Name: clonal analysis of embryonic hematopoietic stem cell precursors using single cell index sorting combined with endothelial cell niche co-culture
Uploaded: 2018-05-08T13:00:00.000+00:00
Duration: 9 min 32 s
Description: Watch this Scientific Journal Video about Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture at JoVE.com

我们要感谢安德鲁. 伯格, 史黛西 Dozono, 和布莱恩 Raden 在弗雷德. 哈钦森流式细胞术核心协助与外地资产管制。这项工作得到了国家卫生研究院 NHLBI UO1 赠款 #HL100395、辅助合作赠款 #HL099997 和 NIDDK 赠款 #RC2DK114777 的支持。布兰登上 Hadland 是支持的亚历克斯的柠檬水站基础和现代希望在车轮基础上。

这里描述的所有方法都得到了弗雷德哈钦森癌症研究中心机构动物护理和使用委员会 (IACUC) 的批准。
1. 为共同文化准备年会-EC 膜
<ol><li>24小时前的年会解剖和排序, 使年会-欧共体文化媒体和过滤消毒。</li>
	<li>在水中加入0.1% 明胶 (100 µL/井), 每井96井板, 并在室温下孵化15分钟. 吸入明胶, 将盘子放在组织培养罩中, 除去盖子, 直到水井干涸为止。</li>
	<li>
将股东大会-EC 单分子膜和板材与96井板分离。 注: 在股东大会-欧共体文化媒体上, 保持前述16所准备的股东大会-ec。为了保持一致性, 应在获得足够数量 (一般为 1-3) 的情况下, 尽快冻结年会的细胞线, 并在定义的通道号 (一般通过 5-12) 中用于共培养实验。欧共体细胞系由 C57BL/6J (CD45.2) 股东大会获得。有关所有媒体组合的材料表, 请参见。<ol>
<li>从75厘米2烧瓶中的股东大会-EC 文化中吸取媒体, 并添加5毫升 PBS。吸入 PBS, 并添加3毫升胰蛋白酶溶液 (见材料表)。</li>
		<li>孵育37°c 2-4 分钟, 直到大多数细胞从盘子上抬起。添加7毫升的年会-EC 培养基和吸管8-10 倍, 10 毫升吸管, 以准备单细胞悬浮, 并转移到15毫升管。</li>
		<li>旋转 300 x g 5 分钟, 移除上清, 在10毫升的年会-EC 培养基中重新悬浮, 并用 hemocytometer 估计细胞数。将细胞稀释成浓度为 1 x 105细胞/毫升的年会-欧共体文化媒体。</li>
	</ol>
</li>
	<li>增加100µL 年会-欧共体细胞悬浮 (1 x 104细胞) 到每个井糊96井板材。在一夜之间放置37°c, 5% CO2组织培养孵化器, 让年会-欧共体附上和形成一个汇合的单层。 注意: 均匀分布的年会-EC 基质细胞, 所以有有限的过度生长或细胞聚集, 以最佳的共同培养。</li>
	<li>
为股东大会共同文化准备年会-EC 单层。
	<ol>
<li>第二天早上, 准备年会无血清的媒体。</li>
		<li>使用12井多通道吸管, 从股东大会上删除欧盟联盟的文化媒体, 并添加200µL/井的年会无细胞因子的无血清介质, 以冲洗掉任何残留的含血清介质。</li>
		<li>删除媒体, 并添加200µL/良好的年会无血清培养基与细胞因子。在移除和添加介质时快速工作, 使内皮层不致受损;例如, 一次移除和重新添加媒体一行。将96井板放在组织培养孵化器中37摄氏度, 直到胚胎细胞准备好进行排序。</li>
	</ol>
</li>
</ol>2. 小鼠胚胎组织单细胞悬浮液的制备
<ol><li>
从定时的交配中剖析股东大会。
	<ol>
<li>设置定时交配, 以生成所需年龄的胚胎组织。</li>
		<li>在9.5 至11.5 天后 coitum (dpc), 根据预 HSC 的阶段进行分析, 从孕妇中收获胚胎。 注意: 胚胎组织是从 C57BL/6J (CD45.2) 小鼠身上收获的, 如先前所描述的, 并基于 somite 计数16精确分期。我们专注于从 somite 分期的 E9.5 到 E11.5 的胚胎周年大会区及其前驱体的种群分析。</li>
		<li>从胚胎20中解剖周年大会。 注: 其他20公布了从小鼠胚胎中剥离周年大会的详细协议。另外, 也可以用这种方法分析卵黄囊或其他组织的可能具有预抗性活性的细胞。</li>
	</ol>
</li>
	<li>
分离解剖的胚胎组织。
	<ol>
<li>收集15毫升圆锥管中的解剖组织, 其中含有10毫升 PBS, 冰上有10% 个血清。重力沉降组织, 吸入 PBS/血清, 然后立即添加1毫升0.25% 胶原酶和放置在37°c 水浴25分钟。</li>
		<li>添加1毫升的 PBS/10%fbs 和吸管约20-30 倍1毫升吸管提示, 以获得单个细胞悬浮。增加8毫升的 PBS/10%fbs 和离心细胞在 300 x g 5 分钟。</li>
	</ol>
</li>
</ol>3. 小鼠胚胎细胞的抗体染色
<ol><li>
准备用于抗体染色的阻塞缓冲器。
	<ol>
<li>添加10µg/毫升抗小鼠 CD16/CD32 (Fc 受体 (FcR) 块) 和1µg/毫升 DAPI (1 毫克/毫升股票在 H2O) 到1毫升 PBS 与10% 血清。</li>
		<li>在3毫升注射器中绘制, 通过一个0.22 µm 注射器过滤器进行杀菌。将细胞颗粒从分离的小鼠胚胎组织 (从步 2.2.2) 中重新悬浮在500µL 阻塞缓冲液中, 在冰上孵育5分钟。</li>
	</ol>
</li>
	<li>准备抗体组合17。<ol>
<li>添加10µg/毫升 FcR 块到1毫升 PBS 与10% 的血清和10µL 的每 fluorochrome 共轭抗 VE 钙粘蛋白抗体 (CD144, 见材料表) 和 fluorochrome 共轭抗 EPCR 抗体 (CD201, 见材料表)。使用1:100 的最终稀释抗体, 除非另有说明根据制造商的建议或根据滴定。 注: 此抗体组合用于定义用于排序的门, 如下所述 (步骤 4.2)。基于 VE 钙黏蛋白和 EPCR 的共表达式排序, 可以显著地丰富包含 HSC 前体活动的股东大会派生细胞的一部分, 如前面所描述的17。</li>
