这项工作得到了 Kinderkrebshilfe Regenbogen 提的支持。

1. 多参数荧光活化细胞分离纯化造血骨髓细胞的研究 <ol> <li> 示例着色 <ol> <li> 在启动进程之前, 需要收集以下项目并准备: <ol> <li> 用于制备单个核细胞 (GM) 的梯度介质, 50 毫升锥形管 </li> <li> 12 x 75 mm 染色细胞的流管 </li> <li> 染色缓冲液: 磷酸缓冲盐水 (PBS) + 3% 胎小牛血清 (FCS) </li> <li> 管我 >> <li> FC 阻断和染色抗体 (CD34 APC, CD36 FITC, CD45 V450, BD 生物科学)。有了这种荧光组合, 就可以忽略溢出到其他渠道, 但任何其他合适的荧光组合也可能. </li>
			<li> 补偿珠子 (如有必要) </li>
			<li> 悬浮缓冲: 汉克 & #39 平衡盐溶液 (HBSS) + 25 mM HEPES + 3% FCS </li>
			<li> 台盼蓝和例 </li>
			<li> 收集管: 任何富含高血清管的培养基 (含1毫升的 FCS + 25 mM HEPES) 可用于收集排序的单元格. </li>
			<li> 骨髓样本: 通常需要从髋部背部的髂骨嵴进行骨髓活检的知情同意。骨髓标本比在 RT 中保存在素注射器, 直到染色。下列协议步骤的执行遵循了机构和 #39; 人类福利研究伦理委员会的指导方针. </li>
		</ol> </li> <li> 生成骨髓样本的单个细胞悬浮。在离心管中加入3毫升的 GM。小心地把4毫升的单细胞悬浮液放到 GM 溶液中。离心机在 550 g 为30分钟在20和 #176; 摄氏 (C) (刹车关闭)。用无菌吸管绘制含有血浆和血小板的上层, 使单个核细胞层在界面上不受干扰。使用无菌吸管将单核细胞层转移到无菌离心管. </li>
		<li> 用5毫升染色缓冲液冲洗后, 在2毫升悬浮缓冲液中重细胞, 测定细胞浓度。将5和 #181; L 在螺帽试管中的细胞悬浮。添加95和 #181; 0.2% 台盼蓝染色的 L。混合彻底。允许在室温下站立5分钟。填充例作为细胞计数。在显微镜下观察不是否被染色并计数存活的细胞. </li>
		<li> 在20和 #176 离心250克的细胞10分钟; C、丢弃上清和重染色缓冲液中的细胞, 浓度可达 1 x 10 6 每100和 #181; L. </li>
		<li> 通过使用2.5 和 #181 阻止 FcR; g Fc 块每 1 x 10 6 冰上的单元格 10-15 min </li>
		<li> 添加适当的10和 #181 的组合; g 单克隆抗体染色 1 x 10 6 细胞和孵化20分钟4和 #176; C 在黑暗中, 其次是洗涤步骤与3毫升染色缓冲区。为获得正确的补偿, 小等分细胞 (或优选补偿珠) 被染色与单一抗体;一个分剩余的不. </li>
		<li> 将浓度调整为 20 x 10 6 单元/mL. </li>
		<li> 设置和优化单元格排序器。建立流式细胞仪和软件的过程是标准化的, 由公司提供的详细说明, 需要由经过适当培训的人员进行. </li>
	</ol> </li> <li> 在单元格排序器 <ol> <li> 上从步骤1.1.1.9 中准备收集管. </li>
		<li> 设置一个包含二元图形的模板, 以显示前向散射 (FSC) 和侧散点 (SSC). </li>
		<li> 运行单元格并调整 FSC/SSC 以将感兴趣的人群放在比例上 </li>
		<li> 记录负控制管. </li>
		<li> 运行单个正向控制管; 记录每个管的数据. </li>
