We thank Dr. C. Geoffrey Burns for fish stock. The authors are grateful to UNC Flow Cytometry Core Facility, UNC-CGIBD AAC core for resources enabling this project, and the ZAC facility for animal care. L.S. is supported by NIH T32 grant HL069768-13 (PI, Nobuyo Maeda). N.F. is supported by NSF Graduate Research Fellowship NSF-DGE-1144081. This study was supported by NIH P30DK034987 grant (to UNC Advanced Analytics Core), American Heart Association Scientist Development Grant 13SDG17060010 and Ellison Medical Foundation New Scholar Grant AG-NS-1064-13 (to Dr. Qian), and NIH R00 HL109079 grant (to Dr. Liu).

该协议要求使用的活，成年斑马鱼产生的胚胎。将胚收获组织收集。重要的是要获得相应的伦理审查委员会批准进行这项实验。 1.获取胚胎上演<ol><li>在实验前一天，繁殖准备健康，成年斑马鱼。放置一个男性和一个养殖池一个明确的分隔两侧一名女性。 </li><li>重复1.1所必需的足够胚胎生产下游应用尽可能多的养殖池。获得从野生型鱼和转基因鱼胚胎表达的感兴趣的细胞类型的荧光蛋白质。 注意:需要在步骤2-8下游应用胚胎数依赖于在感兴趣的时间点感兴趣的细胞的相对丰度。虽然这可能通过细胞类型而异，200胚胎产生2,000-5,000排序细胞当C感兴趣的厄尔代表&lt;细胞总数的1.0％，在24 HPF（小时受精后）。 </li><li>第二天早上，通过转移鱼到一个新的养殖池在面包屑槽换水和删除的分隔符。倾斜槽以一定的角度，鼓励养殖。 </li><li>收集上演胚胎。 <ol><li>每15分钟，由成年人转移到一个新的养殖池，并通过这些留守通过滤茶鸡蛋收集胚胎。 注意:当保持在可比的密度和温度的斑马鱼胚胎的同步发展。 </li><li>冲洗（在1L双蒸水0.21克/ L的即时海洋盐）与蛋水鸡蛋和转移到陪替氏培养皿。与空气循环转移培养皿在潮湿培养箱中于28.5℃。 </li></ol></li><li>最后收集后两小时，排序受精，多细胞胚胎成10cm陪替皿并降低密度，以每皿50胚胎。从一个单一的，15选择胚胎用于下游应用集合分钟时间窗口。孵育28.5℃的胚胎。 注:例如，在8点半，8时45分，9:00 9:15，9:30，9时45，10:00，上午10点15分收集胚胎。跨越时间点相比较，如果受精胚胎人数最多的是从上午9时收集到的离合器，然后只使用这些胚胎下游应用。 </li></ol> 2.设置单细胞分离<ol><li>感兴趣（18 HPF）的时间点前约30分钟，从他们的绒毛膜手动用细镊子取出胚胎。 </li><li>收集并标记为每个条件如下:2发2 ml微量离心管一40微米的细胞过滤网，一次35 MM细胞培养皿中，并用一个35微米的细胞滤网淋上2 FACS管。 </li><li>冰浴的下列试剂:含有蛋水0.21克/ L即时海洋盐1L双蒸水;脱yolking含有55 mM氯化钠，1.8 mM的KCl和1.25毫碳酸氢钠缓冲液;和FACS缓冲液含有Leibovitz的L-15补充了5％热灭活的胎牛血清的介质。 </li><li>每带细胞分离试剂1的样品1毫升RT。解冻1毫升细胞分离试剂2每个样品上冰。把使用前立即RT。 </li></ol> 3.单细胞分离<ol><li>使用大口径玻璃吸管，转移100-300胚胎2ml的微量离心管中蛋水的最小量。 </li><li>通过在冰上用1ml冰冷的蛋水更换水和浸没管20分钟安乐死胚胎。 注意！关键是要取得适当的体制动物保护监督委员会批准，该安乐死方法（在冰上冷却后细胞分离二次安乐死）。标准安乐死通常需要胚胎&lt;3天受精后它们被冷却后，这是不恰当的用于获得活细胞漂白。 </li><li>用1毫升的冰冷蛋水冲洗胚胎两次。要洗，使用大口径玻璃吸管取出鸡蛋水和P1000加入鸡蛋水。 </li><li>通过用1ml Deyolking缓冲器，带有一个P1000尖更换胚胎水和捣碎8-12次去除蛋黄，或直到蛋黄溶解并只胚胎的机构是可见的。 </li><li>收集通过离心组织在300×g离心1分钟。用吸管轻轻地去除上清液而不破坏组织的沉淀。在1毫升鸡蛋水再挂起。 </li><li>重复步骤3.5，共洗涤三次，但在最后的洗涤，在1毫升的RT细胞解离试剂1重新暂停。 </li><li>孵育在细胞解离试剂1在RT 10分钟与管水平放置。每2-3分钟，轻轻地用吸管P1000防止结块磨碎。 注:在研磨步骤，轻拿轻放样品。有时一个单一的，大丛将形成胚体纠结管。这会与温和的研磨辅助附加消化分散。不要旋涡振荡。 </li><li>离心300 XG 3分钟收集组织。在1毫升细胞分离试剂2取出上清，重悬。 </li><li>在室温下孵育5-15分钟。水平放置的管子。每2-3分钟，轻轻磨碎，以防止结块。每5分钟，评估消化的进展。 <ol><li>为了评估消化进度，稀释2微升上清到18微升FACS缓冲液和吸管作为液滴到细胞培养皿。 </li><li>将盖玻片在样品和10X 20X和放大倍率组织培养显微镜下观察。 注:编制应显示为单细胞，小集群，大集群，和偶尔的大多是原封胚胎体的混合。 </li><li>视觉评估是单个细胞和小簇的制剂的比例。 注:在消化会减少可行性;在消化会减少单细胞产量。&lt;/ LI&gt; </ol></li><li>收集通过离心细胞制剂在300×g离心5分钟。弃去上清液并在1ml再悬浮冷FACS缓冲液。 </li><li>用浸湿FACS缓冲液40微米的细胞过滤。穿过40微米的细胞过滤细胞悬液到35毫米的细胞培养皿。用1ml的FACS分选缓冲液洗细胞过滤一次。 </li><li>转移流通到2ml微量离心管中。在300 xg离心收集细胞离心5分钟，并在100μlFACS缓冲液重悬浮。 </li><li>算使用血球细胞产量。进一步稀释样品建议选择的FACS机器每毫升，或最佳浓度5×10 6个细胞。可选当在血球计数细胞产量，用锥虫蓝染剂死细胞以确认之前FACS分选的细胞制剂的可行性。 注:准备被过度稀释，将需要大量的时间过量的排序。准备工作过于Çoncentrated易于聚集成多聚体，并为次优排序纯粹的人口。 </li><li>储备各试样的10〜20％，以使用作为未染色对照。对于剩余的样品，加入染色死细胞荧光活/死（L / D）的歧视染料到每个样品。不洗。 注:穿透细胞用的核酸可以被用作的L / D染料受损细胞膜和污渍不限荧光染料，只要该荧光光谱是与感兴趣的选择和荧光标记的细胞的流式细胞仪机兼容。 <ol><li>加入染料的一些细胞会在运输途中死FACS净化，应从排序人口被排除后，不要洗的准备。 </li></ol></li><li>皑皑浸湿FACS管20微升FACS缓冲的35微米的细胞过滤。加入细胞的过滤器，并通过重力收集。存储在冰上。 </li></ol> 4. FACS富集<ol><li>使用FACS来富集单，表达荧光标记的活细胞。 <ol><li>设置一个门上的前向散射（FSC-A）幅度VS侧散射幅度（SSC-A）的散点图从碎片区分细胞，都与线性缩放。 注意！斑马鱼细胞通常比小鼠或人类细胞小。这反映在细胞和碎片之间的下基底分离。 </li><li>从在4.1.1设置栅极，设置一个门以富集单细胞和排除使用前向散射高度（FSC-H）和低侧散射高度（SSC-H）的散点图多体，既具有线性缩放。 注:具有不成比例的高FSC-H和低SSC-H细胞可能多聚体和排序被排除在外。 </li><li>从4.1.2门设定，使用单一颜色控件，设置一个门包括使用FSA-A的散点图与线性缩放和通道的幅度只活细胞用于检测的L / D与污渍日志缩放。如:L / D阴性细胞。 </li><li>从门在4.1.3设置，使用单一的色彩控制，采用FSA-A的散点图与用于检测荧光蛋白细胞标记物与日志缩放通道的线性缩放和幅度设置一个门只包括活细胞阳性荧光。包括阳性细胞。 </li><li>设置任何补偿控制，如果需要的话，要占的L / D染色和荧光蛋白的光谱之间的干扰。 </li></ol></li><li>设置排序逻辑落入所有步骤设置4.1城门细胞。 <ol><li>采用双标记细胞，确认选通细胞分选。 注意:用于排序将细胞而不是碎屑，单个细胞而不是多聚L / D阴性和荧光阳性细胞的细胞。 </li></ol></li><li>排序2,000-4,000细胞从感兴趣的人口到在冰上的离心管5微升冷的FACS缓冲液中。为了在排序过程中尽量减少对细胞的剪切应变，用最低的压力可能使细胞分拣机。 注:FACS缓冲液的5微升液滴用作退出FACS机器的细胞的缓冲。收集2,000-4,000细胞直接进入FACS缓冲液消除装载到IFC芯片之前需要进行离心分离。建议采用此策略，因为离心可导致多聚形成的，如果细胞坚持颗粒彼此。 </li><li>评估排序后的可行性分析。 <ol><li>转移1微升排序细胞与100微升FACS分类缓冲器和1个新鲜FACS管:1000稀释的L / D污点。通过FACS分拣机与步骤4.1浇注战略传递细胞。使用L的比例/ D负到正来估计分选的细胞的生存力。 </li></ol></li></ol> 5.称重传感器到微流控芯片<ol><li>使用血细胞计数器，测量的浓度和分选的细胞的尺寸。 <ol><li>稀释排序细胞至少10微升。稀释5μ的等分试样; L细胞与5微升台盼蓝。加载到血球和应用盖玻片。 </li><li>收集所有细胞的亮场图像，4×4格血细胞计数。计数活细胞的数量，并计算活细胞/ ml。活细胞不占用台盼蓝。 注意:使用任何标准的图像分析软件来测量所有活细胞的直径。计算小区大小的平均值和标准偏差，并选择适合于感兴趣的细胞尺寸范围内的IFC板。如果单元格大小范围横跨国际金融公司板单元尺寸范围内，使用多个板来捕捉全方位的利益细胞。 </li></ol></li><li>上根据制造商的说明IFC板负载细胞（见材料）。 <ol><li>稀释细胞至1×10 6个细胞/ ml，并执行浮力优化按照制造商的说明。 注:浮力优化确保细胞既不沉底也不漂浮到装载WEL的顶L为取得最佳装载到IFC芯片。缓冲器的细胞的比例可以由细胞类型而有所不同，但通常为6:4-7:3细胞:缓冲器。 </li><li>加入细胞涂上底漆，IFC板。压板到兼容的流体机械，并运行一个小区负载脚本通过微流体电路进入采集通道，推动细胞。 </li></ol></li><li>确认细胞在国际金融公司板捕获网站提出。 <ol><li>卸下流体机械IFC板。安装在配备有板适配器显微镜的IFC板。 </li><li>在放大10倍收集亮场和荧光每次捕捉网站的快照。 注:明场捕获时间可能从10-50毫秒，这取决于灯泡的强度。荧光捕获时间可能从250-750毫秒，这取决于荧光亮度。以免过度曝光细胞光损伤预防。 </li></ol></li></ol> 6. cDNA合成<ol><li>根据我在原地进行细胞裂解FC板制造商的说明。 <ol><li>加入裂解缓冲液对IFC板的孔中。压板上兼容的流体机械及运行细胞裂解脚本按照制造商的说明。 </li></ol></li><li>根据国际金融公司板制造商的说明书进行反转录。 <ol><li>加入反转录试剂IFC板。压板上兼容的流体机械。运行逆转录脚本。样品循环条件:在25℃1个循环10分钟，在42℃下1个周期为1小时，然后在1周期在85℃下5分钟。 </li></ol></li><li>根据国际金融公司板制造商的说明用基因特异性探针进行前置放大。 注:由于他们与低拷贝数更大的特异性，采用荧光标记探针，而不是用于嵌入染料的使用最优化的探头。 <ol><li>池引物和稀释到每个最终180纳米。汇集引物添加到IFC板。压板上兼容fluidiCS机。前置放大运行脚本。 </li></ol></li><li>在95℃下在95℃变性15秒，然后退火和延伸在60℃下4分钟1个循环10分钟，然后18个循环:用下面的循环条件进行前置放大。在4℃直至收获商店预扩增的cDNA。嘉实cDNA的根据，直到使用制造商的说明和储存在-20℃微板。 </li></ol> 7.从单目标定量RT-PCR单细胞基因选择<ol><li>根据制造商的说明在25微升稀释试剂稀释的cDNA。 注:近似最终产量为28微升每单元前置放大cDNA的。保留稀释剂的等分试样用作定量RT-PCR检测的无模板对照。 <ol><li>谈到明场，并记录在步骤5.3.2荧光图像，得分每个捕获点。手动计数并记录细胞的数目。评估和记录每一个细胞是否感orescent。 注:每个单独的网站可能包含0，1，或&gt; 1的细胞，其中&gt; 1单元包括包含多个细胞和/或无法识别的碎片捕获点。如果图像是足够的质量，像素值也可以被分配给量化荧光信号的强度。 </li></ol></li><li>对于定量RT-PCR分析选择样本。 <ol><li>从步骤7.2确定了包含具有积极的荧光确切1细胞捕获地点选择cDNA样品。从每个样品的cDNA的池1微升等份。 注意:这是用来确定是否感兴趣的基因中的任何选定单元格中表达了"池"阳性对照。 </li><li>从包含恰好0细胞作为阴性对照的至少一个捕捉现场选择的cDNA。步骤7.1.2为无模板控制使用稀释剂。 </li></ol></li><li>运行定量RT-PCR检测。 <ol><li>仅使用在步骤6.4.1用于预扩增探针。加载triplicat所有样品即1个循环50℃2分钟，1个循环95℃，10分钟，40个循环，在95℃下15秒的变性，然后退火和延伸，在60℃进行1:用于在384孔板10微升反应循环条件分钟。 </li></ol></li></ol> 8.数据分析<ol><li>验证由标准凝胶电泳定量RT-PCR产物。确认产物是预期的分子量的单一条带（每个探头而异）。 注:如果探测器在不适当的大小产生多个频段或单波段，那么专一是值得怀疑的，这从排除分析基因。 </li><li>检查有无异常14所有扩增曲线和帐号。计算每个样品/基因组合15的平均CT值。 注:阳性对照CT值通常从10-30不等。为阴性对照CT值范围通常为35-40（无放大）。 CT值30-35被认为是非常低的表达应谨慎14来解释。 </li><li>报告数据<ol><li>如果样品倒在CT = 10-30，数据也可以被报告为相对转录丰度对照样品倍数变化。 注:折叠变化等于2 ^（ - ΔΔCT）其中ΔCT是相对于管家基因通过从管家基因减去样品的平均CT和ΔΔCT是样品和阳性对照15之间在ΔCT的差异。 </li></ol></li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+early+development+of+zebrafish.">Genetics and early development of zebrafish.</a> Trends in genetics : TIG. 5, 283-288 (1989).</li><li>Liu, J., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+in+the+study+of+early+cardiac+development.">Zebrafish in the study of early cardiac development.</a> Circulation research. 110, 870-874 (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 203, 253-310 (1995).</li><li>Zhao, S., Fung-Leung, W. P., Bittner, A., Ngo, K., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+RNA-Seq+and+microarray+in+transcriptome+profiling+of+activated+T+cells.">Comparison of RNA-Seq and microarray in transcriptome profiling of activated T cells.</a> PloS one. 9, e78644 (2014).</li><li>McCall, M. N., McMurray, H. R., Land, H., Almudevar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+non-detects+in+qPCR+data.">On non-detects in qPCR data.</a> Bioinformatics. 30, 2310-2316 (2014).</li><li>Schmittgen, T. D., Livak, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+real-time+PCR+data+by+the+comparative+C(T)+method.">Analyzing real-time PCR data by the comparative C(T) method.</a> Nature. 3, 1101-1108 (2008).</li><li>Lyons, 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=Myogenic+and+morphogenetic+defects+in+the+heart+tubes+of+murine+embryos+lacking+the+homeo+box+gene+Nkx2-5.">Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5.