Name: the aortic ring co-culture assay: a convenient tool to assess the angiogenic potential of mesenchymal stromal cells in vitro
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作者感谢以下工作人员和研究人员: Andrée Gauthier-费希尔, 马修 Librach, 坦尼娅先生, Tharsan Velauthapillai 和萨拉 Laronde。

所有涉及动物的研究都是按照到达指南 29 进行的。所有研究都是通过机构研究伦理委员会的批准 (REB 号 4276) 进行的。所有动物程序均由大学卫生网络 (加拿大多伦多) 动物保育委员会批准, 所有动物都接受了符合《 动物保育和使用指南》的人道护理 , 8 th 版 (国立卫生研究院 2011). 
 1. 主动脉环试验装置 <ol> <li> 将大鼠主动脉分离。
	<ol> <l...

<ol>
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D., Laschke, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geraniol+Suppresses+Angiogenesis+by+Downregulating+Vascular+Endothelial+Growth+Factor+(VEGF)/VEGFR-2+Signaling.">Geraniol Suppresses Angiogenesis by Downregulating Vascular Endothelial Growth Factor (VEGF)/VEGFR-2 Signaling.</a> PLoS One. 10 (7), e0131946 (2015).</li><li>Masson, V. V., 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=Mouse+Aortic+Ring+Assay:+A+New+Approach+of+the+Molecular+Genetics+of+Angiogenesis.">Mouse Aortic Ring Assay: A New Approach of the Molecular Genetics of Angiogenesis.</a> Biol Proced Online. 4, 24-31 (2002).</li><li>Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mesenchymal+stem+cells+for+tissue+engineering+versus+regenerative+medicine.">Adult mesenchymal stem cells for tissue engineering versus regenerative medicine.</a> J Cell Physiol. 213 (2), 341-347 (2007).</li><li>Caplan, A. I., Dennis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+as+trophic+mediators.">Mesenchymal stem cells as trophic mediators.</a> J Cell Biochem. 98 (5), 1076-1084 (2006).</li><li>Keating, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells:+new+directions.">Mesenchymal stromal cells: new directions.</a> Cell Stem Cell. 10 (6), 709-716 (2012).</li><li>Kilroy, G. E., 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=Cytokine+profile+of+human+adipose-derived+stem+cells:+expression+of+angiogenic,+hematopoietic,+and+pro-inflammatory+factors.">Cytokine profile of human adipose-derived stem cells: expression of angiogenic, hematopoietic, and pro-inflammatory factors.</a> J Cell Physiol. 212 (3), 702-709 (2007).</li><li>Tang, Y. 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=Paracrine+action+enhances+the+effects+of+autologous+mesenchymal+stem+cell+transplantation+on+vascular+regeneration+in+rat+model+of+myocardial+infarction.">Paracrine action enhances the effects of autologous mesenchymal stem cell transplantation on vascular regeneration in rat model of myocardial infarction.</a> Ann Thorac Surg. 80 (1), 229-236 (2005).</li><li>Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+MSCs+are+pericytes?.">All MSCs are pericytes?.</a> Cell Stem Cell. 3 (3), 229-230 (2008).</li><li>Hong, S. H., 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=Ontogeny+of+human+umbilical+cord+perivascular+cells:+molecular+and+fate+potential+changes+during+gestation.">Ontogeny of human umbilical cord perivascular cells: molecular and fate potential changes during gestation.</a> Stem Cells Dev. 22 (17), 2425-2439 (2013).</li><li>Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+bioscience+research+reporting:+The+ARRIVE+guidelines+for+reporting+animal+research.">Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research.</a> J Pharmacol Pharmacother. 1 (2), 94-99 (2010).</li><li>Sarugaser, R., Ennis, J., Stanford, W. L., Davies, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+propagation,+and+characterization+of+human+umbilical+cord+perivascular+cells+(HUCPVCs).">Isolation, propagation, and characterization of human umbilical cord perivascular cells (HUCPVCs).</a> Methods Mol Biol. 482, 269-279 (2009).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Gelati, M., Aplin, A. C., Fogel, E., Smith, K. D., Nicosia, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+angiogenic+response+of+the+aorta+to+injury+and+inflammatory+cytokines+requires+macrophages.">The angiogenic response of the aorta to injury and inflammatory cytokines requires macrophages.</a> J Immunol. 181 (8), 5711-5719 (2008).</li></ol>