		<li> 使用购置软件提供的功能手动或自动计算补偿. </li>
		<li> 获取实验样本并将它们放在一个双变量 FSC/SSC 点图 ( 图 1A ) 中, 以包括淋巴细胞和髓细胞。通过比较 FSC 的不同信号 ( 即 、高度、宽度和面积) 来排除双峰和聚合体 ( 图 1B )。可视化显示 CD45 和 CD36 ( 图 1C ) 的双变量点图上的单线态单元。积极的 CD45 是白 (包括他们的祖), 那些消极的 CD45 和积极的 CD36 是红祖。使用浇注工具来定义 CD36+ (如果需要, 这些细胞可以进一步划分 CD36 高和低表达细胞) 和 CD45+ 细胞。可视化 CD45+ 事件在另一个点图中显示 CD34 和 SSC 参数。使用浇注工具来定义 CD34+ 细胞 (白细胞祖) 和 SSC 高细胞 (不同分化阶段的髓细胞). </li>
		<li> 一旦确定了闸门, 就可以选择闸门来分拣外部收集管. </li>
		<li> 按4和 #176 进行排序, 直到获得所需的单元格数 </li>
		<li> 执行 post-sort 分析以确定已排序的单元格填充的纯度 ( 图 1E , 1F ). </li>
	</ol> </li> </ol> 2。克隆检测 <ol> <li> 试剂的制备 在开始该过程之前, 需要收集并准备以下项目: <ol> <li> 管, 100 mm 板 </li> <li> 台盼蓝和例 </li> <li> 重建人类重组促红细胞生成素 (EPO) 在 500 U/毫升的无菌 PBS 含有至少0.1% 人血清白蛋白。将此解决方案划分为小等分, 并保持在-20 和 #176; C, 以避免重复的冻结解冻. </li>
		<li> 准备完整的 Iscove 和 #39 的 Dulbecco 和 #39 培养基 (IMDM), 最终浓度为 5% FCS, 2 毫米谷氨酰胺, 100 单位/毫升青霉素/链霉素 (PS)。在不同浓度的 IMDM 中加入人类重组促红细胞生成素 (epo): 3 单位 epo/井, 6 单位 epo/井和 12 epo 单位/井 (1 x, 2 x, 4 x epo 分别). </li>
	</ol> </li> <li> 骨髓细胞的制备 <ol> <li> 10 毫升骨髓样本稀释20毫升 PBS 是缓慢地在15毫升密度梯度培养基上的50毫升锥形管, 保持清晰的界面之间的两个阶段和你下无菌条件。然后离心15毫升圆锥管在 550 x g 的30分钟室温 (RT) 与加速度设置为9和减速设置为 0 (或刹车). </li>
		<li> 离心后, 将接口移到新的50毫升管中, 并将 PBS 添加到50毫升的最终容量中. </li>
		<li> 离心后与250克10分钟, 在10和 #176; C 去除上清和重细胞在完全 IMDM 培养基在大约 0.5 x 10 6 细胞/毫升。采取10和 #181; L 的细胞悬浮成微, 混合与相同体积的台盼蓝溶液 (0.4%) 和计数使用例. </li>
		<li> Optional: 根据制造的指示, CD34+ 细胞通过 Milteny CD34 珠的磁性细胞分类而富集。这一浓缩步骤的主要优点是检测造血干细胞的质量, 这是生长在一个半固态基质添加 50 ng/毫升干细胞因子, 20 ng/毫升粒细胞-巨噬细胞集落刺激因子 (GM-CSF), 20 ng/毫升interleukin-3 (IL-3), 20 ng/毫升 interleukin-6 (IL-6), 20 ng/毫升粒细胞集落刺激因子 (g-csf), 和3不同浓度的 EPO, 在 1.4. </li>
