器官生成的比较研究

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) ...

开发HiPSC衍生的无血清胚胎体用于使用生理相关测定的3-D干细胞培养物的询问

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制备纤维蛋白三维支架干细胞培养应用

从多能干细胞中无血清自由生产三维人类肝球

基于2D和3D人诱导多能干细胞的模型，用于剖析新皮质发育过程中原发性纤毛受累

功能性 3D 球状体细胞培养物的半自动表型分析

异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

人诱导多能干细胞来源的心脏球体的生成、高通量筛选和生物样本库

鼠胚体外发育无血清全胚胎培养系统

基底膜基质用于人类干细胞维持和肠道类器官生成的比较研究

基底膜基质用于人类干细胞维持和肠道类器官生成的比较研究