我们得到了马萨诸塞州总医院的HSCI-CRM流式细胞术设施和哈佛大学Bauer Core设施的专家技术支持。T.K 和 K. G 得到了瑞典研究委员会的支持，C.M. 得到了德国研究基金会的支持。我们感谢 Sergey Isaev 和 I-Hsiu Lee 在分析单细胞 RNA 测序数据方面的帮助。

所有与人体组织有关的工作都是在马萨诸塞州总医院内部审查委员会 （IRB） 批准后进行的。所有动物程序均按照马萨诸塞州总医院机构动物护理和使用委员会 （IACUC） 指南进行。本研究采用C57Bl/6只小鼠，年龄8-10周龄，雄性和雌性均为小鼠。这些动物是从商业来源获得的（见 材料表）。
1.鼠胸腺组织的制备
<ol><li>准备以下缓冲液和溶液。<ol><li>培养基 199 （M199） 与 2% （v/v） 胎牛血清 （FBS）（参见 材料表）。</li>
		<li>小鼠解离混合物：0.5 WU/mL Liberase TM 和 6.3 U/mL DNase I，M199 + 2% FBS（参见 材料表）。</li>
		<li>M199 含 2% （w/v） 牛血清白蛋白 （BSA）。 注意：含有FBS的培养基可以切换到BSA以提高RNA质量。</li>
	</ol></li>
	<li>进行小鼠胸腺解剖<ol><li>通过 CO2 窒息对小鼠实施安乐死（遵循机构批准的方案）并将其放在背上。通过喷洒 70% 乙醇弄湿胸部的毛发，然后继续打开胸腔。</li>
		<li>首先用手术剪刀在肋骨下方做一个横向切口，小心地解剖胸腺。切开小鼠两侧的肋骨，一直切到锁骨。然后切开横膈膜，使胸壁可以释放。用一把镊子提起肋骨，露出胸腔。<ol><li>胸腺是一个白色的双叶器官，位于腔体的顶部，就在心脏上方。用镊子握住胸腺，轻轻切开将胸腺固定到位的结缔组织。将解剖的胸腺组织放入冰上含有M199 + 2%FBS的6孔板的孔中。 注意：当处理老年或辐射条件的小鼠或具有胸腺缺陷的突变品系时，胸腺可能非常小，以至于需要解剖显微镜才能可靠地切除器官。</li>
		</ol></li>
		<li>使用解剖显微镜，使用钝头镊子和微弹簧剪刀去除任何胸腺外组织。</li>
	</ol></li>
	<li>进行小鼠胸腺组织的酶解离（30分钟，增量为10分钟）<ol><li>将清洁的胸腺放入 15 mL 管的盖子中，并用无菌手术剪刀将组织细碎。 注意：将用于消化的管帽中的组织切碎可以最大限度地减少由于从一个容器转移到另一个容器而造成的组织损失。当处理老年或辐射条件的小鼠或具有胸腺缺陷的突变菌株时，这一点非常重要。在这些情况下，胸腺体积较小，因此需要尽可能减少物质损失。</li>
		<li>向 15 mL 试管中加入 2 mL 小鼠解离混合物，连接含有胸腺片的盖子，并倒置 5 次以确保组织重新悬浮在解离混合物中。</li>
		<li>为防止泄漏，请用石蜡膜包裹管帽。然后将试管水平放置在37°C的水浴中，以250rpm的转速摇动。孵育 10 分钟。 注：消化方案的成功取决于在37°C和搅拌下进行的孵育。所描述的协议已经使用振荡水浴进行了优化。如果这不可用，在加热环境中启用搅拌的其他方法可能有效，但我们建议首先针对这些设置优化协议。</li>
		<li>将 15 mL 试管放入架子中，让组织碎片沉淀。小心地取出上清液，并将其通过70μm细胞过滤器放入冰冷的50ml管中。</li>
		<li>重复步骤 2-4 两次，总共 3 轮消化。在最后一次孵育结束时，组织应或多或少完全解离。</li>
		<li>向收集的细胞悬液中加入 20 mL M199 + 2% FBS。在4°C下以500× g 旋转5分钟。</li>
		<li>取出上清液并重悬于适当体积的 M199 + 2% FBS 中。然后继续进行细胞计数。 注意：M199 + 2% FBS 重悬的体积将取决于胸腺的状态。对于 6-10 周龄的小鼠，我们建议将组织重悬于 3-4 mL M199 + 2% FBS 中。然而，对于使用老化或辐射条件的组织或具有胸腺缺陷的突变菌株的实验，小胸腺尺寸将需要 0.1-1 mL 的较低体积。</li>
		<li>通过在 0.4% （w/v） 台盼蓝溶液中按 1：2 稀释细胞悬浮液来计数细胞。将细胞加载到血细胞计数器上，并在明场显微镜下计数。对未染色的细胞进行计数，以确定每毫升活细胞的数量，以及蓝色染色的细胞数量，以评估样品的活力。</li>