		<li> 将单元格稀释到 0.1-0.15 x 10 6 单核细胞/毫升或 2 x 10 3 CD34 + 细胞/ml, 添加完整的 IMDM 培养基, 并辅以 EPO 和转移300和 #181; L 到3毫升 Methocult H4434 培养基。搅拌后, 一个短孵育5分钟板三乘以11毫升在一个35毫米的菜。其中两种文化菜肴与第三个填充的水放在100毫米的盘子里. </li>
		<li> 细胞在湿润的37和 #176 中培养; C 孵化器与 5% CO 2 14 天. </li>
	</ol> </li> <li> 对殖民地的分析 <ol> <li> 的殖民地是根据他们的形态学和一个倒置显微镜在40X 放大倍率在一个培养皿标有评分网格。为了我们的分析目的, 菌落形成单位 (CFU) 分为4类: 多向祖细胞 (CFU-GEMM)、粒细胞-巨噬祖细胞 (CFU-GM)、爆裂形成单元-红 (BFU-E) 和菌落形成单位-红 (CFU-E)。请参阅制造商和 #39 关于进一步殖民地分类的说明. </li>
		<li> 为进一步分析, CFU 检测板的细胞分别根据4种类别进行回收, 悬浮在4毫升室温 PBS 中, 内含 2% FCS/IMDM。离心后, 在400克10分钟, 在4和 #176; C, 细胞悬浮在 PBS, 计数, 并处理 DNA 提取. </li>
	</ol> </li> </ol> 3。嵌的分析 <ol> <li> DNA 隔离 <ol> <li> 通过离心在400克上为10分钟的细胞进行颗粒化。
		<li> 使用血液 dna 提取试剂盒 (QIAmp dna 血液萃取试剂盒) 分离 dna。预热洗脱缓冲剂 (AE) 在37和 #176; C 提高洗 DNA 的收率. </li>
		<li> 用 Nanodrop ND-1000 光谱光度计测定 DNA 浓度。样品与 OD 260/280 在 1.8-2.0 之间使用作进一步分析. </li>
		<li> DNAase 自由 H 的稀释 DNA 2 O 到 0.1 ng/和 #181; l 在总容量为50和 #181; l. </li>
	</ol> </li> <li> STR 分析 <ol> <li> 通过混合反应混合、AmplTaq 金 dna 聚合酶和探查器以及20和 #181 的底漆集设置 PCR 反应; 根据手册 (0.1 ng/和 #181; l) (入门套件包含缓冲区中未标记的底漆, 用来放大 STR 基因座 D3S1358、vWA、FGA、D8S1179、D21S11、D18S51、D5S818、D13S317 和 D7S820, 以及性别标记釉)。包括无核酸的水作为阴性控制和9947A 作为正面控制。捐献者和受体的移植前 DNA 也包括在内. </li>
		<li> 使用离心 mastercycler 梯度进行 PCR 反应, 条件如下:95 和 #176; c 为 11 min, 28 放大周期95和 #176; c 为 1 min、59和 #176; c 为 1 min 和72和 #176; c 为1分钟, 后跟60和 #176; c 为 45 min. </li>
		<li> 设置碎片分析反应, 增加12和 #181; Formamid 和0.5 和 #181; l 基因500火箭尺寸标准 (全部应用生物) 到1和 #181; PCR 产物 l。在第一和最后的位置位梯 (Genotyper 安培 Fl STR 蓝色, 绿色 II 和黄色, 应用生物) 是需要的. </li>
		<li> 使用电泳仪开始电泳和采集. </li>
		<li> 使用软件分析样本和控件的位、等高和峰值高度 13 . </li>
	</ol>
	</li>
</ol>