在建立一个成功的主动脉环试验 MSC 共培养实验的几个关键阶段。首先, 分离和剖胸主动脉的最重要步骤是: 1) 完全获得主动脉的胸椎段;2) 仔细去除分支血管、结缔组织和脂肪组织;3) 切割甚至部分主动脉 (〜 1 mm), 以限制每一个化验之间的变异性。第二, 成功地将主动脉环植入 BME 是该方法的关键。如果 BME 没有完全聚合或聚合不均匀, BME 中嵌入的主动脉环将无法启动内皮发芽, 或者它们可能发育不?...

血管生成的复杂过程改善和维持组织灌注, 促进新的血管发育从预先存在的血管1。它是一个严密调控, 平衡的过程由 pro-angiogenic 和血管因素。该系统的任何缺陷都可能导致血管维持或生长不足, 导致严重的缺血性疾病, 包括心肌病、中风和神经退行性疾病。然而, 夸大的血管发育是典型的条件, 包括癌症和炎症性疾病2。
开发旨在控制血管生成以获得良好组织再生的治疗方法至关重要。尽管进行了广泛的前临床研究, 但使用 pro-angiogenic 因子和 rna 刺激血管生成的尝试未能达到预期的结果3,4,5。暂时性影响的可能原因包括: 血管生成蛋白和核酸的寿命有限, 以及靶生长因子的有限数量6,7。虽然可溶性血管生成因子对启动血管形成至关重要, 但血管的维持和功能依赖于支持的细胞类型, 包括周和平滑肌细胞8。pro-angiogenic 疗法的领域现在正在探索潜在的干细胞和祖细胞来源, 可能提供局部血管生成因子, 而物理支持新发展的血管, 更新甚至分化成内皮样细胞9,10。寻找能够满足这些功能要求的最佳血管生成细胞类型对缺血性组织再生有很大的希望。
为了成功地将可能的细胞疗法转化为临床试验, 前期研究需要证明它们的功效, 并强调潜在的血管生成机制。尽管已经建立了大量的血管生成试验, 但该领域缺乏一个 "金标准" 的体外检测方法, 可以可靠地评估潜在候选细胞类型的有效性11,12, 13. 大多数体外, 血管生成检测 (包括内皮细胞增殖、迁移和试管形成分析) 通常评估细胞或化合物对血管型变化或分化的影响管状和网络结构14,15。虽然这些功能对血管生成至关重要, 但 "可翻译" 的化验还应评估: 1) 增强或替换支持的细胞类型, 包括周或平滑肌细胞, 2) 处理 ECM 和/或基底膜, 和 3)促进功能性微血管形成的效率。在体内血管生成模型, 包括角膜检测和基质塞检测, 概括了独特的体内微环境, 但由于跟踪被管理的细胞观察物理相互作用的困难而面临挑战。此外, 在体内模型中, 异免疫排斥可能发生在测试潜在的异基因细胞治疗候选者16。前体血管生成模型, 特别是主动脉环试验可以提供: 1) 管状结构, 2) 辅助支持细胞, 3) 从主机和人工用品的 ECM, 4) 排斥炎症组件和 5) 快速且价格低廉的设置17,18。通常, 主动脉环试验可以检测小分泌蛋白、药理制剂和转基因啮齿动物模型的血管生成电位19,20,21。
骨髓间充质干细胞主要通过其分泌介导的效果22,23,24, 有望成为血管再生的候选对象。骨髓间充质干细胞已被证实能分泌血管内皮生长因子 (VEGF)、肝细胞生长因子 (HGF)、胰岛素样生长 Factor-1 (IGF-1)、碱性成纤维细胞生长因子 (bFGF) 和 angiopoeitin-1 (Ang-1) 的主要血管生成因子 (25 ,26。MSCs 也可以检测到缺血性或发炎的组织, 但确切的机制仍在调查中。越来越多的文献支持的假说, 大多数 MSCs 产生的血管细胞, co-express 细胞标记, 并可以表现出像周27。HUCPVCs 是人类脐带血管区的一个年轻的骨髓间充质干细胞来源。它们代表了具有细胞样性质的 MSCs 的种群, 并且从 FTM 和足月的脐带中都有特征。FTM HUCPVCs 展示了细胞标记的高表达, 包括 CD146 和 NG2, 高增殖和多向电位, 免疫特权属性, 并显示一个健壮的分泌配置文件28。FTM HUCPVCs 是一个理想的候选细胞类型, 通过促进新的血管, 通过其细胞的性质, 促进损伤组织再生。
为了测试人类骨髓间充质干细胞的血管生成潜能和细胞样的特性, 在积极的 angiotropic 迁移 (以下简称为 "归巢")、ECM 处理和发展物理在获得微血管发育的定量数据的同时, 可以研究细胞类型之间的相互作用。
在此, 我们提出了一个协议, 描述了一个新的应用的主动脉环试验。人骨髓间充质干细胞与开发大鼠主动脉内皮网络共, 以评估其对管的形成、成熟和稳态的贡献。这一版本的主动脉环试验评估细胞治疗候选者的能力和效力的地方, 血管生成, 执行和调解 ECM 处理, 并促进内皮管发展通过建立细胞样的物理相互.除了量化 mscs 对体外内皮网络形成和观察细胞间相互作用的净效应之外, 我们还优化了从 co-cultures 中分离 mscs 的协议。通过执行流式细胞仪和 qPCR, 可以表征 MSC 表型和基因表达在共培养后的变化。作为模型细胞类型, 我们比较了 ontogenetically 早期 (产前) 和晚期 (成人) 的人骨髓间充质干细胞的来源: FTM HUCPVCs 和人骨骨髓间充质干细胞 (骨髓), 分别在主动脉环试验。我们建议, 在研究血管生成再生应用时, 主动脉环法可用于分析任何物理支持细胞类型的血管生成潜能。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Alpha-MEM</td>
			<td >Gibco</td>
			<td >12571071</td>
			<td >For FTM HUCPVC and BMSC culture media.</td>
		</tr>
		<tr >
			<td >APC-conjugated anti human TRA-1-85</td>
			<td >R&#38;D Systems</td>
			<td >FAB3195A</td>
			<td >Human-specific cell marker for flow cytometry and cell sorting</td>
		</tr>
		<tr >
			<td >Basal membrane extract (BME) (Matrigel)</td>
			<td >Corning</td>
			<td >354234</td>
			<td >For aorta embedding</td>
		</tr>
		<tr >
			<td >Bullet-kit</td>
			<td >Lonza</td>