	</ol></li>
	<li>对小鼠胸腺基质进行荧光激活细胞分选<ol><li>在4°C下以500× g 旋转细胞5分钟。 将细胞以 5 x 107 个细胞/mL 的浓度重悬于补充有 25 U/mL Protector RNase 抑制剂的 M199 + 2% FBS 中（参见 材料表）。</li>
		<li>加入浓度为2μg/ mL的小鼠Fc块（参见 材料表），并在4°C下孵育10分钟。</li>
		<li>用以下抗体染色细胞：CD45-PE / Cy7（4μg/ mL），Ter119-PE（4μg/ mL），CD31-BUV737（2μg/ mL）和EpCam-BV711（2μg/ mL）（参见 材料表）在4°C下30分钟。</li>
		<li>在M199 + 2%BSA中洗涤样品，并在4°C下以500× g 旋转5分钟。</li>
		<li>去除上清液并将细胞重悬于含有活性染料（如DAPI（0.5μg/ mL）或7-AAD（1μg/ mL）的M199 + 2%BSA中（参见 材料表）。按照既定的方案进行胸腺基质细胞的FACS分离19。</li>
	</ol></li>
</ol>2.人胸腺组织的制备
<ol><li>准备以下缓冲液和溶液。<ol><li>培养基 199 （M199），含 2% （v/v） FBS。</li>
		<li>M199 含 1.5% （w/v） BSA。</li>
		<li>人解离混合物：1 mg/ml 胶原酶 IV 和 2 mg/mL 分散酶和 6.3 U/mL DNase I，M199 +1.5% BSA 溶液（参见 材料表）。 注：制备人胸腺组织的最后一个消化步骤包括胰蛋白酶。因此，在此之前的所有步骤都必须在含有 BSA 的缓冲液中进行，因为 FBS 会抑制胰蛋白酶活性。</li>
	</ol></li>
	<li>进行人胸腺组织分离<ol><li>将人胸腺（按照先前发表的方法19收集）置于合适的M199 + 1.5%BSA体积中，并将其储存在冰上直至处理开始。</li>
		<li>在处理人类胸腺组织时，通过在组织培养罩中进行工作并根据 CDC 指南20 使用标准预防措施，将其处理为具有潜在传染性的工作。</li>
		<li>将组织放入装有 10 mL 冰冷 M199 + 1.5% BSA 的 10 cm 培养皿中。使用无菌镊子和剪刀，小心地清除在摘除器官时受损的胸腺外脂肪或组织。</li>
		<li>使用手术剪刀和一把镊子，将胸腺切成 1 cm3 立方体，然后使用 10 mL 注射器的柱塞轻轻向下推到碎片上。这将释放一些发育中的胸腺细胞，从而减少要消化的组织体积。 注意：如果需要对胸腺细胞发育阶段进行后续分析，请保存释放的造血部分。但是，不要将该部分与酶消化过程中产生的基质细胞部分混合。据观察，将两种馏分合并显着增加了细胞悬液中形成堵塞的风险，最终降低了基质细胞的产量。在轻轻粉碎组织的过程中，很少有基质细胞丢失。</li>
	</ol></li>
	<li>进行人胸腺组织的酶解离（90分钟，增量为30分钟）<ol><li>将约 5 mg 轻度压碎的胸腺组织块转移到 50 mL 锥形管的盖子中。然后使用无菌手术剪刀将组织切细。</li>
		<li>将 8 mL 人解离混合物移入 50 mL 试管中，连接含有切碎的人胸腺组织的盖子，然后轻轻倒置试管 5 次，以确保所有组织都与酶混合物混合。</li>
		<li>用石蜡膜固定管帽，防止泄漏。将试管在37°C的振荡（250rpm）水浴中水平孵育30分钟。</li>
		<li>将 50 mL 试管置于架子中，让人胸腺组织碎片沉淀，然后取出上清液并将其通过 70 μm 细胞过滤器放入放置在冰上的新 50 ml 试管中。</li>
		<li>重复步骤2.3.2-1.3.4，用人解离混合物总共进行两轮消化。</li>
		<li>第二轮消化完成后，向剩余组织中再加入 8 mL 人解离混合物。然后将 2 mL 0.25% 胰蛋白酶移液到组织解离管中。在37°C下再孵育30分钟，以250rpm振荡。 注意：人体组织的消化时间至少为 90 分钟是绝对必要的，因为较短的孵育时间会导致基质细胞产量急剧降低。</li>