<ol>
	<li>Angelucci, 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=Hematopoietic+stem+cell+transplantation+in+thalassemia+major+and+sickle+cell+disease:+indications+and+management+recommendations+from+an+international+expert+panel.">Hematopoietic stem cell transplantation in thalassemia major and sickle cell disease: indications and management recommendations from an international expert panel.</a> Haematologica. 99, 811-820 (2014).</li><li>Lucarelli, G., Isgro, A., Sodani, P., Gaziev, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell+transplantation+in+thalassemia+and+sickle+cell+anemia.">Hematopoietic stem cell transplantation in thalassemia and sickle cell anemia.</a> Cold Spring Harb Perspect Med. 2, a011825 (2012).</li><li>Andreani, M., Testi, M., Lucarelli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed+chimerism+in+haemoglobinopathies:+from+risk+of+graft+rejection+to+immune+tolerance.">Mixed chimerism in haemoglobinopathies: from risk of graft rejection to immune tolerance.</a> Tissue Antigens. 83, 137-146 (2014).</li><li>Andreani, 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=Persistence+of+mixed+chimerism+in+patients+transplanted+for+the+treatment+of+thalassemia.">Persistence of mixed chimerism in patients transplanted for the treatment of thalassemia.</a> Blood. 87, 3494-3499 (1996).</li><li>Andreani, 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=Long-term+survival+of+ex-thalassemic+patients+with+persistent+mixed+chimerism+after+bone+marrow+transplantation.">Long-term survival of ex-thalassemic patients with persistent mixed chimerism after bone marrow transplantation.</a> Bone Marrow Transplant. 25, 401-404 (2000).</li><li>Kumar, A. J., 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=Pilot+study+of+prophylactic+ex+vivo+costimulated+donor+leukocyte+infusion+after+reduced-intensity+conditioned+allogeneic+stem+cell+transplantation.">Pilot study of prophylactic ex vivo costimulated donor leukocyte infusion after reduced-intensity conditioned allogeneic stem cell transplantation.</a> Biol Blood Marrow Transplant. 19, 1094-1101 (2013).</li><li>Lawler, S. D., Harris, H., Millar, J., Barrett, A., Powles, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetic+follow-up+studies+of+recipients+of+T-cell+depleted+allogeneic+bone+marrow.">Cytogenetic follow-up studies of recipients of T-cell depleted allogeneic bone marrow.</a> Br J Haematol. 65, 143-150 (1987).</li><li>McCann, S. R., Crampe, M., Molloy, K., Lawler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemopoietic+chimerism+following+stem+cell+transplantation.">Hemopoietic chimerism following stem cell transplantation.</a> Transfus Apher Sci. 32, 55-61 (2005).</li><li>Dewald, G., 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+multicenter+investigation+with+interphase+fluorescence+in+situ+hybridization+using+X-+and+Y-chromosome+probes.">A multicenter investigation with interphase fluorescence in situ hybridization using X- and Y-chromosome probes.</a> Am J Med Genet. 76, 318-326 (1998).</li><li>Andreani, 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=Quantitatively+different+red+cell/nucleated+cell+chimerism+in+patients+with+long-term,+persistent+hematopoietic+mixed+chimerism+after+bone+marrow+transplantation+for+thalassemia+major+or+sickle+cell+disease.">Quantitatively different red cell/nucleated cell chimerism in patients with long-term, persistent hematopoietic mixed chimerism after bone marrow transplantation for thalassemia major or sickle cell disease.</a> Haematologica. 96, 128-133 (2011).</li><li>Andreani, M., Testi, M., Battarra, M., Lucarelli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split+chimerism+between+nucleated+and+red+blood+cells+after+bone+marrow+transplantation+for+haemoglobinopathies.">Split chimerism between nucleated and red blood cells after bone marrow transplantation for haemoglobinopathies.</a> Chimerism. 2, 21-22 (2011).</li><li>Kropshofer, G., Sopper, S., Steurer, M., Schwinger, W., Crazzolara, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+management+of+mixed+chimerism+after+bone+marrow+transplant+in+beta-thalassemia+major.">Successful management of mixed chimerism after bone marrow transplant in beta-thalassemia major.</a> Am J Hematol. 91, E357-E358 (2016).</li><li>Kimpton, C. 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=Automated+DNA+profiling+employing+multiplex+amplification+of+short+tandem+repeat+loci.">Automated DNA profiling employing multiplex amplification of short tandem repeat loci.</a> PCR Methods Appl. 3, 13-22 (1993).</li><li>Centis, 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=The+importance+of+erythroid+expansion+in+determining+the+extent+of+apoptosis+in+erythroid+precursors+in+patients+with+beta-thalassemia+major.">The importance of erythroid expansion in determining the extent of apoptosis in erythroid precursors in patients with beta-thalassemia major.</a> Blood. 96, 3624-3629 (2000).</li><li>Miccio, 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=In+vivo+selection+of+genetically+modified+erythroblastic+progenitors+leads+to+long-term+correction+of+beta-thalassemia.">In vivo selection of genetically modified erythroblastic progenitors leads to long-term correction of beta-thalassemia.</a> Proc Natl Acad Sci U.S.A. 105, 10547-10552 (2008).</li></ol>