		<li>添加额外的 fluorochrome 共轭抗体, 进一步表型分析的个别指数排序前体。 注意: 这些抗体不一定用于定义用于排序的门。在这个样本协议中, 我们包括了 PE 共轭 anti-CD41 抗体和 FITC 共轭 anti-CD45 抗体, 以分析这些造血标记物的相对表达, 这些基因在从 hemogenic 内皮细胞出现前 HSC 的发生过程中演化为4 ,5,16,17。其他感兴趣的标记可以按需要包括在内。用 fluorochrome 共轭同种抗体控制染色的样品用于定义阴性和阳性门进行分析。</li>
		<li>在3毫升注射器中绘制抗体组合, 通过一个0.22 µm 注射器过滤器进行杀菌。添加500µL 的抗体混合到细胞悬浮在阻塞缓冲, 吸管混合, 并在冰上孵化至少15-20 分钟。</li>
	</ol>
</li>
	<li>
清洗染色的细胞。
	<ol>
<li>加入8毫升 PBS/10%fbs。离心细胞在 300 x g 5 分钟, 吸入, 并重新悬浮细胞颗粒与1毫升 PBS/10%fbs。</li>
		<li>通过在5毫升管上通过35微米细胞过滤器盖吹打细胞悬浮液来去除细胞团簇。用另外的3毫升 PBS/10%fbs 清洗细胞过滤器。离心细胞在 300 x g 5 分钟, 吸入, 重新悬浮在500µL PBS/10%fbs, 并放置在冰上, 直到外地资产管制。</li>
	</ol>
</li>
</ol>4. 对96井的单 Hemogenic 前体进行指数排序, 并与年会-EC 基质共培养
<ol><li>根据制造商的指示, 准备用于板模式的流式机, 并对齐96井板。在软件中选择 "单个单元格模式" 和 "索引排序" 模式, 以实现最大纯度掩码设置, 并为每个已排序的单元格获取索引参数。</li>
	<li>
从抗体染色样品中获取细胞样本, 以设置门进行排序。
	<ol>
<li>将染色的细胞样本加载到该机器上, 并以最低的流速获取细胞。情节 FSC-一节, 并选择一个门, 其中包括不同大小的细胞 (图 1B) (这是基于观察, 预 HSC 活动是在股东大会17的不同大小配置文件的单元格中标识)。剧情 fsc-一节 fsc-w 和 ssc-一节的经文-w, 并选择门排除双峰。然后绘制 FSC-一节 DAPI 到门活单元格 (DAPI 的负数) (图 1B)。</li>
		<li>绘制 PECy7 (或相关 fluorochrome 的 VE 钙黏蛋白染色) 与 PerCP (或 EPCR 染色相关 fluorochrome)。对 VE 钙黏蛋白 (包括内皮细胞混合种群以及造血祖和预 HSC) 具有高 EPCR 表达 (从 P Sp/周年大会中丰富的前 hsc) 的门细胞 (由 p--17) 组成。这是将用于在步骤4.3 中对单个单元格进行索引的最后一个门 (图 1B)。</li>
		<li>绘制用于染色的附加荧光。 注意: 根据要分析的亚群, 可以根据检测到的其他标记对染色细胞进行设置。但是, 索引排序可以记录每个已排序单元的荧光参数, 以便对这些参数进行回顾性分析。因此, 在这一点上不需要额外的浇口。对于所有的外地管理装置, 使用单一抗体染色的样本应用于调整补偿, 以说明重叠荧光的排放溢出, 以及同种控制抗体, 以说明背景染色并确定负/正门。</li>
	</ol>
</li>
	<li>
索引对年会单元进行排序。
	<ol>
<li>放置一个96井板与年会-EC 基质在年会上与细胞因子 (从步骤 1.5.3) 在板块块的流媒体分拣机。加载示例并开始采样获取。选择索引排序/单细胞模式, 并开始 sortingsingle 单元格到单个96井。使用最低流速最小化胚胎细胞的剪切应力。</li>
		<li>确保在索引排序过程中记录所有事件。这将使在排序门中分析的单元格总数相对于与单个96井排序的实际数字进行计数, 因为并非所有记录的事件都将被分类为水井。</li>
		<li>一旦细胞被整理成一个完整的96井板, 把盘子放在一个组织培养孵化器, 在37摄氏度设置在 5% CO2。重复每个实验所需的额外板材。</li>
		<li>共培养96口水井中的个体分类细胞 (从步骤 4.3) 到7天 (从 E11-11.5 胚胎排列的细胞5天, 从 E10-10.5 胚胎的6天, 7 天 E9-9. 5 个胚胎)。在共培养期之后, 在100X 放大倍数 (图 2B) 上, 可视化各种大小和形态的造血菌落。 注意: 在共用文化期间不需要更改媒体。排序的 hemogenic 前体最初可能会与 ec 类似的形态结合到股东大会-ec 层, 并且最初不能与股东大会-ec 基质 (图 2A) 区分开来。然而, 在共培养24-48 小时后, 可以检测到小的、圆形的造血细胞或细胞簇。在共培养 (5-7 天) 结束时, 可以在96井的子集中可视化造血细胞的个体菌落, 该群接收到具有克隆造血电位的有序细胞。如果目视检查的水井包含多个不同的菌落, 则此样本被排除在下游分析之外, 以排除可能接收了多个单元格的样本。</li>
	</ol>
</li>
</ol>5. 共培养后克隆造血后代的分析
<ol><li>
从每 96-井中收获克隆, 以进行资产管制分析。
	<ol>
<li>大力吸管用200µL 吸管贴士的多通道吸管将造血细胞从内皮层中分离出来。尽量不要分离内皮层。去除100µL (约50% 的样本), 用于表型分析的造血后代通过外地资产管制署。</li>
		<li>转移到一个96井 V 底板和离心机在 300 x g 2 分钟, 以颗粒细胞。当板块反转时, 用单片快速运动将介质从板上弹出来, 将介质移开 (轻拍板)。</li>
		<li>将剩余的100µL 细胞放在原来的96井板中, incubatorfor 在随后的植入化验中使用37°c (步骤 6)。</li>
	</ol>
</li>
	<li>
用适当的抗体对细胞进行造血分析。
	<ol>
<li>在96井 V 底的每个井中重新悬浮细胞小球, 50 µL PBS/2%fbs, 其中10µg/毫升 FcR 块和1µg/毫升 DAPI。在4摄氏度孵化出5分钟的盘子。</li>