		<li>完成最后一个孵育步骤后，通过70μm细胞过滤器，将细胞悬浮液和剩余的组织片段与先前步骤中收集的上清液混合在一起。向样品中加入 3 mL FBS 以破坏胰蛋白酶反应并置于冰上。</li>
		<li>继续按照步骤 1.3 中所述对细胞进行计数。</li>
	</ol></li>
	<li>对人胸腺基质进行荧光激活细胞分选<ol><li>旋转人胸腺的细胞悬浮液，并以 5 x 107 个细胞/mL 的浓度重悬于补充有 25 U/mL Protector RNase 抑制剂的 M199 + 2% FBS 中。</li>
		<li>与人Fc模块（5μg/ mL）（参见 材料表）在4°C下孵育10分钟。</li>
		<li>用CD45-BV711（2.5μg/ mL），CD235a-BV711（2.5μg/ mL），Lineage-cocktail-FITC（3.76μg/ mL），CD66b-FITC（20μL / 100μL样品），CD8-APC / Cy7（5μL / 100μL样品），CD4-BV605（5μL / 100μL样品），CD31-PE / Dazzle594（10μg/ mL）和EpCam-BV421（2.5μg/ mL）（参见 材料目录）染色30分钟。</li>
		<li>在M199 + 1.5%BSA中洗涤样品，并在4°C下以500× g 旋转5分钟。</li>
		<li>去除上清液并将细胞重悬于含有活性染料（如DAPI（0.5μg/ mL）或7-AAD（1μg/ mL）的M199 + 1.5%BSA中。按照既定的方案进行胸腺基质细胞的FACS分离19。</li>
	</ol></li>
</ol>3.鼠骨和骨髓组织的制备
注：骨和骨髓部分在两个单独的消化反应中制备，以获得基质细胞的最大纯度和组织的最佳解离。在酶解步骤之后，可以将样品合并为一个基质室进行分类。
<ol><li>准备以下缓冲液和溶液。<ol><li>M199 与 2% （v/v） FBS。</li>
		<li>小鼠 B&M（骨和骨髓）解离混合物：M199 +2% FBS 中的 Stemxyme 2 mg/mL、分散酶 1 mg/mL 和 10 U/mL DNase I（参见 材料表）。 注意：准备新鲜的 B&M 解离混合物，并将混合物放在冰上直至使用。</li>
		<li>M199 含 0.5% （w/v） BSA。</li>
		<li>含 0.5% （w/v） BSA 的磷酸盐缓冲盐水 （PBS）。</li>
		<li>M199 含 2% （w/v） 牛血清白蛋白 （BSA） 和 2 mM EDTA。 注：含有FBS的培养基可以切换到BSA以提高RNA质量。</li>
	</ol></li>
	<li>进行小鼠骨髓分离<ol><li>通过 CO2 窒息对小鼠实施安乐死（遵循机构批准的方案）。喷洒 70% 乙醇以弄湿毛皮。</li>
		<li>小心地从两侧取出股骨，胫骨，骨盆和肱骨，并将它们放入含有M199 + 2%FBS的冰21,22孔中。</li>
		<li>用组织湿巾去除所有肌肉、韧带和软骨组织，以尽量减少对骨骼的损伤并保留骨膜层。将它们放入冰上含有 M199 + 2% FBS 的新孔中。</li>
		<li>用手术刀切开长骨的生长板，切去骨骺，将其放入10厘米的培养皿中，在冰上加入M199 + 2%FBS。</li>
		<li>将骨髓从骨头中冲出，直到呈淡白色。使用 28 G 针头和装满 10 mL M199 + 2% FBS 的 10 mL 注射器将骨髓冲洗到冰上的 15 mL 管中。将冲洗的骨头与骨骺一起放入 10 厘米的培养皿中。 注意：完全冲洗骨髓以完全分离组织将导致更好的样品制备。</li>
	</ol></li>
	<li>进行小鼠骨髓组织的酶解离（总共 30 分钟，增量为 10 分钟）<ol><li>将 15 mL 试管直立在冰上，直到骨髓沉入试管底部。通过移液缓慢去除 10 mL M199 + 2% FBS。</li>
		<li>向15mL管中加入4mL B&M解离混合物，并将其置于37°C水浴（不摇晃）中10分钟。</li>
		<li>5分钟后，将试管倒置三次，然后将其放回水浴中。</li>