作者没有什么可透露的。

本研究的目的是为观众提供两种方法的结合, 分析红祖细胞的供者/受体嵌在造血治疗的 hemoglobinopathies 患者中的应用: 1.) 荧光活化电池分选骨髓标本造血祖细胞, 其次为短串联重复和2。骨髓细胞的菌落形成单元, 不同祖类型的菌落分类, 其次是短串联重复的分析。这种方法的新颖之处在于将一项协议中的技术结合在一起, 来评估个别殖民地的捐助者/接受嵌。
这种方法的特殊重要性, 可以找到在混合嵌后, 造血治疗 hemoglobinopathies 患者的情况。有几项研究表明, 有相当比例的患者表达了与红前体细胞相关的捐献者髓细胞百分比低的情况, 结果是持久稳定的混合造血嵌3, 11;与此相反, 在相同的患者中, 有很高百分比的供血红细胞 (2-5 倍以上的成熟白细胞) 被注意到, 并建议无效的红细胞发生在红发展的后期阶段。这些观察被归因于在施主 erythroblasts 加速的细胞凋亡的倾向和持续的 T 和 B 残余淋巴细胞负责允许一个混杂的同种异体移植14,15。在造血干细胞移植后免疫的背景下, 我们最近证明了？-地中海贫血造血移植术后免疫抑制剂的改良对基因校正的红室, 给 2-到2.5 倍的剩余供体干细胞的放大12。这一观察支持确定捐助者对残红祖的效用, 因为它提供了对这一现象的理解, 并支持今后研究基因治疗 hemoglobinopathies 的临床试验。事实上, 为达到治疗水平的循环红细胞所需的基因修饰的有核细胞的比例可能与在 hemoglobinopathies 干细胞移植治疗的患者中所观察到的相似。
为了确定捐赠者红细胞的全貌, 我们修改了协议, 并提供了一个详细的, step-by 步骤的实验方法。该协议的关键步骤是建立一个流式细胞仪和软件的过程, 这是标准化的详细说明, 必须由适当的培训人员执行。以可视化的格式表示我们的协议可以让观众很容易地遵循我们的协议。我们比较了我们的 PCR 结果使用的基因组 dna 从红祖细胞获得从殖民地形成单位的 BM 样本与 DNA 从红的骨髓造血干细胞分类的祖先。两种方法的供植入数据基本一致。然而, 在菌落形成的样品中, 通常会发现较少的变异。这可能是因为与未处理和排序的捐赠者相比, 在体外检测中, 供者红细胞前体的存活情况有所改善。在这种情况下, 这个协议可以通过人工创建混合嵌合体组合与已知比例的健康祖和祖, 从一系列不同的 hemoglobinopathies 患者。对嵌的克隆测定和评价应准确反映其投入比例。
虽然缺乏对这一特定环境所涉及的机制的确切理解, 但这里提出的方法对于提供有关信息以用于例行监测植入和预后信息至关重要。临床实践。试图挽救移植功能是有限的风险, 发展移植物抗宿主疾病, 可以指导的串行植入监测。最后, 这种方法还可以为致力于改善 hemoglobinopathies 患者的护理的研究领域提供有用的依据, 特别是那些受急性移植物抗宿主病和自身免疫性疾病影响的病人。

异基因造血干细胞移植 (造血) 是唯一可用的治疗方法, 为各种先天遗传性疾病的造血系统, 实现无疾病存活率超过 90%, 否则高度妥协和生命受限患者1。这一重要的治疗工具的功效通过限制前移植方案2的毒性进行了优化, 而且还通过旨在维持稳定的移植功能的干预, 通过密切监测捐助者派生的单元格3,4,5。
一般情况下, 完整的嵌 (CC) 意味着由供体衍生的细胞完全替换 lymphohematopoietic 室, 而混合嵌 (MC) 则用于在各种比例.分裂嵌 (SC) 表示共存的混合嵌观察在单细胞血统, 如在红室。迅速确定嵌的状态后, 造血是至关重要的, 因为它可能有助于确定患者易复发的疾病, 并启动后续免疫策略, 如供体淋巴细胞输注或减少免疫抑制治疗6。
为监测植入后的造血, 开发了几种方法。免疫球蛋白的 Isotyping 和细胞遗传学分析有很差的灵敏度, 并在其检测多态性7,8的能力受到限制。引入荧光原位杂交 (fish) 可以提高造血后嵌监测的灵敏度, 但仅限于性不匹配的同种异体移植9。目前, 聚合酶链反应 (PCR) 是最广泛的方法用于检测嵌, 是基于传统的琼脂糖-丙烯酰胺凝胶电泳的可变数串联重复 (VNTRs) 或短串联重复 (可疑交易)。常规使用定量 PCR 能够在造血后检测到极小部分的残留供体细胞。到目前为止, 研究的主要限制是 MC 检测几乎完全局限于有核细胞的存在, 而不是成熟的红细胞, 即对受 hemoglobinopathies 影响的患者功能至关重要的细胞。在不同血型的患者中, 值得记住的是, cytofluorometric 分析能够利用针对红细胞抗原 ABO 和 c、c、D、e 和 e10的单克隆抗体来鉴别红细胞中的嵌,11. 在红谱系中, 一种不同但非常有趣的评估嵌的方法是红祖细胞的流细胞分选和各种红祖类型的选择, 并在克隆测定中进行培养,接着分析 STR12。这种方法能够量化红室中供者与受体嵌的相对比例, 并可用于维持骨髓移植的策略。