		<li>添加50µL PBS/2%fbs 含有10µg/毫升 FcR 块和一个抗体组合, 包括 PE-Cyanine7-conjugated 抗 VE 钙黏素, PerCP 共轭 anti-CD45, PE 共轭抗 EPCR, APC 共轭 anti-Sca1, 和 FITC 共轭 anti-Gr1 和anti-F4/80 (每个抗体在1:100 最终稀释, 除非另有说明根据制造商的建议或根据滴定)。在4摄氏度孵化出至少20分钟的板材。</li>
	</ol>
</li>
	<li>
用顺序稀释去除未绑定抗体, 并对细胞进行分析。
	<ol>
<li>添加200µL/井 PBS/2%fbs 和离心机在 300 x g 2 分钟, 以颗粒细胞。然后, 轻拍板丢弃上清, 并添加200µL PBS/2%fbs 到每个细胞颗粒和离心机在 300 x g 2 分钟的颗粒细胞。</li>
		<li>轻拍板丢弃上清, 并添加50µL/井 PBS/2%fbs 进行分析。使用装有平板阅读器的流式细胞仪, 进行单元格的分析。 注: 获取固定体积的细胞 (例如, 35 µL 50 µL 用于重新悬浮), 以列举造血后代的子集。</li>
	</ol>
</li>
	<li>
分析每个含有造血后代的井的数据, 以筛选包含 HSC 电位的菌落 (图 3)。
	<ol>
<li>门首先 CD45 阳性的人群是阴性的髓系标记 Gr1 和 F480。不要门外的细胞仍然表达低浓度的 VE 钙黏蛋白。在造血菌落的收获过程中被打乱的股东大会-欧共体层细胞是 VE 钙黏素阳性和 CD45 阴性。</li>
		<li>门 CD45+VE-Cad-/低Gr1 F4/80-单元格, 该子集表示高级别的 EPCR 和 Sca1 (图 3A -C). 注意: 在以前的研究中, 缺乏单元格的 CD45+VE-Cad-/低Gr1 F4/80 Sca1hiEPCRhi表型 (图 3D) 重现性未能提供可检测的多向外周血植入在辐照受体小鼠中的作用17 (未显示数据);因此, 在步骤6中不需要将这些菌落包括在后续的移植分析中。</li>
	</ol>
</li>
</ol>6. 单个无性系的植入特性分析及其与表型性质的关系通过指数排序阐明
<ol><li>(如果可能的话) 或早晨在步骤5中的表型分析后, 重新暂停其余100µL 细胞从每个96井包含造血菌落后, 共培养 (从步骤 5.1) 的积极吹打使用200µL 吸管提示。</li>
	<li>
从同类系菌株 B6 的全骨髓中制备并添加抢救细胞。SJL-PtprcaPepcb/BoyJ (B6 CD45.1) 成年小鼠16,17。
	<ol>
<li>在 hemocytometer 下对土耳其人溶液中有核的骨髓细胞进行计数。稀释到 5 x 105有核细胞/毫升的浓度, 在 PBS 与2% 的血清。</li>
		<li>将100µL 的骨髓细胞 (5 x 104) 添加到每个含有造血子代的井中, 用于移植分析和吹打混合。</li>
	</ol>
</li>
	<li>
移植后代的个体克隆成一个单一的致命辐照接受同类系菌株 B6。SJL-PtprcaPepcb/BoyJ (B6 CD45.1) 成体鼠标。
	<ol>
<li>致命照射相同数量的 B6 CD45.1 小鼠作为克隆的数量, 以单独注入一个单一克隆的后裔 (使用 1000 cGy 从铯源)。</li>
		<li>绘制200µL 总细胞悬浮 (这包括从克隆的细胞以及 5 x 104 B6 CD45.1 抢救骨髓细胞) 成½毫升胰岛素注射器与29G ½英寸针。</li>
		<li>致命照射 B6 CD45.1 成人接受者在注射前2-24 小时。</li>
		<li>使用一个塑料鼠标抑制剂, 允许尾部访问, 注射通过尾静脉200µL 卷包含两个细胞从每个克隆和 5 x 104 B6 CD45.1 成人骨髓救援细胞, 以帮助接受者生存。</li>
		<li>耳朵标记和体重的受体小鼠, 并检查他们的体重/健康定期 (例如, 3-4 次/周后照射/移植, 或每一个机构标准操作程序), 以确保良好的健康。</li>
	</ol>
</li>
	<li>
在移植后定期对外周血植入进行分析 (例如、2、16、24周)。
	<ol>
<li>清除50-100 µL 的血液通过复古轨道出血或另一种道德上批准的血液允许方法。</li>
		<li>添加3毫升裂解缓冲液 (16.6 克 NH4Cl, 2 克 NaHCO3和74.4 毫克 EDTA 到2升 H2O) 对血液和孵育在室温下为5分钟离心机在 300 x g 为5分钟抽吸和再暂停球团与2毫升 PBS/2%fbs。离心机在 300 x g 5 分钟。</li>
		<li>重新悬浮颗粒与150µL PBS/2%fbs 含有10µg/毫升 FcR 块和1µg/毫升 DAPI, 并分配1/3 的体积为一个包含50µL 同种控制的捐助者和血统, 1/3 到一个含有50µL 抗体的供方和同种控制的血统, 1/3 到含有50µL 抗体的油井, 以检测捐献者和血统。 注: 检测多向植入的抗体: APCeFluor780-conjugated 抗 CD45.2、Pe-Cyanine7-conjugated 抗 CD45.1、FITC 共轭 anti-CD3、Pe 共轭 anti-F4/80、PerCP 共轭 anti-Gr1 和 APC 共轭 anti-CD19, 或相关的同种控制。</li>
		<li>通过对 CD45.2 (供体) 衍生造血细胞与髓系 (Gr1 和/或 F4/80 阳性)、B 细胞 (CD19 阳性) 和 T 细胞 (CD3 阳性) 的时间进行评估外周血贡献来确定长期植入的克隆 (图4A -b)。 注: 造血植入也可以通过 CD45.2 (供体) 对从骨髓、脾脏、胸腺和腹膜获得的组织中的造血细胞的贡献, 或继骨髓干细胞的二次移植来分析。到 B6 CD45.1 鼠标来分析串行植入属性17。</li>
	</ol>
</li>
	<li>将每个克隆的表单属性与初始单个单元格索引排序所确定的植入属性关联起来。使用流分析软件, 上载每个索引排序的单元格的排序参数 (相关的 96-好, 它被排序)。将功能数据映射到流图, 以评估单个单元格的表单属性 (例如, 长期、多向植入) (图 4C)。</li>
</ol>

