部分支持这项工作是由美国国立卫生研究院授予K99EB009096 - 01A1 JM - M。

1。制备<ol><li> 设为EGM - 2培养基（500毫升） <ul><li>加入100毫升胎牛血清（FBS），395毫升的内皮细胞基础培养基（EBM - 2）。 </li><li>添加5毫升100X谷氨酰胺青霉素，链霉素溶液（GPS）。 </li><li>除了氢化可的松（即血管内皮生长因子，hFGF - B，R - IGF - 1，人表皮生长因子，肝素，抗坏血酸，GA - 1000）加入临时股东大会2 SingleQuots补充。 </li><li> 0.2微米孔径真空过滤机过滤消毒。 </li></ul></li><li> 请MSCGM液（500毫升） <ul><li>加入5毫升至440毫升间质干细胞基础培养基（MSCBM）100X的GPS。 </li><li>添加MSCGM SingleQuots套件的所有内容。 </li><li>添加EGM - 2 SingleQuots补充hFGF - B的等份。 </li><li> 0.2微米孔径真空过滤机过滤消毒。 </li></ul></li><li> 请DMEM培养液（500毫升） <ul><li>添加50毫升胎牛血清（FBS），440毫升的1X高血糖贝科的改良Eagle培养基（DMEM）。 </li><li>添加5毫升的100X GPS。 </li><li>添加不必要的氨基酸溶液5毫升。 </li><li> 0.2微米孔径真空过滤机过滤消毒。 </li></ul></li><li> 使胶原蛋白/纤维连接蛋白溶液（3.6毫升;注射当天 ） <ul><li>加入0.4 mL至10倍的DMEM 150μL蒸馏水H 2 O </li><li>加入100μL1M HEPES 25毫米（决赛）。 </li><li>细心，加入2.4 mL的牛胶原（5毫克/毫升的股票;最后的3毫克/毫升），并在冰轻轻混匀。 </li><li>调整pH值至中性加入1N氢氧化钠溶液（约30μL）。 </li><li>使用酚红指示剂，以评估中性pH值，替代，使用pH试纸监测pH值。 </li><li>添加120μL人纤维连接蛋白（1 mg / mL的股票; 30微克/毫升最终） </li><li> FB的0.4毫升（10％最终） </li><li>继续，直到用冰的解决方案。 </li></ul></li><li> 使纤维蛋白原溶液（1毫升，注射当天） <ul><li>添加30毫克粉状纤维蛋白原1毫升0.9N氯化钠（pH 7.4）的溶液（30毫克/毫升的决赛）。 </li><li>在使用之前，纤维蛋白原溶液在37 ° C 30分钟。 </li><li>轻轻混匀，无震荡，除了胶原蛋白/纤维连接蛋白凝胶前。 </li></ul></li><li> 使凝血酶溶液（200毫升） <ul><li>新增1 200毫升0.9％氯化钠生理盐水（50 U / mL）的粉状凝血酶的Ku。 </li><li>过滤0.2微米的孔径注射器过滤器和存储在-20 ° C，直到使用的消毒。 </li><li>使用前，用0.9％NaCl到终浓度为10 U / ml的生理盐水稀释。 </li></ul></li><li> 1％的明胶溶液（500毫升 ） <ul><li>添加5克粉状明胶500毫升贝科磷酸盐缓冲液（PBS）。 </li><li>蒸压在121 ° C为30分钟。 </li><li> 0.2微米孔径真空过滤机过滤消毒。 </li></ul></li><li> 大衣 100毫米的1％明胶涂层溶液组织培养板<ul><li>加入10毫升1％的明胶溶液，以每100毫米的组织培养板。 </li><li>孵育板在37 ° C下60分钟。 </li><li>在使用之前，删除明胶溶液，并用PBS洗一次板块。 </li></ul></li></ol> 2。文化的人脐血血源性内皮细胞集落形成细胞（ECFCs） 这个协议假定ECFC冷冻小瓶是在此之前实验的实验室。 ECFC可以隔离要么脐带血或如前面描述的7成人外周血单个核细胞的一小部分。 <ol><li>解冻ECFCs小瓶（通常为0.5 - 1 × 10 6细胞在1毫升冻结媒体）从液氮储存罐，并立即采取淡化其内容中含有10毫升的DMEM培养基的15 mL锥形管。 1200 5分钟的转速旋转。去除上清液，重悬细胞沉淀在温暖的EGM - 2中的10毫升。 </li><li>添加到1％明胶包100毫米的组织培养板ECFC悬浮液10毫升。在37 ° C和5％的CO 2，放置在培养箱板。