			<td >CC-3162</td>
			<td >Includes: Gentamicin/Amphotericin-B 
			(GA)human Epidermal 
			Growth Factor (hEGF); Vascular 
			Endothelial Growth Factor (VEGF); R3- 
			Insulin-like Growth Factor-1 (R3-IGF-1); 
			Ascorbic Acid; Hydrocortisone; 
			human Fibroblast Growth Factor-Beta (hFGF-&#946;); Heparin; Fetal Bovine Serum 
			(FBS). Required to prepare EGM</td>
		</tr>
		<tr >
			<td >CellTracker Green CMFDA Dye</td>
			<td >Thermo-fisher</td>
			<td >C2925</td>
			<td >For staining MSCs, green is picked up optimally by MSCs</td>
		</tr>
		<tr >
			<td >CKX53 Culture Microscope</td>
			<td >Olympus</td>
			<td ></td>
			<td >For bright-field imaging of endothelial network development</td>
		</tr>
		<tr >
			<td >Countess automated cell counter</td>
			<td >Invitrogen</td>
			<td >C10227</td>
			<td >Cell counting for MSC culture, flow cytometry and qPCR</td>
		</tr>
		<tr >
			<td >Dispase</td>
			<td >StemCell technologies</td>
			<td >7923</td>
			<td >For dissociating aortic ring-MSC co-cultures (pre-warm at 37 &#176;C)</td>
		</tr>
		<tr >
			<td >Disposable sterile scalpels</td>
			<td >VWR</td>
			<td >21909-654</td>
			<td >For sectioning aorta</td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s phosphate buffered saline</td>
			<td >Sigma-Aldrich</td>
			<td >D8537</td>
			<td >PBS. 1X, Without calcium chloride and magnesium chloride</td>
		</tr>
		<tr >
			<td >Endothelial basal media (EBM)</td>
			<td >Lonza</td>
			<td >CC-3156</td>
			<td >Basal media required for culturing aortic ring assay-MSC co-cultures (warm at 37 &#176;C before use). Required for EGM and EBM-FBS</td>
		</tr>
		<tr >
			<td >Ethanol, 70%, Biotechnology Grade</td>
			<td >VWR</td>
			<td >97064-768</td>
			<td >To sterilize surfaces</td>
		</tr>
		<tr >
			<td >EVOS</td>
			<td >Life Technologies</td>
			<td ></td>
			<td >In-house fluorescent microscope to track MSC migration and integration</td>
		</tr>
		<tr >
			<td >Fetal bovine serum (FBS) (Hyclone)</td>
			<td >GE Healthcare</td>
			<td >SH3039603</td>
			<td >Serum component of cell culture medium</td>
		</tr>
		<tr >
			<td >FITC-conjugated anti-CD31 antibody</td>
			<td >BD</td>
			<td >558068</td>
			<td >Human endothelial marker for flow cytometry</td>
		</tr>
		<tr >
			<td >FITC-conjugated anti-CD146antibody</td>
			<td >BD</td>
			<td >560846</td>
			<td >Human pericyte marker for flow cytometry</td>
		</tr>
		<tr >
			<td >Forceps</td>
			<td >Almedic</td>
			<td >7727-A10-704</td>
			<td >For handing rat tissue. Can use any similar forceps</td>
		</tr>
		<tr >
			<td >Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td >Life Technologies</td>
			<td >14175-094</td>
			<td >1X Without calcium chloride and magnesium chloride</td>
		</tr>
		<tr >
			<td >HERAcell 150i CO2 Incubator</td>