		<li>第一个 10 分钟后，确保沉淀位于底部并收集上清液，同时通过 70 μm 细胞过滤器过滤到冰冷的 50 mL 管中。将 2 mL 新鲜的 B&M 解离混合物加入剩余的 BM 沉淀中，然后将其放回水浴中。</li>
		<li>通过使用 1 mL 移液管剧烈移液来解离任何剩余的骨髓碎片。将所有产生的细胞悬液收集在同一管中，就像在前面的步骤一样。向收集的细胞悬液中加入 20 mL M199 + 0.5% BSA + 2 mM EDTA。 注：在步骤3.3.5中，在最终消化样品后添加的缓冲液含有EDTA，以帮助抑制进一步的酶活性。</li>
	</ol></li>
	<li>对小鼠骨髓组织进行造血细胞耗竭。<ol><li>在4°C下以500× g 离心细胞5分钟。 用靶向 CD45、Ter119、CD11b、Gr1、B220 和 CD3 的生物素抗体 （5 mg/mL） 重悬于 500 μL PBS + 0.5% BSA 中（参见 材料表）。转移到带盖的圆底管中。</li>
		<li>在室温下，在黑暗中在轨道振荡器上孵育10分钟。</li>
		<li>彻底涡旋磁珠，并向试管中加入 25 μL 磁珠。</li>
		<li>在室温下，在黑暗中在轨道振荡器上孵育5分钟。</li>
		<li>将试管放入匹配的磁铁中 2-3 分钟，并将上清液收集在新鲜的试管中。 注意：在此步骤之后，骨髓可以合并或与骨组分开。如果谱系去除成功，则所得细胞悬液应不含红细胞。</li>
	</ol></li>
	<li>进行小鼠骨组织的酶解离。<ol><li>用 50 mL 管的扁平盖端将骨头在 10 cm 培养皿中切成小块。通过70μm细胞过滤器过滤上清液，以分离过滤器内的骨碎片。 注意：或者，可以使用研钵和研杵进行骨碎裂。然而，当骨头被磨碎而不是破碎时，这种选择往往会产生更多的碎片和更低的基质细胞活力。基质细胞仍然附着在骨骼上和骨骼中，含有造血细胞的上清液应被丢弃。</li>
		<li>用剪刀剪开细胞过滤器中的骨头碎片。这将增加表面积，从而实现更有效的酶解离。用 5 mL M199 + 2% FBS 清洗骨头。</li>
		<li>将骨部分放入 5 mL 小鼠 B&M 解离液（参见 材料表）混合物中，放入新的 50 mL 管中。</li>
		<li>为防止泄漏，请用石蜡膜包裹管帽。然后将试管水平置于37°C水浴中，以120rpm振荡30分钟。</li>
		<li>消化后，加入 25 mL M199 + 0.5% BSA + 2 mM EDTA，并通过 70 μm 细胞过滤器过滤含有基质细胞的上清液到冰冷的 50 ml 管中。 注意：检查产生的骨头碎片是否因消化而明显减少。在步骤3.5.5中，在最终消化样品后添加的缓冲液含有EDTA，以帮助抑制进一步的酶活性。</li>
	</ol></li>
	<li>对小鼠骨骼和骨髓基质进行荧光激活细胞分选。<ol><li>在4°C下以500× g 旋转细胞5分钟。</li>
		<li>以10μg/ mL的浓度在PBS + 0.5%BSA + 2mM EDTA中重悬于小鼠Fc块中，并在4°C下孵育10分钟。</li>
		<li>用以下抗体对细胞（多达 1000 万个细胞/100 μL 总染色体积）进行染色：CD45-PE/Cy7 （1 μg/mL）、Ter119-PE/Cy7 （1 μg/mL）、CD31-BV421 （0.66 μg/mL）、CD140a-APC （2 μg/mL）、Sca1-AF700 （0.66 μg/mL）、CD51-PE （2 μg/mL）、CD105-PE/dazzle （2 μg/mL）（参见 材料表）。</li>
		<li>让Cal黄绿素-AM平衡至室温，并重悬于50μL DMSO中。为每次使用准备新鲜的。重悬后在室温下保存。在加入已经重悬于抗体染色混合物中的样品（20μg/ mL）之前，在PBS（0.1mg / mL）中预稀释钙黄绿素，并在4°C下在黑暗中孵育20-30分钟。</li>
		<li>在PBS + 0.5%BSA + 2mM EDTA中洗涤样品，并在4°C下以500× g 离心5分钟。</li>
		<li>取出上清液并将细胞重悬于PBS + 0.5%BSA + 2mM EDTA中。按照既定的方案进行骨和骨髓基质细胞的FACS分离19。</li>
	</ol></li>
</ol>