第二天，轻轻吸出未结合的细胞和介质，和饲料用10毫升新鲜EGM - 2培养基势必细胞。 </li><li>饲料板块EGM - 2培养基，每2-3天。允许细胞的扩大等，是一个融合的细胞单层覆盖板。在汇合，传代培养细胞如下: </li><li>吸出培养液中，用10毫升的PBS洗细胞。 </li><li>删除PBS和胰蛋白酶EDTA溶液加2毫升，每100毫米板。轻轻摇动板均匀分布的胰蛋白酶- EDTA溶液。在37 ° C和5％CO 2为3-5分钟。轻轻敲击板倒置显微镜下看到的悬浮液中分离的细胞。 </li><li>当细胞完全分离，EGM - 2培养基中添加8 mL和收集的细胞溶液TION到15 mL锥形管。取10μL，血球计数细胞和收获细胞总数。 </li><li>板细胞在1％明胶涂层的组织在5000细胞/厘米的播种密度的培养板2使用EGM - 2培养基。在孵化器板和饲料用EGM - 2培养基，每隔2-3天。 </li></ol>后续段落重复此过程。细胞群扩大，保持传代次数的轨道。 ECFCs将用于通道4-8之间。 3。人体骨骼的文化骨髓间充质干细胞（MSCs） 这个协议假定人类MSC的冷冻小瓶在此之前实验的实验室。 MSC可以分离出骨髓吸如先前所描述的11。 <ol><li>解冻后的MSCs的小瓶（通常为0.50 - 1 × 10 6细胞在1 mL冻结媒体）采取从液氮储存罐，并立即淡化其内容中含有10毫升的DMEM培养基的15 mL锥形管。 1200 5分钟的转速旋转。去除上清液和细胞沉淀重新悬浮在10毫升的温暖MSCGM介质。 </li><li>添加到一个无涂层100毫米的组织培养板MSC的悬浮液10毫升。在37 ° C和5％的CO 2，放置在培养箱板。第二天，轻轻吸出未结合的细胞和介质，用10毫升新鲜MSCGM培养基和饲料绑定细胞。 </li><li>饲料板块MSCGM中期每2-3天。允许细胞扩大等板块达到80％的融合细胞单层。在80％汇合，传代培养细胞如下: </li><li>吸出培养液中，用10毫升的PBS洗细胞。 </li><li>删除PBS和胰蛋白酶EDTA溶液加2毫升，每100毫米板。轻轻摇动板均匀分布的胰蛋白酶- EDTA溶液。在37 ° C和5％CO2为3-5分钟。轻轻敲击板倒置显微镜下看到的悬浮液中分离的细胞。 </li><li>当细胞完全分离，加入8毫升MSCGM介质和细胞溶液收集到15 mL锥形管。取10μL，血球计数细胞和收获细胞总数。 </li><li>板在10,000细胞/厘米2使用MSCGM中期播种密度在无涂层的组织培养板的细胞。在孵化器板和饲料MSCGM中期每2-3天。 </li></ol>后续段落重复此过程。细胞群扩大，保持传代次数的轨道。干细胞将被用于通道4-8之间。 4。再悬浮在胶原蛋白/纤维连接蛋白/纤维蛋白原解决方案的单元格（0天） 实验之前，确保有足够ECFCs和文化的MSCs; 0.8x10 6 ECFCs和1.2x10 6细胞将需要为每个植入和鼠标。 <ol><li>吸出的每一种文化板的媒介，并用10毫升的PBS洗细胞。删除PBS和胰蛋白酶EDTA溶液加2毫升，每100毫米板。轻轻摇动板均匀分布的胰蛋白酶- EDTA溶液。孵育3-5分钟。轻轻敲击板倒置显微镜下看到的悬浮液中分离的细胞。 </li><li>当细胞完全分离，加8 mL DMEM培养基中，收集到15 mL锥形管的细胞溶液。取10μL，血球计数细胞和工作ECFCs和MSCS收获总数。 </li><li>传输4 × 6 ECFCs（5倍0.8x10 6细胞）和6x10 6储值卡（5倍1.2x10 6细胞）成一个单一的50 mL锥形管。这是五个人的植入物和小鼠所需的细胞的总金额。在1200转离心，去除上清。 </li><li>轻轻地，新增100 UL纤维蛋白原溶液0.9毫升胶原蛋白/纤维连接蛋白溶液（3 mg / mL的最终纤维蛋白原浓度）;保持冰的混合物。 </li><li>重悬细胞沉淀1毫升的冰冷的胶原蛋白/纤维连接蛋白/纤维蛋白原的解决方案;混合细胞非常轻柔，以避免气泡。装入1 mL无菌注射器的混合物，其注射器的针尖上的第一个26号针头的地方。继续，直到注射冰装入注射器。 </li></ol> 5。注射到免疫缺陷的裸鼠（0天） 所有的动物实验将进行6周岁的无胸腺裸小鼠（NU / NU）。 <ol><li>注射前，放置成气室提供异氟醚麻醉的免疫缺陷小鼠。让小鼠吸入约2分钟，直到他们的异氟醚麻醉，反应迟钝到脚趾捏（监督检查他们的心跳）。 </li><li>对于每个鼠标，皮下上部背地区使用的26号针头注入凝血酶溶液50μL（10 U / ml的）。 </li><li&gt;在同一个地方，凝血酶注射，注入200μL的细胞混合使用的26号针头。胶原凝胶将在37 ° C和纤维蛋白原形成纤维蛋白凝胶中存在的凝血酶。因此，植入应该形成一个小的，但明显的，凹凸的皮肤下。 </li><li>注射后，小鼠放在一层纱布的舒适和温暖，并观察他们，直到他们成为日间。再经过前三天每天观察小鼠。 </li></ol> 6。收获（7日） <ol><li>注射一个星期后，安乐死的小鼠，由他们提供压缩的CO 2气体转化为气体室。 </li><li>一旦安乐死，切开皮肤注射区附近，手术取出凝胶插件。数码照片的检索与规模凝胶插头建议。 </li><li>病理卡带放入收获的凝胶插头，并深入到10％中性缓冲福尔马林室温过夜。 </li><li>固定后，洗净中性缓冲福尔马林，用蒸馏水H 2 O，10％和病理磁带放置在4 ° C直到在PBS组织学评价。 </li></ol> 7。评价:病理切片（H＆E）和免疫​​组织化学（hCD31） <ol><li>对于组织的评价，植入嵌入式石蜡切片（7微米厚的部分）使用标准的组织学程序。 </li><li> 量化评价从中间部分植入Hematoxilin和曙红染色（H＆E）微血管密度。 H＆E染色的标准协议，可以在别处找到。微血管，可以被认定为含有红细胞lumenal结构。报告微血管密度平均数量的红血细胞填充的微血管，从分析和船舶/毫米2表示等领域。 </li><li>为了证明人性的微血管血管，检索植入部分应免疫组织化学染色与一个人类特有的CD31（hCD31）抗体，使用标准的染色协议。我们建议，使用DakoCytomation（JC70A克隆猫＃M0823）。单克隆鼠抗人CD31抗体在1:100稀释。 7,11并行染色切片小鼠组织的多样性得到的负面反应已被证实的人类特异性抗体。值得注意的是，往往是看到鼠标血管内植入物（特别是周围的边界），但鼠标的船只不会弄脏这种抗体阳性。此外，人类MSC的存在可以与人类抗CD90或α-平滑肌肌动蛋白（α- SMA）的特异性抗体的免疫组织化学染色检测。人类的MSC发现，无论是在新形成的血管，血管周围地区和间隙位于整个植入11。 </li></ol> 8。代表性的成果<img alt="图1" src="/files/ftp_upload/3065/3065fig1.jpg" /> 图1。 ECFC和MSC文化的典型外观。相衬显微照片显示的ECFCs和干细胞在培养典型外观。 （一）聚合单层显示的内皮细胞的形态特征的鹅卵石石的脐血源性ECFCs。 （b）人类骨髓来源的MSCs显示纺锤形形态。倍线器酒吧，200微米。 <img alt="图2" src="/files/ftp_upload/3065/3065fig2.jpg" /> 图2。第7天explanted插头外观 。人脐血衍生ECFCs和骨骨髓源性干细胞包埋在胶原蛋白/纤维连接蛋白/纤维蛋白凝胶，植入到裸鼠皮下，如文中所述。 （一）经过7天，一旦鼠标已经安乐死，切开皮肤注射区附近和揭露翻转皮肤细胞/凝胶插件。 （二）插件的外观从鼠标和前福尔马林固定手术切除。红色的植入是一个血管的迹象。 <img alt="图3" src="/files/ftp_upload/3065/3065fig3.jpg" /> 图3。 explanted插头的血管网的组织学鉴定。 Hematoxilin和曙红染色（H＆E）采取从explanted插头的中间部分。 （一）低倍率（10倍）的显微照片显示在宿主组织周围的背景下（即，脂肪组织和骨骼肌）的种植体（由黄色虚线标示）。 （b）高倍率（40倍）显微照片显示多个微血管（黄色箭头指向其中的一些）插头内微血管，可以被认定为含有红细胞lumenal结构。 <img alt="图4" src="/files/ftp_upload/3065/3065fig4.jpg" /> 图4。人类的免疫组化鉴定流明。免疫组织化学染色切片explanted插头的中间部分。染色进行DakoCytomation（JC70A克隆猫＃M0823）。使用单克隆鼠抗人CD31（hCD31）抗体在1:100稀释;细胞核与hematoxilin counterstained。 （一）低倍率（10倍）的显微照片显示在周围宿主组织中植入（由黑色虚线划定）。人权的具体化，CD31阳性的微血管被染成棕色（过氧化物酶染色）。 （二）高倍率显微镜（40X）插头内显示多个人类微血管（黑箭头指向一些hCD31阳性流明）。