<table><tbody><tr><td>Ficoll-Paque</td><td>GE Healthcare</td><td>GE17-1440-02&amp;nbsp;</td><td>Remove RBC</td></tr><tr><td>50 mL conical tubes</td><td>Falcon</td><td>14-432-22</td><td>Sample preparation</td></tr><tr><td>12 x 75 mm flow tubes</td><td>Falcon</td><td>352002</td><td>FACS sorting</td></tr><tr><td>Phosphate buffered saline</td><td>Gibco</td><td>10010023</td><td>PBS</td></tr><tr><td>Fetal calf serum</td><td>Invitrogen Inc.</td><td>16000-044</td><td>FCS (heat-inactivated)</td></tr><tr><td>CD34 APC</td><td>BD Bioscience</td><td>561209</td><td>FACS-Ab</td></tr><tr><td>CD36 FITC</td><td>BD Bioscience</td><td>555454</td><td>FACS-Ab</td></tr><tr><td>CD45 V450</td><td>BD Bioscience</td><td>642275</td><td>FACS-Ab</td></tr><tr><td>Trypan blue</td><td>Gibco</td><td>15250061</td><td></td></tr><tr><td>Hemocytometer</td><td>Invitrogen Inc.</td><td>C10227</td><td>Automatic cell counting</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Gibco</td><td>14025092</td><td>Suspension buffer in FACS analysis</td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630080</td><td>Component of suspension buffer</td></tr><tr><td>FcR</td><td>BD Bioscience</td><td>564220</td><td>Block FCR</td></tr><tr><td>FACS Aria I</td><td>BD Bioscience</td><td>23-11539-00</td><td>FACS Sorter</td></tr><tr><td>Recombinant&amp;nbsp; human erythropoietin</td><td>Affimetrix eBioscience</td><td>14-8992-80</td><td>EPO</td></tr><tr><td>Isocove&amp;rsquo;s Modified Dulbecco&amp;rsquo;s Medium</td><td>Gibco</td><td>12440053</td><td>IMDM</td></tr><tr><td>L-Glutamine</td><td>Invitrogen</td><td>25030-081</td><td>Component of Culture Medium</td></tr><tr><td>CD34+ magnetic beads</td><td>Milteny Biotech</td><td>130-046-702</td><td>CD34+ purification</td></tr><tr><td>Recombinant&amp;nbsp; human G-CSF</td><td>Gibco</td><td>PHC2031</td><td>CFU-Assay</td></tr><tr><td>Recombinant&amp;nbsp; human SCF</td><td>Gibco</td><td>CTP2113</td><td>CFU-Assay</td></tr><tr><td>Recombinant&amp;nbsp; human GM-CSF</td><td>Gibco</td><td>PHC2015</td><td>CFU-Assay</td></tr><tr><td>Recombinant&amp;nbsp; human IL-3</td><td>BD Bioscience</td><td>554604</td><td>CFU-Assay</td></tr><tr><td>Recombinant&amp;nbsp; human IL-6</td><td>BD Bioscience</td><td>550071</td><td>CFU-Assay</td></tr><tr><td>Methocult H4434 Medium</td><td>Stemcell Technologies</td><td>4444</td><td>CFU-Assay</td></tr><tr><td>QiAmp DNA Blood extraction kit</td><td>Qiagen</td><td>51306</td><td>DNA Isolation</td></tr><tr><td>Nanodrop ND-1000 spectra photometer</td><td>Thermo Scientific</td><td>ND 1000</td><td>DNA Quantification</td></tr><tr><td>DNAase free H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Thermo Scientific</td><td>FEREN0521</td><td>DNA Preparation</td></tr><tr><td>AmplTaq Gold DNA Polymerase</td><td>Applied Bioscience</td><td>N8080240</td><td>PCR</td></tr><tr><td>Eppendorf mastercycler gradient</td><td>Eppendorf</td><td>6321000019</td><td>PCR&amp;nbsp;</td></tr><tr><td>Hi-Di Formamid</td><td>Applied Bioscience</td><td>4311320</td><td>PCR</td></tr><tr><td>GeneScan 500 ROX Size Standard</td><td>Applied Bioscience</td><td>4310361</td><td>PCR</td></tr><tr><td>3130 Genetic Analyzer</td><td>Applied Bioscience</td><td>313001R</td><td>PCR</td></tr></tbody></table>

detection of residual donor erythroid progenitor cells after hematopoietic stem cell transplantation for patients with hemoglobinopathies