优化从胸腺和骨髓中分离基质细胞

<ol>
	<li>Baryawno, 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=A+cellular+taxonomy+of+the+bone+marrow+stroma+in+homeostasis+and+leukemia.">A cellular taxonomy of the bone marrow stroma in homeostasis and leukemia.</a> Cell. 177 (7), 1915-1932 (2019).</li><li>Severe, 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=Stress-induced+changes+in+bone+marrow+stromal+cell+populations+revealed+through+single-cell+protein+expression+mapping.">Stress-induced changes in bone marrow stromal cell populations revealed through single-cell protein expression mapping.</a> Cell Stem Cell. 25 (4), 570-583 (2019).</li><li>Han, J., Zuniga-Pflucker, 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=A+2020+view+of+thymus+stromal+cells+in+t+cell+development.">A 2020 view of thymus stromal cells in t cell development.</a> J Immunol. 206 (2), 249-256 (2021).</li><li>Park, J. 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=A+cell+atlas+of+human+thymic+development+defines+t+cell+repertoire+formation.">A cell atlas of human thymic development defines t cell repertoire formation.</a> Science. 367 (6480), (2020).</li><li>Denisenko, 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=Systematic+assessment+of+tissue+dissociation+and+storage+biases+in+single-cell+and+single-nucleus+rna-seq+workflows.">Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus rna-seq workflows.</a> Genome Biol. 21 (1), 130 (2020).</li><li>Lischetti, U., 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=Dynamic+thresholding+and+tissue+dissociation+optimization+for+cite-seq+identifies+differential+surface+protein+abundance+in+metastatic+melanoma.">Dynamic thresholding and tissue dissociation optimization for cite-seq identifies differential surface protein abundance in metastatic melanoma.</a> Commun Biol. 6 (1), 830 (2023).</li><li>Ding, L., Saunders, T. L., Enikolopov, G., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+and+perivascular+cells+maintain+haematopoietic+stem+cells.">Endothelial and perivascular cells maintain haematopoietic stem cells.</a> Nature. 481 (7382), 457-462 (2012).</li><li>Mendez-Ferrer, 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=Mesenchymal+and+haematopoietic+stem+cells+form+a+unique+bone+marrow+niche.">Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.</a> Nature. 466 (7308), 829-834 (2010).</li><li>Raaijmakers, M. 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=progenitor+dysfunction+induces+myelodysplasia+and+secondary+leukaemia.">progenitor dysfunction induces myelodysplasia and secondary leukaemia.</a> Nature. 464 (7290), 852-857 (2010).</li><li>Himburg, H. 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=Distinct+bone+marrow+sources+of+pleiotrophin+control+hematopoietic+stem+cell+maintenance+and+regeneration.">Distinct bone marrow sources of pleiotrophin control hematopoietic stem cell maintenance and regeneration.</a> Cell Stem Cell. 23 (3), 370-381 (2018).</li><li>Zhou, B. O., 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=marrow+adipocytes+promote+the+regeneration+of+stem+cells+and+haematopoiesis+by+secreting+scf.">marrow adipocytes promote the regeneration of stem cells and haematopoiesis by secreting scf.</a> Nat Cell Biol. 19 (8), 891-903 (2017).</li><li>Steinmann, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+human+thymus+during+aging.">Changes in the human thymus during aging.</a> Curr Top Pathol. 75, 43-88 (1986).</li><li>Steinmann, G. G., Klaus, B., Muller-Hermelink, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+involution+of+the+ageing+human+thymic+epithelium+is+independent+of+puberty.+A+morphometric+study.">The involution of the ageing human thymic epithelium is independent of puberty. A morphometric study.</a> Scand J Immunol. 22 (5), 563-575 (1985).</li><li>Ambrosi, T. 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=Adipocyte+accumulation+in+the+bone+marrow+during+obesity+and+aging+impairs+stem+cell-based+hematopoietic+and+bone+regeneration.">Adipocyte accumulation in the bone marrow during obesity and aging impairs stem cell-based hematopoietic and bone regeneration.</a> Cell Stem Cell. 20 (6), 771-784 (2017).</li><li>Ho, Y. 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=Remodeling+of+bone+marrow+hematopoietic+stem+cell+niches+promotes+myeloid+cell+expansion+during+premature+or+physiological+aging.">Remodeling of bone marrow hematopoietic stem cell niches promotes myeloid cell expansion during premature or physiological aging.</a> Cell Stem Cell. 25 (3), 407-418 (2019).</li><li>Kusumbe, A. 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=Age-dependent+modulation+of+vascular+niches+for+haematopoietic+stem+cells.">Age-dependent modulation of vascular niches for haematopoietic stem cells.</a> Nature. 532 (7599), 380-384 (2016).</li><li>Seach, N., Wong, K., Hammett, M., Boyd, R. L., Chidgey, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+enzymes+improve+isolation+and+characterization+of+the+adult+thymic+epithelium.">Purified enzymes improve isolation and characterization of the adult thymic epithelium.</a> J Immunol Methods. 385 (1-2), 23-34 (2012).</li><li>Stoeckle, C., 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=Isolation+of+myeloid+dendritic+cells+and+epithelial+cells+from+human+thymus.">Isolation of myeloid dendritic cells and epithelial cells from human thymus.</a> J Vis Exp. (79), e50951 (2013).</li><li>Gustafsson, K., Scadden, D. T. <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+thymus+stromal+cells+from+human+and+murine+tissue.">Isolation of thymus stromal cells from human and murine tissue.</a> Methods Mol Biol. 2567, 191-201 (2023).</li><li>Amend, S. R., Valkenburg, K. C., Pienta, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+hind+limb+long+bone+dissection+and+bone+marrow+isolation.">Murine hind limb long bone dissection and bone marrow isolation.</a> J Vis Exp. (110), e53936 (2016).</li><li>Zhu, 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=A+protocol+for+isolation+and+culture+of+mesenchymal+stem+cells+from+mouse+compact+bone.">A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone.</a> Nat Protoc. 5 (3), 550-560 (2010).</li><li>Calvi, L. 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=Osteoblastic+cells+regulate+the+haematopoietic+stem+cell+niche.">Osteoblastic cells regulate the haematopoietic stem cell niche.</a> Nature. 425 (6960), 841-846 (2003).</li><li>Kode, 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=Leukaemogenesis+induced+by+an+activating+beta-catenin+mutation+in+osteoblasts.">Leukaemogenesis induced by an activating beta-catenin mutation in osteoblasts.</a> Nature. 506 (7487), 240-244 (2014).</li><li>Agarwal, 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=Mesenchymal+niche-specific+expression+of+cxcl12+controls+quiescence+of+treatment-resistant+leukemia+stem+cells.">Mesenchymal niche-specific expression of cxcl12 controls quiescence of treatment-resistant leukemia stem cells.</a> Cell Stem Cell. 24 (5), 769-784 (2019).</li><li>Duarte, D., 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=Inhibition+of+endosteal+vascular+niche+remodeling+rescues+hematopoietic+stem+cell+loss+in+aml.">Inhibition of endosteal vascular niche remodeling rescues hematopoietic stem cell loss in aml.</a> Cell Stem Cell. 22 (1), 64-77 (2018).</li><li>O'flanagan, C. 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=Dissociation+of+solid+tumor+tissues+with+cold+active+protease+for+single-cell+rna-seq+minimizes+conserved+collagenase-associated+stress+responses.">Dissociation of solid tumor tissues with cold active protease for single-cell rna-seq minimizes conserved collagenase-associated stress responses.</a> Genome Biol. 20 (1), 210 (2019).</li><li>Stoeckius, 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=Cell+hashing+with+barcoded+antibodies+enables+multiplexing+and+doublet+detection+for+single+cell+genomics.">Cell hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics.</a> Genome Biol. 19 (1), 224 (2018).</li></ol>