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	<li>Melero-Martin, J. M., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+13.+An+in+vivo+experimental+model+for+postnatal+vasculogenesis.">Chapter 13. An in vivo experimental model for postnatal vasculogenesis.</a> Methods Enzymol. 445, 303-329 (2008).</li><li>Koike, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering:+creation+of+long-lasting+blood+vessels.">Tissue engineering: creation of long-lasting blood vessels.</a> Nature. 428, 138-139 (2004).</li><li>Schechner, 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=In+vivo+formation+of+complex+microvessels+lined+by+human+endothelial+cells+in+an+immunodeficient+mouse.">In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse.</a> Proc Natl Acad Sci USA. 97, 9191-9196 (2000).</li><li>N&amp;ouml, r., E, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+and+characterization+of+functional+human+microvessels+in+immunodeficient+mice.">Engineering and characterization of functional human microvessels in immunodeficient mice.</a> Lab Invest. 81, 453-463 (2001).</li><li>Lin, Y., Weisdorf, D. J., Solovey, A., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+circulating+endothelial+cells+and+endothelial+outgrowth+from+blood.">Origins of circulating endothelial cells and endothelial outgrowth from blood.</a> J Clin Invest. 105, 71-77 (2000).</li><li>Ingram, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+hierarchy+of+endothelial+progenitor+cells+using+human+peripheral+and+umbilical+cord+blood.">Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.</a> Blood. 104, 2752-2760 (2004).</li><li>Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+vasculogenic+potential+of+human+blood-derived+endothelial+progenitor+cells.">In vivo vasculogenic potential of human blood-derived endothelial progenitor cells.</a> Blood. 109, 4761-4768 (2007).</li><li>Au, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+in+vivo+potential+of+endothelial+progenitor+cells+from+human+umbilical+cord+blood+and+adult+peripheral+blood+to+form+functional+long-lasting+vessels.">Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels.</a> Blood. 111, 1302-1305 (2008).</li><li>Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Traktuev, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+Functional+Vascular+Network+Formation+In+Vivo+by+Cooperation+of+Adipose+Progenitor+and+Endothelial+Cells.">Robust Functional Vascular Network Formation In Vivo by Cooperation of Adipose Progenitor and Endothelial Cells.</a> Circ Res. 104, 1410-1420 (2009).</li><li>Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+robust+and+functional+vascular+networks+in+vivo+with+human+adult+and+cord+blood-derived+progenitor+cells.">Engineering robust and functional vascular networks in vivo with human adult and cord blood-derived progenitor cells.</a> Circ Res. 103, 194-202 (2008).</li><li>Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+Myeloid+Cells+Are+Necessary+for+Creating+Bioengineered+Human+Vascular+Networks+In+Vivo.">Host Myeloid Cells Are Necessary for Creating Bioengineered Human Vascular Networks In Vivo.</a> Tissue Eng Part A. 16, 2457-2466 (2010).</li></ol>