			<td >Thermo Fisher Scientific</td>
			<td >51026410</td>
			<td >For incubating cells</td>
		</tr>
		<tr >
			<td >Human Angiogenesis RT2 profiler PCR array</td>
			<td >Qiagen</td>
			<td >PAHS-024Z</td>
			<td >Human specific and includes primers for 84 genes involved in angiogenesis. Each well is 1 primer reaction</td>
		</tr>
		<tr >
			<td >ImageJ</td>
			<td ></td>
			<td ></td>
			<td >Open source image processing software. Require Angiogenesis analyzer plugin</td>
		</tr>
		<tr >
			<td >LSR II</td>
			<td >BD</td>
			<td ></td>
			<td >UHN SickKids FC Facility. For flow cytometry.</td>
		</tr>
		<tr >
			<td >MoFlo Astrios</td>
			<td >Beckman Coulter</td>
			<td ></td>
			<td >UHN SickKids FC Facility. For cell sorting.</td>
		</tr>
		<tr >
			<td >Penicillin/streptomycin</td>
			<td >Gibco</td>
			<td >15140122</td>
			<td >Antibiotic component to buffers and cell culture medium</td>
		</tr>
		<tr >
			<td >RNeasy Mini Kit</td>
			<td >Qiagen</td>
			<td >74104</td>
			<td >For RNA purification. Includes cell lysis buffer</td>
		</tr>
		<tr >
			<td >RT2Easy First Strand Kit</td>
			<td >Qiagen</td>
			<td >330421</td>
			<td >For preparation of cDNA for qPCR</td>
		</tr>
		<tr >
			<td >RT2PreAMP cDNA Synthesis Kit</td>
			<td >Qiagen</td>
			<td >330451</td>
			<td >Pre-amplification of cDNA if low-yield RNA</td>
		</tr>
		<tr >
			<td >Surgical scissors</td>
			<td >Fine Science Tools</td>
			<td >14059-11</td>
			<td >For cutting skin, muscle and aorta</td>
		</tr>
		<tr >
			<td >Sterile gauze</td>
			<td >VWR</td>
			<td >3084</td>
			<td >To dampen and sterilize chest fur</td>
		</tr>
		<tr >
			<td >TrypleE</td>
			<td >Thermo Fisher Scientific</td>
			<td>12605036</td>
			<td >MSC dissociation enzyme pre-warm at 37 &#176;C</td>
		</tr>
		<tr >
			<td >0.2 &#956;m pore filtration unit</td>
			<td >Thermo Fisher Scientific</td>
			<td >566-0020</td>
			<td >To sterilize tissue culture media</td>
		</tr>
		<tr >
			<td >0.25% Trypsin/EDTA</td>
			<td >Gibco</td>
			<td >25200056</td>
			<td >For cell dissociation, pre-warm at 37 &#176;C</td>
		</tr>
		<tr >
			<td >10 cm tissue culture dishes</td>
			<td >Corning</td>
			<td >25382-428</td>
			<td >For cleaning and sectioning aorta and MSC cell culture</td>
		</tr>
		<tr >
			<td >12 well-cell culture plates</td>
			<td >Corning-Sigma Aldrich</td>
			<td >CLS3513</td>
			<td >For setting up aortic ring assay-MSC co-cultures</td>
		</tr>
		<tr >
			<td >15 mL tube</td>
			<td >BD Falcon</td>
			<td >352096</td>
			<td >For general tissue culture procedures</td>
		</tr>
		<tr >
			<td >70 &#956;m cell strainer</td>
			<td >Fisherbrand</td>
			<td >22363548</td>
			<td >To ensure a single cell suspension before flow cytometry or sorting</td>
		</tr>
	</tbody>
</table>