</a> Genes &amp; development. 9, 1654-1666 (1995).</li><li>Chen, J. N., Fishman, 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=Zebrafish+tinman+homolog+demarcates+the+heart+field+and+initiates+myocardial+differentiation.">Zebrafish tinman homolog demarcates the heart field and initiates myocardial differentiation.</a> Development. 122, 3809-3816 (1996).</li><li>Zhou, Y., 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=Latent+TGF-beta+binding+protein+3+identifies+a+second+heart+field+in+zebrafish.">Latent TGF-beta binding protein 3 identifies a second heart field in zebrafish.</a> Nature. 474, 645-648 (2011).</li><li>Paffett-Lugassy, N., 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=Heart+field+origin+of+great+vessel+precursors+relies+on+nkx2.5-mediated+vasculogenesis.">Heart field origin of great vessel precursors relies on nkx2.5-mediated vasculogenesis.</a> Nature cell biology. 15, 1362-1369 (2013).</li><li>McCurley, A. T., Callard, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+housekeeping+genes+in+zebrafish:+male-female+differences+and+effects+of+tissue+type,+developmental+stage+and+chemical+treatment.">Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment.</a> BMC molecular biology. 9, 102 (2008).</li><li>Tang, R., Dodd, A., Lai, D., McNabb, W. C., Love, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+zebrafish+(Danio+rerio)+reference+genes+for+quantitative+real-time+RT-PCR+normalization.">Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization.</a> Acta biochimica et biophysica Sinica. 39, 384-390 (2007).</li><li>Serbedzija, G. N., Chen, J. N., Fishman, 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=Regulation+in+the+heart+field+of+zebrafish.">Regulation in the heart field of zebrafish.</a> Development. 125, 1095-1101 (1998).</li><li>de Pater, 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=Distinct+phases+of+cardiomyocyte+differentiation+regulate+growth+of+the+zebrafish+heart.">Distinct phases of cardiomyocyte differentiation regulate growth of the zebrafish heart.</a> Development. 136, 1633-1641 (2009).</li><li>Hami, D., Grimes, A. C., Tsai, H. J., Kirby, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+cardiac+development+requires+a+conserved+secondary+heart+field.">Zebrafish cardiac development requires a conserved secondary heart field.</a> Development. 138, 2389-2398 (2011).</li><li>Yelon, D., Horne, S. A., Stainier, D. 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The authors have nothing to disclose.