没有利益冲突的声明。

这是一个生物工程人体血管网络的实验模型。这种模式的主要特点是:1）微血管形成从产后组织（即血液和骨髓）中分离出的人类细胞; 2）微血管形成成年动物; 3）微血管不会出现从前期现有船舶（主机），而是他们形成， 从头 ，从单细胞悬浮在一个合适的凝胶。 血管生成中起着重要的作用，因为在这个实验中小鼠血管连接，以实现红细胞充满血管，在这个实验中的功能读出。此外，主机髓细胞招募初期移植后植入，他们的新的血管床12的形成所必需的。 这个实验模型提供了一个多功能的，量化的，和相对简单的的模型系统， 研究产后体内人体血管网络的形成。此外，这个实验很简单，执行的，它并不需要一个切口或手术。该模型可以用来研究的不同来源的人类内皮细胞和血管周围的血管生成潜力。该模型可以用于筛选抗和/或亲血管生成化合物。最后，该模型可以用来研究人类内皮细胞组成的血管网的形成和功能的特定基因的作用在（S）。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>内皮细胞基础培养基，EBM - 2 </td><td>龙沙</td><td> CC - 3156 </td><td></td></tr><tr><td> EGM - 2 SingleQuot补充剂</td><td>龙沙</td><td> CC - 3162 </td><td></td></tr><tr><td>谷氨酰胺青霉素，链霉素溶液，100倍的GPS </td><td> Mediatech，公司</td><td> 30 - 009 - CI </td><td></td></tr><tr><td>间质干细胞生长的培养基BulletKit，MSCBM / MSCGM </td><td>龙沙</td><td> PT - 3001 </td><td></td></tr><tr><td> 1X的DMEM高糖贝科的改良Eagle培养基， </td><td> Invitrogen公司</td><td> 11995-073 </td><td></td></tr><tr><td> MEM非必需氨基酸溶液，100X NEAA </td><td> Invitrogen公司</td><td> 11140-050 </td><td></td></tr><tr><td>高血糖贝科的改良Eagle，粉状的DMEM </td><td> Invitrogen公司</td><td> 12100-046 </td><td></td></tr><tr><td> HEPES缓冲液</td><td> Invitrogen公司</td><td> 15630-080 </td><td></td></tr><tr><td> Cultrex牛胶原我的解决方案</td><td> Trevigen </td><td> 3442-050-01 </td><td></td></tr><tr><td> Cultrex人纤维连接蛋白</td><td> Trevigen </td><td> 3420-001-01 </td><td></td></tr><tr><td>从牛血浆纤维蛋白原</td><td> Sigma - Aldrich公司</td><td> F8630 - 1G </td><td></td></tr><tr><td>明胶</td><td>费舍尔</td><td> DF0143 - 17 - 9 </td><td></td></tr><tr><td>贝科的磷酸盐缓冲液，PBS </td><td> Invitrogen公司</td><td> 14190-250 </td><td></td></tr><tr><td>胰蛋白酶- EDTA溶液，1个</td><td> Invitrogen公司</td><td> 25300-054 </td><td></td></tr><tr><td>异氟醚吸入液体</td><td> Buxter医疗保健公司</td><td> NDC 10019-360-40 </td><td></td></tr><tr><td>从牛凝血酶等离子</td><td> Sigma - Aldrich公司</td><td> T4648 - 1千片</td><td></td></tr><tr><td>中性缓冲福尔马林溶液中， </td><td> Sigma - Aldrich公司</td><td> HT501128 </td><td></td></tr><tr><td>单克隆鼠抗人CD31抗体</td><td> DakoCytomation </td><td> M0823 </td><td>克隆JC70A </td></tr></tbody></table>