D.T.S. 是 Agios Therapeutics 和 Editas Medicines 的董事和股东;Magenta Therapeutics 和 LifeVault Bio 的创始人、董事、股东和科学顾问委员会成员，Fate Therapeutics 和 Garuda Therapeutics 的股东和创始人，Clear Creek Bio 的董事、创始人和股东，FOG Pharma、Inzen Therapeutics 和 VCanBio 的顾问，以及 Sumitomo Dianippon 赞助研究资金的接受者。D.T.S和K.G是专利US20220143099A1的发明者。

造血器官中的基质细胞对于正常造血至关重要，而造血基质扰动可导致造血维持和对应激反应的严重损害 9,23,24。深入了解造血基质细胞对于理解血液疾病至关重要 7,9,10,24 以及开发对抗它们的治疗方法的能力 25,26</sup...

在健康的成年人中，血细胞的从头产生发生在骨髓和胸腺中。这些部位的基质细胞对于维持造血至关重要，但基质占组织的不到 1%，2,3,4。因此，获得支持基质的造血干细胞的纯分离株是一个重大挑战，特别是对于单细胞多组学而言，需要权宜之计才能获得高质量的样本。不同消化混合物的成分可能会干扰多组学分析中的某些步骤5,6。本文介绍的方案详细介绍了从骨髓和胸腺组织中分离出多种基质细胞的方法。
骨髓和胸腺中基质成分的扰动会导致血细胞发育的严重破坏，并可能导致恶性肿瘤 7,8,9。在细胞毒性调理和骨髓移植后，支持基质的造血功能受到损害，导致维持造血干细胞和祖细胞 （HSPC） 的细胞因子和生长因子的分泌减少2,10,11。此外，衰老会影响骨髓和胸腺基质细胞，可能导致造血表型老化。胸腺是第一个经历广泛的年龄相关退化的器官。早在青春期开始时，脂肪和纤维化组织就开始取代 T 细胞支持基质12,13。在骨髓中，脂肪细胞含量随着年龄的增长而增加，血管和骨内膜壁龛显着重塑14,15,16。
为了能够在多种应激状态下以及人和小鼠组织的胸腺中研究造血支持基质，我们优化了先前发表的消化方案1、2、8、17、18。这些方案确保了细胞的高效和可重复的分离，并且它们与单细胞RNA测序（scRNAseq）和其他类型的多组学兼容。