本文所描述的方法使用荧光蛋白的表达下的细胞类型特异性启动子的控制，以丰富从斑马鱼胚胎中的微流体辅助单细胞捕获系统中使用的心脏祖细胞群，以评估在单个心动基因的一个子集的表达细胞。只要FACS激光激发和发射能力与所选择的荧光团（多个）相容，可以使用任何荧光报道线路此方法。许多斑马鱼记者线已经存在，并且可以在少至3个月后将产生携带新颖记者转基因鱼。此外，此工作?...

细胞和分子生物学目前大多数研究是基于人群的平均水平。然而，重要的生物事件可能由这些传统的以人群为基础的分析被屏蔽，因为较小的群体可以在生物过程和疾病预后扮演着重要的角色。理解在不同的人群的基因表达在单细胞水平可以（有）导致相关的生物和临床见解1,2。关注胚胎发育研究，在细胞的人口较多，祖细胞往往代表性不足，使得它具有挑战性的检测基因表达微妙的变化，最终引发细胞命运决定3。同样地，一个单一的细胞类型可响应于微环境4具有不同的表达谱。例如，在相同的器官或在不同的器官（ 例如 ，主动脉或肾）驻地内皮细胞表现显著异质尽管共享共同morphological和功能特点5。此外，癌细胞填充相同的肿瘤还可以在单细胞水平6不同分子型材或突变。 在模型系统中，单个细胞转录组已成功确定了新的细胞群，其特征细胞分化过程中发生的中间状态，并透露差细胞对刺激7,8,9。这样的见解会被掩盖了传统的基于人口的研究。斑马鱼的胚胎是干，祖的极大充分利用的源代码，并在开发过程中探索单细胞的异质性和细胞身份的分子调控问题分化细胞。他们的高度千篇一律， 体外发育和易于遗传操作使其成为这种方法10,11极好的模型系统。具体而言，主要的限制单细胞根的解释Ë表达数据是在开发过程中新的中间细胞状态的可靠的鉴定需要组织收集9非常小心时机。这是必要的，以确保捕获的细胞之间的异质性表示在单个时间点的组织内的异质性，而不是异质由年龄依赖性细胞分化呈现的基因表达。相比小鼠，斑马鱼胚胎发育可以精确在大量的胚胎12的同步。此外，具有大的离合器的尺寸，斑马鱼的胚胎可以用作干细胞和祖细胞的丰富来源。 这个协议描述来隔离从斑马鱼胚胎细胞和使用用于QRT-PCR的基因表达分析的市售集成微流体回路（IFC）芯片和Autoprep设备系统捕获单细胞的方法。该协议可迅速移植到任何高通量测定复包括全转录组测序，使细胞的异质性13的更全面的分析。它还提供了多种优势，传统的基因表达分析。单细胞分离协议产生的FACS后高存活率，这降低了包含在下游应用中受损细胞的比例。通过使用IFC，捕获细胞可以直接观察来评估捕获速率和形态学评估细胞的健康。此外，该协议是广泛适用于斑马鱼研究界，只需要一个标有转基因鱼线，并获得微流体细胞捕获技术。 作为一个原则的证明，从心脏祖细胞衍生的单细胞分离和IFC芯片上捕获，然后心脏分化标志物的相对丰度被定量RT-PCR检测。在单细胞水平的基因表达分析表明，心脏祖细胞与各色共存ntiating后代。从心脏祖细胞的单细胞分析的洞察力可以在脊椎动物的发育过程中心脏祖细胞中的基因表达模式的异质性，这可能在传统的基于人群的分析被掩盖线索。