bioengineering human microvascular networks in immunodeficient mice

组织工程和组织再生的细胞疗法的未来很可能会依靠我们的能力，在体内产生的功能的血管网络。在这方面，实验模型建立在体内的血管网络搜索是最重要的 1 。在体内的生物工程微血管网络的可行性，首次使用人体组织派生成熟的内皮细胞 （ECs）2-4所示，然而，这种自体血管内皮细胞，为临床广泛使用存在的问题，因为它们很难获得足够的数量，并要求从现有的血管收获。这些限制煽动其他来源的内皮细胞的搜索。 （ECFCs）在血液中内皮细胞集落形成细胞的识别非侵入性的获得提供了一个机会内皮5-7 。我们和其他作者已经表明，成人和脐带血衍生ECFCs的能力， 形成 体内 7-11功能性血管网络。更重要的是，这些研究还表明，获得稳定和持久的血管网络，ECFCs需要与血管周围细胞植入合作。我们在这里描述的检测说明了这一概念:我们显示人类脐带血衍生ECFCs可与骨髓间充质干细胞（MSCs）合并为一个单细胞悬液，在胶原蛋白/纤维连接蛋白/纤维蛋白原凝胶，形成一个功能的人类后7日内到免疫缺陷小鼠植入的血管网。植入的指示，不仅形成一个血管网（ 从头 ），而且还与主机循环系统的功能吻合的发展可以看出整个人类ECFC内衬流明包含主机红细胞的存在。这非常适合对人体血管网形成的细胞和分子机制研究及血管设计组织的战略，以发展生物工程人血管网络的小鼠模型。

Watch this Scientific Journal Video about Bioengineering Human Microvascular Networks in Immunodeficient Mice at JoVE.com

Bioengineering Human Microvascular Networks in Immunodeficient Mice

在这里，我们描述了一种方法来提供人脐血源性内皮细胞集落形成细胞（ECFCs）和骨髓来源的间质干细胞（MSCS），胶原蛋白/纤维连接蛋白凝胶中嵌入皮下到免疫缺陷小鼠，。这种细胞/凝胶相结合，产生一个人的血管网，用鼠标血管连接。

生物人类免疫缺陷小鼠的微血管网络

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

bioengineering-human-microvascular-networks-in-immunodeficient-mice

Research

JoVE Journal

Biology

12.2K 次观看.  Harvard Medical School. 在这里，我们描述了一种方法来提供人脐血源性内皮细胞集落形成细胞（ECFCs）和骨髓来源的间质干细胞（MSCS），胶原蛋白/纤维连接蛋白凝胶中嵌入皮下到免疫缺陷小鼠，。这种细胞/凝胶相结合，产生一个人的血管网，用鼠标血管连接。

视频: Bioengineering Human Microvascular Networks in Immunodeficient Mice

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

使用微机电系统（MEMS）开发诊断工具

科研

生物学

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + T淋巴细胞艾滋病毒的捕获使用一次性微流体芯片

Title Cell Encapsulation by Droplets

通过飞沫标题细胞封装

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

组织工程细胞在三维凝胶封装

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

卡哈尔（ICC）的网络的间质细胞在小鼠体内的可视化

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

共培养内皮细胞介导成熟的人类胚胎干细胞向胰腺中胰岛素生成细胞在定向分化的方法

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

RNA-seq的凝血酶处理和控制人肺微血管内皮细胞的转录组分析

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

选择反应监测质谱法进行绝对定量蛋白质

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

测定野生型肝脏和牛奶样品中的唾液酸<em&gt; CMAH</em&gt;敲出小鼠

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...