<ol>
	<li>Copley, M. R., Beer, P. A., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell+heterogeneity+takes+center+stage.">Hematopoietic stem cell heterogeneity takes center stage.</a> Cell Stem Cell. 10 (6), 690-697 (2012).</li><li>Medvinsky, A., Rybtsov, S., Taoudi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origin+of+the+adult+hematopoietic+system:+advances+and+questions.">Embryonic origin of the adult hematopoietic system: advances and questions.</a> Development. 138 (6), 1017-1031 (2011).</li><li>Kumaravelu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+developmental+anatomy+of+definitive+haematopoietic+stem+cells/long-term+repopulating+units+(HSC/RUs):+role+of+the+aorta-gonad-mesonephros+(AGM)+region+and+the+yolk+sac+in+colonisation+of+the+mouse+embryonic+liver.">Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver.</a> Development. 129 (21), 4891-4899 (2002).</li><li>Taoudi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+hematopoietic+stem+cell+generation+in+the+AGM+region+via+maturation+of+VE-cadherin+CD45++pre-definitive+HSCs.">Extensive hematopoietic stem cell generation in the AGM region via maturation of VE-cadherin+CD45+ pre-definitive HSCs.</a> Cell Stem Cell. 3 (1), 99-108 (2008).</li><li>Rybtsov, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+organization+and+early+hematopoietic+specification+of+the+developing+HSC+lineage+in+the+AGM+region.">Hierarchical organization and early hematopoietic specification of the developing HSC lineage in the AGM region.</a> J Exp Med. 208 (6), 1305-1315 (2011).</li><li>Rybtsov, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+the+origin+of+the+HSC+hierarchy+reveals+an+SCF-dependent,+IL-3-independent+CD43(-)+embryonic+precursor.">Tracing the origin of the HSC hierarchy reveals an SCF-dependent, IL-3-independent CD43(-) embryonic precursor.</a> Stem Cell Reports. 3 (3), 489-501 (2014).</li><li>McGrath, K. 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+Sources+of+Hematopoietic+Progenitors+Emerge+before+HSCs+and+Provide+Functional+Blood+Cells+in+the+Mammalian+Embryo.">Distinct Sources of Hematopoietic Progenitors Emerge before HSCs and Provide Functional Blood Cells in the Mammalian Embryo.</a> Cell Rep. 11 (12), 1892-1904 (2015).</li><li>Lin, Y., Yoder, M. C., Yoshimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoid+progenitor+emergence+in+the+murine+embryo+and+yolk+sac+precedes+stem+cell+detection.">Lymphoid progenitor emergence in the murine embryo and yolk sac precedes stem cell detection.</a> Stem Cells Dev. 23 (11), 1168-1177 (2014).</li><li>Medvinsky, A., Dzierzak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definitive+hematopoiesis+is+autonomously+initiated+by+the+AGM+region.">Definitive hematopoiesis is autonomously initiated by the AGM region.</a> Cell. 86 (6), 897-906 (1996).</li><li>Sheridan, J. M., Taoudi, S., Medvinsky, A., Blackburn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+generation+of+reaggregated+organotypic+cultures+that+permits+juxtaposition+of+defined+cell+populations.">A novel method for the generation of reaggregated organotypic cultures that permits juxtaposition of defined cell populations.</a> Genesis. 47 (5), 346-351 (2009).</li><li>Yoder, M. C., Hiatt, K., Mukherjee, P. <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+repopulating+hematopoietic+stem+cells+are+present+in+the+murine+yolk+sac+at+day+9.0+postcoitus.">In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus.</a> Proc Natl Acad Sci U S A. 94 (13), 6776-6780 (1997).</li><li>Arora, 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=Effect+of+developmental+stage+of+HSC+and+recipient+on+transplant+outcomes.">Effect of developmental stage of HSC and recipient on transplant outcomes.</a> Dev Cell. 29 (5), 621-628 (2014).</li><li>Butler, J. M., Rafii, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+vascular+niche+for+studying+stem+cell+homeostasis.">Generation of a vascular niche for studying stem cell homeostasis.</a> Methods Mol Biol. 904, 221-233 (2012).</li><li>Butler, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+are+essential+for+the+self-renewal+and+repopulation+of+Notch-dependent+hematopoietic+stem+cells.">Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells.</a> Cell Stem Cell. 6 (3), 251-264 (2010).</li><li>Kobayashi, 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=Angiocrine+factors+from+Akt-activated+endothelial+cells+balance+self-renewal+and+differentiation+of+haematopoietic+stem+cells.">Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells.</a> Nat Cell Biol. 12 (11), 1046-1056 (2010).</li><li>Hadland, B. K., 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=Endothelium+and+NOTCH+specify+and+amplify+aorta-gonad-mesonephros-derived+hematopoietic+stem+cells.">Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derived hematopoietic stem cells.</a> J Clin Invest. 125 (5), 2032-2045 (2015).</li><li>Hadland, B. K., 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=A+Common+Origin+for+B-1a+and+B-2+Lymphocytes+in+Clonal+Pre-+Hematopoietic+Stem+Cells.">A Common Origin for B-1a and B-2 Lymphocytes in Clonal Pre- Hematopoietic Stem Cells.</a> Stem Cell Reports. 8 (6), 1563-1572 (2017).</li><li>Wilson, N. K., 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=Combined+Single-Cell+Functional+and+Gene+Expression+Analysis+Resolves+Heterogeneity+within+Stem+Cell+Populations.">Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations.</a> Cell Stem Cell. 16 (6), 712-724 (2015).</li><li>Schulte, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Index+sorting+resolves+heterogeneous+murine+hematopoietic+stem+cell+populations.">Index sorting resolves heterogeneous murine hematopoietic stem cell populations.</a> Exp Hematol. 43 (9), 803-811 (2015).</li><li>Morgan, K., Kharas, M., Dzierzak, E., Gilliland, 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=Isolation+of+early+hematopoietic+stem+cells+from+murine+yolk+sac+and+AGM.">Isolation of early hematopoietic stem cells from murine yolk sac and AGM.</a> J Vis Exp. (16), (2008).</li><li>deBruijn, M. F., 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=Hematopoietic+stem+cells+localize+to+the+endothelial+cell+layer+in+the+midgestation+mouse+aorta.">Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta.</a> Immunity. 16 (5), 673-683 (2002).</li><li>Lorsbach, R. B., 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=Role+of+RUNX1+in+adult+hematopoiesis:+analysis+of+RUNX1-IRES-GFP+knock-in+mice+reveals+differential+lineage+expression.">Role of RUNX1 in adult hematopoiesis: analysis of RUNX1-IRES-GFP knock-in mice reveals differential lineage expression.</a> Blood. 103 (7), 2522-2529 (2004).</li><li>Holmes, R., Z&#250;&#241;iga-Pfl&#252;cker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OP9-DL1+system:+generation+of+T-lymphocytes+from+embryonic+or+hematopoietic+stem+cells+in+vitro.">The OP9-DL1 system: generation of T-lymphocytes from embryonic or hematopoietic stem cells in vitro.</a> Cold Spring Harb Protoc. 2009 (2), pdb.prot5156 (2009).</li><li>Yoshimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+wave+of+B+lymphopoiesis+develops+independently+of+stem+cells+in+the+murine+embryo.">The first wave of B lymphopoiesis develops independently of stem cells in the murine embryo.</a> Ann N Y Acad Sci. 1362, 16-22 (2015).</li><li>Kobayashi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+B-1+progenitor+cells+are+present+in+the+hematopoietic+stem+cell-deficient+embryo+and+depend+on+Cbf&#946;+for+their+development.">Functional B-1 progenitor cells are present in the hematopoietic stem cell-deficient embryo and depend on Cbf&#946; for their development.</a> Proc Natl Acad Sci U S A. 111 (33), 12151-12156 (2014).</li><li>Yoshimoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+murine+T-cell+progenitor+production+in+the+extra-embryonic+yolk+sac+before+HSC+emergence.">Autonomous murine T-cell progenitor production in the extra-embryonic yolk sac before HSC emergence.</a> Blood. 119 (24), 5706-5714 (2012).</li><li>Inlay, M. A., 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=Identification+of+multipotent+progenitors+that+emerge+prior+to+hematopoietic+stem+cells+in+embryonic+development.">Identification of multipotent progenitors that emerge prior to hematopoietic stem cells in embryonic development.</a> Stem Cell Reports. 2 (4), 457-472 (2014).</li><li>Palis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell-independent+hematopoiesis:+emergence+of+erythroid,+megakaryocyte,+and+myeloid+potential+in+the+mammalian+embryo.">Hematopoietic stem cell-independent hematopoiesis: emergence of erythroid, megakaryocyte, and myeloid potential in the mammalian embryo.</a> FEBS Lett. 590 (22), 3965-3974 (2016).</li></ol>