the aortic ring co-culture assay: a convenient tool to assess the angiogenic potential of mesenchymal stromal cells in vitro

血管生成是一个复杂的, 高度规范的过程, 负责提供和维持足够的组织灌注。血管维持不足和病理性畸形可能导致严重的缺血性疾病, 而过度丰富的心血管发育与癌症和炎症失调有关。pro-angiogenic 治疗的一种很有希望的形式是使用血管生成细胞源, 它可以为新发展的血管提供调节因子和物理支持。
骨髓间质细胞 (msc) 是广泛研究的候选血管再生由于其分泌的影响和能力, 以检测和家庭缺血性或发炎组织。特别是, 第一个四个月的人脐带血管细胞 (FTM HUCPVCs) 是一个非常有希望的候选者, 由于其细胞性质, 高增殖和多向电位, 免疫特权的属性, 和强健的分泌配置.为了有效地评估潜在的血管生成再生细胞, 这是一个必要的测试他们在可靠和 "可翻译的" 前临床化验。主动脉环试验是一种体外血管生成模型, 可以方便地量化管状内皮结构, 提供宿主的辅助支持细胞和胞内基质 (ECM), 不包括炎症成分, 并且快速和低廉的设置。这是有利的, 当与在体内模型 (如: 如, 角膜检测, 基质插头检测);主动脉环试验可以跟踪所管理的细胞, 观察细胞间的相互作用, 同时避免异免疫排斥反应。
我们提出了一个新的应用的主动脉环试验的协议, 其中包括人骨髓间充质干细胞在 co-cultures 发育大鼠主动脉内皮网络。这种方法可以分析 MSC 对导管的形成和发展的贡献, 通过物理细胞样的相互作用及其效力, 积极迁移到血管生成的部位, 并评估其执行和调解的能力ECM 处理。该协议提供了进一步的信息, 在 MSC 表型的变化和基因表达后共培养。

在图 1中演示了建立主动脉环/MSC 共培养试验的示意图工作流程。主要步骤包括: 大鼠主动脉的分离、主动脉环的切片和嵌入、监测内皮的萌发和网络发育, 最后对 MSCs 进行标记和管理。内皮网分析的时间线概述了窗口为分析可行为每期间大块: 1 天, 5 和 #38; 7。附加注释由虚线框突出显示。
主动脉环内皮细...

Watch this Scientific Journal Video about The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro at JoVE.com

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

在这里, 我们提出了一个新的应用的主动脉环法 prelabelled 间充质细胞共大鼠主动脉源性内皮网络。这种新的方法可以可视化间质基质细胞 (MSCs) 归巢和与内皮网络整合, 网络属性的量化, 并评价 MSC 免疫和基因表达。

主动脉环共培养法: 一种方便的工具, 可以评估骨髓间质细胞的血管生成潜能体外

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