<table><tbody><tr><td>0.25% Trypsin-EDTA</td><td>Thermo Fisher Scientific</td><td>25200-072</td><td></td></tr><tr><td>7AAD (7-aminoactinomycin D)</td><td>BD Biosciences</td><td>559925</td><td></td></tr><tr><td>Anti-Human Lineage Cocktail 3-FITC</td><td>BD Biosciences</td><td>643510</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Millipore Sigma</td><td>A9647</td><td></td></tr><tr><td>C57Bl/6 mice</td><td>Jackson</td><td>664</td><td>Males or females, 8-12 weeks old</td></tr><tr><td>Calcein&amp;nbsp;</td><td>Fisher Scientific</td><td>65-0853-78</td><td></td></tr><tr><td>Collagenase IV</td><td>Millipore Sigma</td><td>C5138</td><td></td></tr><tr><td>Corning Sterile Cell Strainers, White, Mesh Size: 70 &amp;micro;m</td><td>Fisher Scientific</td><td>08-771-2</td><td></td></tr><tr><td>Dispase II</td><td>Thermo Fisher Scientific</td><td>17105041</td><td></td></tr><tr><td>Dnase I Solution</td><td>Thermo Fisher Scientific</td><td>90083</td><td>&amp;nbsp;2500 U/mL</td></tr><tr><td>Easysep mouse streptavidin RapidSpheres Isolation kit</td><td>StemCell Technologies</td><td>19860</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>A31605-01</td><td>Qualified One Shot</td></tr><tr><td>Human Fc Block</td><td>BD Biosciences</td><td>564220</td><td></td></tr><tr><td>Liberase TM&amp;nbsp;</td><td>Millipore Sigma</td><td>5401127001</td><td>Research Grade</td></tr><tr><td>Medium 199</td><td>Gibco</td><td>12350</td><td></td></tr><tr><td>Mouse anti-human CD235a-BV77</td><td>BD Biosciences</td><td>740785</td><td></td></tr><tr><td>Mouse anti-human CD31-PE/Dazzle594</td><td>Biolegend</td><td>303130</td><td></td></tr><tr><td>Mouse anti-human CD45-BV77</td><td>Biolegend</td><td>304050</td><td></td></tr><tr><td>Mouse anti-human CD4-BV605</td><td>BD Biosciences</td><td>562658</td><td></td></tr><tr><td>Mouse anti-human CD66b-FITC</td><td>BD Biosciences</td><td>555724</td><td></td></tr><tr><td>Mouse anti-human CD8-APC/Cy7</td><td>BD Biosciences</td><td>557760</td><td></td></tr><tr><td>Mouse anti-human EpCam-BV421</td><td>Biolegend</td><td>324220</td><td></td></tr><tr><td>Protector RNase Inhibitor</td><td>Millipore Sigma</td><td>3335402001</td><td></td></tr><tr><td>Rat anti-mouse CD105-PE /dazzle594</td><td>Biolegend</td><td>120424</td><td></td></tr><tr><td>Rat anti-mouse CD11b-Biotin</td><td>Biolegend</td><td>101204</td><td></td></tr><tr><td>Rat anti-mouse CD140a-APC</td><td>Fisher Scientific</td><td>17-1401-81</td><td></td></tr><tr><td>Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)</td><td>BD Biosciences</td><td>553142</td><td></td></tr><tr><td>Rat anti-mouse CD31-BUV737</td><td>BD Biosciences</td><td>612802</td><td></td></tr><tr><td>Rat anti-mouse CD31-BV421</td><td>Biolegend</td><td>102424</td><td></td></tr><tr><td>Rat anti-mouse CD3-Biotin</td><td>Biolegend</td><td>100244</td><td></td></tr><tr><td>Rat anti-mouse CD45.2-Biotin</td><td>Biolegend</td><td>109804</td><td></td></tr><tr><td>Rat anti-mouse CD45-PE/Cy7</td><td>Biolegend</td><td>103114</td><td></td></tr><tr><td>Rat anti-mouse CD45-PE/Cy7</td><td>Biolegend</td><td>103114</td><td></td></tr><tr><td>Rat anti-mouse CD45-PE/Cy7</td><td>Biolegend</td><td>103114</td><td></td></tr><tr><td>Rat anti-mouse CD45R/B220-Biotin</td><td>Biolegend</td><td>103204</td><td></td></tr><tr><td>Rat anti-mouse CD51-PE</td><td>Biolegend</td><td>104106</td><td></td></tr><tr><td>Rat anti-mouse EpCam-BV711</td><td>BD Biosciences</td><td>563134</td><td></td></tr><tr><td>Rat anti-mouse Ly-6A/E(Sca-1)-AF700</td><td>Biolegend</td><td>108142</td><td></td></tr><tr><td>Rat anti-mouse Ly-6G/Ly-6C(Gr1)-Biotin</td><td>Biolegend</td><td>108404</td><td></td></tr><tr><td>Rat anti-mouse Ter119-Biotin</td><td>Biolegend</td><td>116204</td><td></td></tr><tr><td>Rat anti-mouse Ter119-PE</td><td>Biolegend</td><td>116208</td><td></td></tr><tr><td>Rat anti-mouse Ter119-PE/Cy7</td><td>Biolegend</td><td>116222</td><td></td></tr><tr><td>Stemxyme&amp;nbsp;</td><td>Worthington Biochemical</td><td>LS004107</td><td></td></tr></tbody></table>