作者没有什么可透露的。

研究胚胎发育过程中的 hsc 成因, 需要检测 hemogenic 前体中的 hsc 电位, 但在移植成人接受者中缺乏提供长期多向造血重建的能力。在本议定书中, 我们提出了一种克隆方法的胚胎 hemogenic 前体之间的基质共培养的血管位, 从大会, 支持成熟的前体对 HSC, 并随后功能分析的移植化验。纳入索引排序允许回顾性分析的表型参数, 以表征前 HSC 的性质, 由表面标记的定义包括在化验。因此, 这种方法比其他依?...

克隆研究揭示了成人 HSCs 长期植入特性的异质性, 为 hsc 亚型和在老化的1中的 hsc 行为变化提供了新的见解。然而, 对胚胎 HSCs 及其前体的类似研究更具挑战性。在早期胚胎发育过程中, HSCs 产生于一种称为 hemogenic 内皮的前体, 在一个瞬态过程中称为内皮细胞到造血转换2。第一个 HSC, 定义的能力, 提供强健, 长期多向植入后移植到条件成年接受者, 直到胚胎天 10.5 (E10.5) 后, 在小鼠胚胎, 在非常低频率3.在它们的发展过程中, hemogenic 内皮引起的 hsc 前体 (hsc) 必须在获得成年 HSC 的特性之前进行成熟, 从而在移植化验中允许有效的植入4,5,6. 模糊了罕见的 HSC 起源的研究, 在从前 hsc7、8出现之前, 已经检测到大量具有红、髓系和淋巴电位的造血祖细胞。因此, 区分前 hsc 与其他造血祖细胞需要的方法, 以无性分离的干细胞, 并提供足够的信号, 他们成熟的 hsc, 以检测其植入的性质, 在移植化验。
已经描述了许多方法, 允许通过 " 体外" 或体内成熟度检测 hsc。"前体" 方法依赖于胚胎组织的区域性, 例如, 在开发9中检测到第一个 HSC 的 "年会" 区域。在这些方法的基础上, 结合了周年大会组织的分离、分类和再聚合的协议, 允许在 E9.5 到 E11.5 的准主动脉的发育过程中含有 HSC 前体的排序种群的特征。splanchnopleura (P Sp)/股东大会区域4,5,10;然而, 这些方法不适用于克隆分析所需的单细胞水平的前体高通量分析。同样,在体内通过移植进入新生小鼠, 在这种情况下, 假设微环境更适合于早期的 HSC 前体的支持, 也使从蛋黄囊中分类的种群的研究和周年大会/p sp (p sp 是年度股东大会的前体区域), 具有预 HSC 的特点, 但这些方法也未能为单个单元分析1112提供一个健壮的平台。
从 Rafii et . 的研究表明, Akt 活化内皮细胞 (EC) 基质可以提供一个利基基质支持成人 HSC 自我更新体外13,14,15。我们最近确定, 由年会 (Akt) 衍生的欧共体 (年会) 提供了一个适当的体外利基, 以成熟的 hemogenic 前体, 早在 E9 的发展, 对成年嫁接 HSC, 以及随后自更新生成的 HSC16。鉴于该系统采用了简单的2维共培养, 它很容易适应克隆分析的 hemogenic 前体独立的 HSC 潜力。
我们最近报告了一种方法来检测克隆 hemogenic 前体的 HSC 潜力, 结合 hemogenic 前体的小鼠胚胎的指数排序与年会-欧共体共培养和随后的功能分析移植化验17。索引排序是一种荧光活化细胞分选模式, 它记录 (索引) 所有表型参数 (即、正散射 (FSC a)、侧向散射 (SSC a)、荧光参数), 从而使这些特征可以追溯到后续功能分析后的排序。资产管制系统软件记录每个单元的表单信息, 以及放置在其中的96孔板的位置/井。这种技术以前很优雅地用于鉴定成人 hsc 中的异质性, 确定进一步丰富 hsc 长期嫁接子集的表型参数, 并将 hsc 的表形参数与转录相关联。单个单元格级别的属性18,19。在这里, 我们提供了这种方法的详细方法, 使识别独特的表型参数和谱系贡献的前 HSC 在胚胎发育的早期阶段。

