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>
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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. 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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>AGM-EC culture media </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials for culture of endothelial cells</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&#39;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>AGM-EC culture media (500 mls)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;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>Materials for AGM index sort</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 &#181;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>Antibodies for AGM index sort</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>AGM-serum free media (10mls)</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>Materials for Staining co-cultured cells</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 colspan="3">Antibodies for analysis of Index sorted clones co-cultured with endothelial cells</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>Materials for tail vein injection for transplant</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1/2 ml insulin syringes with 29G 1/2&#34; needles</td>
			<td>BD Biosciences</td>
			<td>309306</td>
			<td>Use for tail vein injection for transplantation</td>
		</tr>
		<tr>
			<td>Antibodies for Peripheral Blood analysis following translant</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>Equipment</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

Universidad de Cartagena UDEC

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

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

通过在胚胎梅克尔软骨Zebrabow克隆细胞分析软骨插层和定向扩散的可视化

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

科研

发育生物学

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

转基因器官皮肤培养使用灭活人真皮代

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

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, ...

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

竞争性移植评价造血干细胞健身

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness following surgical intervention are key experimental strengths. Mica plates were the first barriers used to prevent chick limb bud initiation1. Protocols that use aluminum foil as an impermeable barrier to wing bud or leg bud induction and or initiation are described. We combine this technique with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The results are analyzed using in situ hybridization of subsequent gene expression. Our main focus is on the role of retinoic acid signaling in the induction and later initiation of the chick embryo fore and hindlimb. We use BMS 493 (an inverse agonist of retinoic acid receptors (RAR)) soaked beads implanted in the lateral plate mesoderm (LPM) to mimic the effect of a barrier placed between the somites (a source of retinoic acid (RA)) and the LPM from which limb buds grow. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues. Provided the region of the chick embryo is accessible at the relevant developmental stage, a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in the developing brain, axis extension and in organ development, such as liver or kidney induction.

Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

在小鸡学习感应信号与候选因子浸泡珠结合防渗屏障中的应用

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

Hemoglobinopathies 患者造血干细胞移植后残余供红祖细胞的检测

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

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 ...