在骨髓移植治疗地中海贫血或镰状细胞疾病的患者中, 有很大比例的病人出现了不完全的嵌。这种观察具有巨大的影响, 因为随后的治疗免疫策略可以改善临床结果。传统上, 聚合酶链 reaction-based 分析短串联重复是用来确定嵌的供体来源的血细胞。然而, 这种方法只限于有核细胞, 不能区分分离的单细胞血统。我们将短串联重复的分析应用于流细胞排列的造血祖细胞, 并将其与从骨中采集的选定的突发形成单元-红菌落所获得的短串联重复的分析进行比较。骨髓.通过这种方法, 我们能够证明红室中供体细胞的不同增殖和分化。这项技术有资格完成目前的监测嵌在干细胞移植的设置, 从而可以应用于未来的临床研究, 干细胞研究和基因治疗的设计试验。

lymphohematopoietic 祖细胞的分离
我们在这里展示的结果从排序的必要细胞数量的下游 STR 分析。骨髓细胞染色 V450-conjugated anti-CD45, FITC 共轭 anti-CD36 和 APC 共轭 anti-CD34。有兴趣的人群是巨红祖细胞, 是负责红细胞发育的细胞核。这些细胞表达 CD36, 但是阴性的白细胞共同抗原 CD45 (图 1C)。如有必要, 可根据其 CD36 表达水平进一步区分这些单元格。可以对其他几个填充进行排序, 以用作控件。我们将 CD45+CD34+ 淋巴/髓系前体作为另一种祖细胞群和 CD45+ 细胞, 作为成熟的髓细胞 (图 1D)。排序是由 BD FACSAria I 仪器。分类后, 红祖细胞纯度和 #62; 85% (图 1E) 和髓细胞纯度和 #62; 95% (图 1F)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56002/56002fig1.jpg"> 
图 1.红祖细胞和髓祖细胞的分类.图 1 a/1 b显示在 fsc-a vs. SSC-a 和 fsc-a 和使用多边形门工具来选择感兴趣的人群。1C指示 CD45 vs. CD36;1D显示成熟的髓细胞。显示1E和1F中阳性单元格的百分比。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56002/56002fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
生成突发形成单元-红殖民地
一些细胞来源于骨髓, 由 CD34-specific 磁珠分离出来, 出现了增殖和分化。在半固态培养基上形成14天的潜伏期后。刺激与不同浓度的 EPO 大大增强了突出红色 (红) 殖民地的形成 (图 2)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56002/56002fig2.jpg"> 
图 2。生成一个突发形成单元-红 (BFU-E)。显示的是一个具有代表性的低功耗显微的 BFU-E 群。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56002/56002fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">跨国公司:</td>
		</tr>
		<tr>
			<td></td>
			<td>1X EPO</td>
			<td>2X EPO</td>
			<td>4X EPO</td>
		</tr>
		<tr>
			<td>CFU-E</td>
			<td>2</td>
			<td>2</td>
			<td>2</td>
		</tr>
		<tr>
			<td>BFU-E</td>
			<td>9</td>
			<td>10</td>
			<td>8</td>
		</tr>
		<tr>
			<td>CFU-通用</td>
			<td>30</td>
			<td>30</td>
			<td>29</td>
		</tr>
		<tr>
			<td>CFU-GEMM</td>
			<td>1</td>
			<td>1</td>
			<td>2</td>
		</tr>
		<tr>
			<td colspan="4">CD34+单元格:</td>
		</tr>
		<tr>
			<td></td>
			<td>1X EPO</td>
			<td>2X EPO</td>
			<td>4X EPO</td>
		</tr>
		<tr>
			<td>CFU-E</td>
			<td>1</td>
			<td>1</td>
			<td>0</td>
		</tr>
		<tr>
			<td>BFU-E</td>
			<td>5</td>
			<td>11</td>
			<td>6</td>
		</tr>
		<tr>
			<td>CFU-通用</td>
			<td>25</td>
			<td>26</td>
			<td>32</td>
		</tr>
		<tr>
			<td>CFU-GEMM</td>
			<td>0</td>
			<td>2</td>
			<td>3</td>
		</tr>
	</tbody>
</table>
表1。对未排序骨髓细胞和 CD34+ 细胞的菌落分析。菌落是根据其形态学, 在一个带有评分网格的培养皿中用倒置显微镜在40X 放大倍数下得分的。为我们的分析目的, 殖民地被分类四类别: 殖民地形成单位-红 (CFU), 爆发形成单位-红 (BFU-e), 粒细胞巨噬祖细胞 (CFU-GM) 和多向祖细胞 (CFU-GEMM)。
嵌分析
嵌分析是使用 AmpFlSTR 分析器加套件 (应用生物, 加利福尼亚州) 根据制造商的协议, ABI 棱镜310遗传分析仪 (应用生物, 加利福尼亚州)。分析是执行 GeneMapper 软件 (应用生物, 加利福尼亚州)。基因座 D21S11、D7S820、FGA 和 vWA 被用来计算结果 (接受者和供体特异 DNA 的百分比)。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>示例</td>
			<td>受体 DNA</td>
			<td>供体 DNA</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>25%</td>
			<td>75%</td>
		</tr>
		<tr>
			<td>CD36hi</td>
			<td>25%</td>
			<td>75%</td>
		</tr>
		<tr>
			<td>CD36lo</td>
			<td>55%</td>
			<td>45%</td>
		</tr>
		<tr>
			<td>CD34</td>
			<td>25%</td>
			<td>75%</td>
		</tr>
	</tbody>
</table>
表2。从骨髓细胞的荧光活化细胞中提取的细胞中受体和供体 DNA 的百分比。在 lymphohematopoietic 室的特定细胞谱系中, 给出了供体细胞的嵌。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>示例</td>
			<td>受体 DNA</td>
			<td>供体 DNA</td>
		</tr>
		<tr>
			<td>BM-跨国公司</td>
			<td>24.71%</td>
			<td>75.29%</td>
		</tr>
		<tr>
			<td>BM-CD34</td></tr></tbody></table>/强
			<td>22.62%</td>
			<td>77.38%</td>
		