<table><tbody><tr><td>&lt;strong&gt;AGM-EC culture media &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials for culture of endothelial cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>TrypLE Express</td><td>Gibco</td><td>12605-028</td><td>Use to dissociate AGM-EC monolayers</td></tr><tr><td>Gelatin 0.1% in water</td><td>StemCell Technologies</td><td>7903</td><td>Use to adhere AGM-EC monolayers to plastic</td></tr><tr><td>96-well tissue culture plates</td><td>Corning</td><td>3599</td><td>Use to co-culture AGM-EC monolayers and index sorted clones</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>14190-144</td><td>Use for dissection and FACS staining</td></tr><tr><td>500 ml filter bottles</td><td>Fisher Scientific</td><td>9741202</td><td>Use to sterile filter Endothelial media</td></tr><tr><td>&lt;strong&gt;AGM-EC culture media (500 mls)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>Gibco</td><td>12440-053</td><td>400 mls</td></tr><tr><td>Hyclone Fetal Bovine Serum</td><td>Fisher Scientific</td><td>SH30088.03</td><td>100 mls</td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140-122</td><td>5 mls</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3149</td><td>50 mg dissolved in 10 mls media</td></tr><tr><td>L-glutamine (200 mM)</td><td>StemCell Technologies</td><td>7100</td><td>5 mls</td></tr><tr><td>Endothelial Mitogen (ECGS)</td><td>Alfa Aesar</td><td>J64516 (BT-203)</td><td>Add 10 mls media to dissolve and add</td></tr><tr><td>&lt;strong&gt;Materials for AGM index sort&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Collagenase (0.25%)</td><td>StemCell Technologies</td><td>7902</td><td>Use for dissociation of embryonic tissues</td></tr><tr><td>3ml syringe</td><td>BD Biosciences</td><td>309657</td><td>Use to sterile filter antibody solutions for FACS</td></tr><tr><td>0.22 &amp;micro;M syringe-driven filter</td><td>Millipore</td><td>SLGP033RS</td><td>Use to sterile filter antibody solutions for FACS</td></tr><tr><td>5 ml polystyrene tube with cell-strainer cap</td><td>Corning</td><td>352235</td><td>Use to remove cell clumps prior to FACS</td></tr><tr><td>DAPI (prepared as 1 mg/ml stock in H2O)</td><td>Millipore</td><td>268298</td><td>Use to exclude dead cells from sort</td></tr><tr><td>anti-mouse CD16/CD32 (FcR block)</td><td>BD Biosciences</td><td>553141</td><td>Use to block non-specific staining to Fc receptors</td></tr><tr><td>&lt;strong&gt;Antibodies for AGM index sort&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse CD144 PE-Cyanine7</td><td>eBioscience</td><td>25-1441-82</td><td>Staining</td></tr><tr><td>Rat IgG1 kappa Isotype Control, PE-Cyanine7</td><td>eBioscience</td><td>25-4301-81</td><td>Isotype control for CD144 PE-Cyanine7</td></tr><tr><td>Anti-mouse CD201 (EPCR) PerCP-eFluor710</td><td>eBioscience</td><td>46-2012-80</td><td>Staining</td></tr><tr><td>Rat IgG2b kappa Isotype Control, PerCP-eFlour710</td><td>eBioscience</td><td>46-4031-80</td><td>Isotype control for CD201 PerCP-eFluor710</td></tr><tr><td>Anti-mouse CD41 PE</td><td>BD Biosciences</td><td>558040</td><td>Staining</td></tr><tr><td>Rat IgG1 kappa Isotype Control, PE</td><td>BD Biosciences</td><td>553925</td><td>Isotype control for CD41 PE</td></tr><tr><td>Anti-mouse CD45 FITC</td><td>eBioscience</td><td>11-0451-85</td><td>Staining</td></tr><tr><td>Rat IgG2b kappa Isotype Control, FITC</td><td>eBioscience</td><td>11-4031-81</td><td>Isotype control for CD45 FITC</td></tr><tr><td>&lt;strong&gt;AGM-serum free media (10mls)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>X-Vivo 20</td><td>Lonza</td><td>04-448Q</td><td>10 mls</td></tr><tr><td>recombinant murine stem cell factor (SCF)</td><td>Peprotech</td><td>250-03</td><td>10 ml (100 mg/ml stock) Final concentration 100 ng/ml</td></tr><tr><td>recombinant human FLT3 Ligand (FLT3L)</td><td>Peprotech</td><td>300-19</td><td>10 ml (100 mg/ml stock) Final concentration 100 ng/ml</td></tr><tr><td>recombinant human thrombopoietin (TPO)</td><td>Peprotech</td><td>300-18</td><td>2 ml (100 mg/ml stock) Final concentration 20 ng/ml</td></tr><tr><td>recombinant murine interleukin-3 (IL3)</td><td>Peprotech</td><td>213-13</td><td>2 ml (100 mg/ml stock) Final concentration 20 ng/ml</td></tr><tr><td>&lt;strong&gt;Materials for Staining co-cultured cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96 well plate, V-bottom</td><td>Corning</td><td>3894</td><td>Use for FACS analysis</td></tr><tr><td>&lt;strong&gt;Antibodies for analysis of Index sorted clones co-cultured with endothelial cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse CD45 PerCP-Cyanine5.5</td><td>eBioscience</td><td>45-0451-82</td><td>Staining</td></tr><tr><td>Rat IgG2b kappa Isotype Control, PE-Cyanine5.5</td><td>eBioscience</td><td>35-4031-80</td><td>Isotype control for CD45 PerCP-Cyanine5.5</td></tr><tr><td>Anti-mouse CD201 (EPCR) PE</td><td>eBioscience</td><td>12-2012-82</td><td>Staining</td></tr><tr><td>Rat IgG2b kappa Isotype Control, PE</td><td>eBioscience</td><td>12-4031-81</td><td>Isotype control for CD201 PE</td></tr><tr><td>Anti-mouse Ly-6A/E (Sca-1) APC</td><td>eBioscience</td><td>17-5981-83</td><td>Staining</td></tr><tr><td>Rat IgG2a kappa Isotype Control, APC</td><td>eBioscience</td><td>17-4321-81</td><td>Isotype control for Ly-6A/E APC</td></tr><tr><td>Anti-mouse F4/80 FITC</td><td>eBioscience</td><td>11-4801-81</td><td>Staining</td></tr><tr><td>Rat IgG2a kappa Isotype Control, FITC</td><td>eBioscience</td><td>11-4321-41</td><td>Isotype control for F4/80 FITC</td></tr><tr><td>Anti-mouse Ly-6G/C (Gr1) FITC</td><td>BD Biosciences</td><td>553127</td><td>Staining</td></tr><tr><td>Rat IgG2b kappa Isotype Control, FITC</td><td>BD Biosciences</td><td>556923</td><td>Isotype control for Ly-6G/C and CD3 FITC</td></tr><tr><td>&lt;strong&gt;Materials for tail vein injection for transplant&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1/2 ml insulin syringes with 29G 1/2&quot; needles</td><td>BD Biosciences</td><td>309306</td><td>Use for tail vein injection for transplantation</td></tr><tr><td>&lt;strong&gt;Antibodies for Peripheral Blood analysis following translant&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse Ly-6G/C (Gr1) PerCP</td><td>Biolegend</td><td>108426</td><td>Staining</td></tr><tr><td>Rat IgG2b kappa Isotype Control, PerCP</td><td>Biolegend</td><td>400629</td><td>Isotype control for Ly-6G/C PerCP</td></tr><tr><td>Anti-mouse F4/80 PE</td><td>eBioscience</td><td>12-4801-82</td><td>Staining</td></tr><tr><td>Rat IgG2a kappa Isotype Control, PE</td><td>eBioscience</td><td>12-4321-80</td><td>Isotype control for F4/80 PE</td></tr><tr><td>Anti-mouse CD3 FITC</td><td>BD Biosciences</td><td>555274</td><td>Staining</td></tr><tr><td>Anti-mouse CD19 APC</td><td>BD Biosciences</td><td>550992</td><td>Staining</td></tr><tr><td>Rat IgG2a kappa Isotype Control, APC</td><td>BD Biosciences</td><td>553932</td><td>Isotype control for CD19 APC</td></tr><tr><td>Anti-mouse CD45.1 (A20) PE-Cyanine7</td><td>eBioscience</td><td>25-0453-82</td><td>Staining</td></tr><tr><td>Rat IgG2a kappa Isotype Control, PE-Cyanine7</td><td>eBioscience</td><td>25-4321-81</td><td>Isotype control for CD45.1 PE-Cyanine7</td></tr><tr><td>Anti-mouse CD45.2 (104) APC-eFluor780</td><td>eBioscience</td><td>47-0454-82</td><td>Staining</td></tr><tr><td>Rat IgG2a kappa Isotype Control, APC-eFluor780</td><td>eBioscience</td><td>47-4321-80</td><td>Isotype control for CD45.2 APC-eFluor780</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BD FACSAria II with DIVA software</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BD FACSCanto II with plate reader</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Haemocytometer</td><td>Fisher Scientific</td><td>S17040</td><td>For counting cells</td></tr><tr><td>Multi-channel pipette</td><td>Fisher Scientific</td><td>14-559-417</td><td>For dispensing cells</td></tr><tr><td>FACS analysis software</td><td>FlowJo/BD Biosciences</td><td></td><td>https://www.flowjo.com/solutions/flowjo</td></tr></tbody></table>