<table border="0" cellpadding="0" cellspacing="0" width="816">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Supplies</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td style="width:388px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dumont #5 forceps</td>
			<td style="width:111px;">Fine Science Tools</td>
			<td style="width:103px;">11254-20</td>
			<td>For removing the chorion from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge tube 2 mL</td>
			<td style="width:111px;">GeneMate</td>
			<td style="width:103px;">C-3261-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40 um cell strainer</td>
			<td style="width:111px;">Biobasic</td>
			<td style="width:103px;">SP104151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm culture dish</td>
			<td style="width:111px;">Falcon</td>
			<td align="right" style="width:103px;">351008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACS tubes topped with 35 um cell strainer</td>
			<td style="width:111px;">Falcon</td>
			<td align="right" style="width:103px;">352235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P1000 and tips</td>
			<td style="width:111px;">Rainin</td>
			<td align="right" style="width:103px;">17005089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P20 and tips</td>
			<td style="width:111px;">Rainin</td>
			<td align="right" style="width:103px;">17005091</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">IFC chip manufacturer&#39;s protocol</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-6117</td>
			<td>Version 100-6117 E1 was used in representative experiment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wide bore pastuer glass pippette</td>
			<td style="width:111px;">VWR</td>
			<td style="width:103px;">14673-010</td>
			<td>For transferring embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult wild type zebrafish</td>
			<td style="width:111px;">N/A</td>
			<td style="width:103px;"></td>
			<td>We used AB line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult transgenic zebrafish</td>
			<td style="width:111px;">N/A</td>
			<td style="width:103px;"></td>
			<td>We used Tg(nkx2.5:ZsYellow)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reagents for cell dissociation</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Double distilled water</td>
			<td style="width:111px;">N/A</td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Instant Ocean Sea Salt</td>
			<td style="width:111px;">Instant Ocean</td>
			<td style="width:103px;">SS15-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:111px;">FisherScientific</td>
			<td style="width:103px;">S271-3</td>
			<td>make stock in water and use for de-yolking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:103px;">P5405</td>
			<td>make stock in water and use for de-yolking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaHCO3</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:103px;">S6014</td>
			<td>make stock in water and use for de-yolking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leibovitz&#39;s L-15</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">21083-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:111px;">FisherScientific</td>
			<td style="width:103px;">03-600-511</td>
			<td>Heat inactivate; any brand of FBS should be fine</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell Dissociation Reagent 1 -TrypLE</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:103px;">12605-010</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell Dissociation Reagent 2 - FACSmax</td>
			<td style="width:111px;">Genlantis</td>
			<td style="width:103px;">T200100</td>
			<td>Store -20; thaw on ice; bring to room temp before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pronase (optional)</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:103px;">P5147</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L/D Dye - Sytox Blue</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:103px;">S34857</td>
			<td>Any live/dead stain suitable for flow cytometry will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Reagents for IFC plate use and qRT-PCR</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gene-specific probes</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>Probes will vary by experiment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe ef1a</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03432748_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe gata4</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03443262_g1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe nk2-5</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03074126_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe myl7</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03105700_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe vmhc</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03431136_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe isl1</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03425734_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Gene Expression Master Mix</td>
			<td style="width:111px;">Life Technologies</td>
			<td align="right" style="width:103px;">4369016</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Reagents listed in IFC manufacturer&#39;s protocol</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C1 Reagent Kit</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-5319</td>
			<td>Reagents for loading cells onto IFC plate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ambion Single Cell-to-CTTM</td>
			<td style="width:111px;">Life Technologies</td>
			<td align="right" style="width:103px;">4458237</td>
			<td>Reagents for reverse transcription and pre-amplification steps</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Molecular Biology Quality Water</td>
			<td style="width:111px;">Corning</td>
			<td style="width:103px;">46-000CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C1 IFC for PreAmp (5-10 um)</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-5757</td>
			<td>IFC plate for small cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipment</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C1 AutoPrep machine</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-5477</td>
			<td>For IFC plate use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer&#160;</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:103px;">Z359629</td>
			<td>For counting cells and assessing cell size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting microscope</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>For removing embryos from chorion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue culture microscope</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>For assessing single cell digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS machine</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>For isolating cells of interest</td>
		</tr>
	</tbody>
</table>