stromal cell isolation from hematopoietic organs

单细胞测序使得造血器官基质中异质细胞群的定位成为可能。这些方法提供了一个视角，通过它来研究以前未解决的稳态异质性，以及由外在应激或衰老过程中诱导的细胞类型表示的变化。在这里，我们提出了从小鼠和人胸腺以及小鼠骨骼和骨髓中分离高质量基质细胞群的逐步方案。通过这些方案分离的细胞适用于生成高质量的单细胞多组学数据集。本文将讨论样本消化、造血谱系耗竭、FACS分析/分选的影响，以及这些因素如何影响与单细胞测序的兼容性。通过FACS谱的示例，表明测序后分析中成功和低效的解离以及下游基质细胞产量，为用户提供了可识别的指针。考虑基质细胞的特定要求对于获得高质量和可重复的结果至关重要，这些结果可以推动该领域的知识发展。

这些方案从胸腺和骨髓中产生可重复的基质细胞品种，适用于流式细胞术分析，以及单细胞多组学，如scRNA测序。小鼠胸腺组织在应激源的作用下经历了显著的重塑，例如骨髓移植前的细胞毒性调节或自然衰老过程。因此，在这两种情况下，胸腺细胞性都急剧降低（图1A）。虽然来自8周大的野生型小鼠的胸腺含有大约1亿个细胞，但2岁小鼠的细胞密度可以预期是其中的一半，...

Watch this Scientific Journal Video about Stromal Cell Isolation From Hematopoietic Organs at JoVE.com

作者聚焦：使用基质细胞分离进行单细胞测序推进造血研究

在这里，我们提出了能够从小鼠骨骼、骨髓、胸腺和人胸腺组织中分离基质细胞的方案，这些组织与单细胞多组学兼容。

从造血器官中分离基质细胞

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

骨髓和胸腺中血细胞的发育高度依赖基质细胞的支持。造血基质细胞功能的扰动可导致血液生成效率低下甚至恶性。我们的实验室专注于了解造血的基质细胞调节以及如何操纵它以获得治疗效果。单细胞测序的出现推动了许多研究领域的最新进展。在我们的领域也是如此，单细胞分辨率使得能够识别出以前未知的造血器官中的异质基质细胞群。我们的协议已经过优化以解决这个问题，我们希望它们能够对基质细胞进行更多的研究，以解决该领域的关键问题。

stromal-cell-isolation-from-hematopoietic-organs

Research

JoVE Journal

Immunology and Infection

Stromal Cell Isolation From Hematopoietic Organs

1.0K 次观看.  Massachusetts General Hospital. 骨髓和胸腺中血细胞的发育高度依赖基质细胞的支持。造血基质细胞功能的扰动可导致血液生成效率低下甚至恶性。我们的实验室专注于了解造血的基质细胞调节以及如何操纵它以获得治疗效果。单细胞测序的出现推动了许多研究领域的最新进展。在我们的领域也是如此，单细胞分辨率使得能够识别出以前未知的造血器官中的异质基质细胞群。我们的协议已经过优化以?...

视频: 作者聚焦：使用基质细胞分离进行单细胞测序推进造血研究

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

分离和造血干细胞移植（造血干细胞）

科研

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

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, ...

发育生物学

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

从人类心脏组织血管周围多能前体细胞群的分离

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

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 ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

生物工程

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 ...

<meta charset="utf8"></head><body>骨类器官衍生细胞在辐照小鼠中进行有效 HSCT

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

<meta charset="utf8"></head><body>用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

从造血器官中分离基质细胞

副本

摘要

探索更多视频

分离和造血干细胞移植（造血干细胞）

从小鼠胚胎干细胞的造血干细胞的推导

小鼠胚胎Hemogenic内皮细胞的分离

从人类心脏组织血管周围多能前体细胞群的分离

小鼠卵黄囊和AGM早期造血干细胞的分离

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

表型分析和分离小鼠造血干细胞和传承的承诺祖

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法