clonal analysis of embryonic hematopoietic stem cell precursors using single cell index sorting combined with endothelial cell niche co-culture

在胚胎发育过程中, 研究造血干细胞 (hsc) 成因的能力受到了早期胚胎中 HSC 前体稀有性的限制, 缺乏检测功能, 可以识别长期的多向植入潜能。个人假定的 HSC 前体。在这里, 我们描述的方法, 使功能验证的 HSC 前体在单细胞水平的分离和鉴定。首先, 我们利用索引排序来编目每个单独排序的细胞的精确表型参数, 使用表型标记的组合, 以丰富的 HSC 前体和附加标记的实验分析。其次, 每个指数排序细胞都是由主动脉-性腺-中 (嫁接) 区的血管位质基质共同培养的, 它支持非 hsc 前体的成熟与多向的功能性 hsc, 长期植入电位在移植化验。这种方法使克隆 hemogenic 前体的表型性质与其功能植入电位或其他特性 (如转录剖面) 的相关性, 为 HSC 前驱物的详细分析提供了手段。在单个单元格级别上进行开发。

图 1A显示了实验设计的示意图。一旦在胶原酶中解剖、汇集和分离了 p-Sp/年会组织, 就会对 VE 钙黏蛋白和 EPCR 的抗体染色, 以进行指数排序。在 VE 钙黏蛋白+EPCR高(图 1B) 中排序的单元格中, 预 HSC 被丰富。其他 fluorochrome 共轭抗体可包括在回顾性分析其他表型参数, 这是记录的每个细胞在索引排序。在 E11 年会的这...