isolation and characterization of single cells from zebrafish embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

作为一个原则的证明，基因表达进行了评估心脏发育过程中，探索差异化的动态。在斑马鱼，心脏祖细胞从迁移到前侧板中胚层，他们融合形成线性心脏管细胞的中胚层人口出现。之前的融合，心脏祖细胞开始表达转录因子NKX2.5（NK 2同源5），它被认为是心脏祖16,17的最早特异性标记。在这里，先前描述的BAC转基因鱼的Tg（NKX2.5:ZsYellow）18，...

Watch this Scientific Journal Video about Isolation and Characterization of Single Cells from Zebrafish Embryos at JoVE.com

Isolation and Characterization of Single Cells from Zebrafish Embryos

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

isolation-and-characterization-of-single-cells-from-zebrafish-embryos

Research

JoVE Journal

Developmental Biology

14.4K 次观看.  University of North Carolina at Chapel Hill. This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells. 

视频: Isolation and Characterization of Single Cells from Zebrafish Embryos

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

隔离和卫星细胞的表征鼠头Branchiomeric肌肉

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

从斑马鱼胚胎内源性受体亲和标记检测

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

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

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

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

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

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

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

致心律失常心肌病患者心内膜心肌活检样间质基质细胞的分离与鉴定

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.

早产儿和足月婴儿脐带源性间充质干细胞的分离与鉴定

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

从骨骼肌活检程序分离、培养、表征和分化人类肌肉祖细胞

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...