Watch this Scientific Journal Video about Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture at JoVE.com

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

单细胞指数分型与内皮细胞小生境联合培养对胚胎造血干细胞前体的克隆分析

本文介绍了小鼠胚胎发育过程中造血干细胞前体的克隆分析方法。我们结合内皮细胞共培养和移植对胚胎主动脉-性腺-中区单细胞的指标进行分类, 以表征单造血前体的表型性质和植入电位。

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

clonal-analysis-embryonic-hematopoietic-stem-cell-precursors-using

Research

JoVE Journal

Developmental Biology

8.5K 次观看.  Fred Hutchinson Cancer Research Center. 本文介绍了小鼠胚胎发育过程中造血干细胞前体的克隆分析方法。我们结合内皮细胞共培养和移植对胚胎主动脉-性腺-中区单细胞的指标进行分类, 以表征单造血前体的表型性质和植入电位。

视频: Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

科研

发育生物学

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

遗传学

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

小鼠卵黄囊和AGM早期造血干细胞的分离

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

生物学

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

血源性内皮细胞与人多能干细胞的定向分化

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

医学

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able to generate large datasets, necessitate the destruction of the cell of interest, making it impossible to assess its functional fate. Heterogeneous biological differentiating systems like hematopoiesis are therefore difficult to describe. Building on cell division tracking dyes, we further developed a protocol to simultaneously determine kinship, division number, and differentiation status for many single hematopoietic progenitors. This protocol allows the assessment of the ex vivo differentiation potential of murine and human hematopoietic progenitors, isolated from various biological sources. Moreover, as it is based on flow cytometry and a limited number of reagents, it can quickly generate a large amount of data, at the single-cell level, in a relatively inexpensive manner. We also provide the analytical pipeline for single-cell analysis, combined with a robust statistical framework. As this protocol allows the linking of cell division and differentiation at the single-cell level, it can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. Altogether, this protocol can be used in experimental designs aiming to unravel the biological differences between hematopoietic progenitors, from a single-cell perspective.

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework.

通过流式细胞术 同时 评估造血干细胞和祖细胞的亲缘关系、分裂数和表型

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able ...

免疫与感染

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

小鼠骨髓造血干细胞和祖细胞以及基质生态位细胞的流式细胞术分析

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As ...

人间充质干细胞的荧光激活单细胞分选

Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and are known for their immunomodulatory and anti-inflammatory properties. The use of these stem cells is a boon to the field of regenerative biology, but at the same time, a bane to regenerative medicine and therapeutics owing to the multiple cellular ambiguities associated with them. These ambiguities may arise from the diversity in the source of these stem cells and from their in vitro growth conditions, both of which reflect upon their functional heterogeneity.
This warrants methodologies to provide purified, homogeneous populations of MSCs&#160;for therapeutic applications. Advances in the field of flow cytometry have enabled the detection of single-cell populations using a multiparametric approach. This protocol outlines a way to identify and purify&#160;stem cells from&#160;human exfoliated deciduous teeth (SHEDs) through fluorescence-assisted single-cell sorting. Simultaneous expression of surface markers, namely, CD90-fluorescein isothiocyanate (FITC), CD73-peridinin-chlorophyll-protein (PerCP-Cy5.5), CD105-allophycocyanin (APC), and CD44-V450, identified the &#34;bright,&#34; positive-expressors of MSCs&#160;using multiparametric&#160;flow cytometry. However, a significant drop was observed in percentages of quadruple expressors of these positive markers from passage 7 onwards to the later passages.
The immunophenotyped subpopulations were sorted using the single-cell sort mode where only two positive and one negative marker constituted the inclusion criteria. This methodology ensured the cell viability of the sorted populations and maintained cell proliferation post sorting. The downstream application for such sorting can be used to evaluate lineage-specific differentiation for the gated subpopulations. This approach can be applied to other single-cell systems to improve isolation conditions and for acquiring multiple cell surface marker information.

作者聚焦：单细胞分选在分离间充质干细胞纯化群体中的重要性 

This protocol describes the use of fluorescence-activated cell sorting of human mesenchymal stem cells using the single-cell sorting method. Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when combined with a multiparametric flow cytometry-based approach.

人脱落乳牙免疫表型间充质干细胞的单细胞分选

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and ...

单细胞指数分型与内皮细胞小生境联合培养对胚胎造血干细胞前体的克隆分析

摘要

探索更多视频

此视频中的章节

小鼠胚胎Hemogenic内皮细胞的分离

一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性

小鼠卵黄囊和AGM早期造血干细胞的分离

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

血源性内皮细胞与人多能干细胞的定向分化

的肿瘤特异性内皮细胞的分离与培养扩展

表型分析和分离小鼠造血干细胞和传承的承诺祖

通过流式细胞术同时评估造血干细胞和祖细胞的亲缘关系、分裂数和表型

小鼠骨髓造血干细胞和祖细胞以及基质生态位细胞的流式细胞术分析

人脱落乳牙免疫表型间充质干细胞的单细胞分选

单细胞指数分型与内皮细胞小生境联合培养对胚胎造血干细胞前体的克隆分析

摘要

探索更多视频

此视频中的章节

小鼠胚胎Hemogenic内皮细胞的分离

一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性

小鼠卵黄囊和AGM早期造血干细胞的分离

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

血源性内皮细胞与人多能干细胞的定向分化

的肿瘤特异性内皮细胞的分离与培养扩展

表型分析和分离小鼠造血干细胞和传承的承诺祖

通过流式细胞术 同时 评估造血干细胞和祖细胞的亲缘关系、分裂数和表型

小鼠骨髓造血干细胞和祖细胞以及基质生态位细胞的流式细胞术分析

人脱落乳牙免疫表型间充质干细胞的单细胞分选

通过流式细胞术同时评估造血干细胞和祖细胞的亲缘关系、分裂数和表型