		<tr>
			<td>BFU-E (BM-跨国公司)</td>
			<td>80.99%</td>
			<td>19.01%</td>
		</tr>
		<tr>
			<td>CFU-通用 (BM-跨国公司)</td>
			<td>0.00%</td>
			<td>100.00%</td>
		</tr>
		<tr>
			<td>CFU-GEMM (BM-跨国公司)</td>
			<td>5.73%</td>
			<td>94.27%</td>
		</tr>
		<tr>
			<td>BFU-E (BM CD34)</td>
			<td>30.13%</td>
			<td>69.87%</td>
		</tr>
		<tr>
			<td>CFU-GM (BM CD34)</td>
			<td>0.00%</td>
			<td>100.00%</td>
		</tr>
		<tr>
			<td>CFU-GEMM (BM CD34)</td>
			<td>9.87%</td>
			<td>90.13%</td>
		</tr>
	

表3。从克隆测定的细胞中获得的受体和供体 DNA 的百分比。在 lymphohematopoietic 室的特定细胞谱系中, 给出了供体细胞的嵌。

Watch this Scientific Journal Video about Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies at JoVE.com

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

对 hemoglobinopathies 患者进行干细胞移植后, 需要对供体细胞进行定量监测植入。流 cytometry-based 细胞分选、菌落形成检测和随后的短串联重复分析的结合可用于评估红室中祖体的增殖和分化。

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 ...

detection-residual-donor-erythroid-progenitor-cells-after

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.2K 次观看.  Medical University Innsbruck. 对 hemoglobinopathies 患者进行干细胞移植后, 需要对供体细胞进行定量监测植入。流 cytometry-based 细胞分选、菌落形成检测和随后的短串联重复分析的结合可用于评估红室中祖体的增殖和分化。

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

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

流式细胞仪估测原发性急性髓系白血病和患者源性移植中白血病干细胞的诊断和随访

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

科研

癌症研究

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 ...

发育生物学

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

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

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

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

应用造血干细胞移植评估骨髓增生异常综合征的起源

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

使用CD71/TER119流流式细胞仪检测小鼠红系祖细胞的识别和分析

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

生物学

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

<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 ...

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

摘要

探索更多视频

此视频中的章节

流式细胞仪估测原发性急性髓系白血病和患者源性移植中白血病干细胞的诊断和随访

竞争性移植评价造血干细胞健身

人多能干细胞对原始和最终造血祖细胞定向分化的研究

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

应用造血干细胞移植评估骨髓增生异常综合征的起源

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

使用CD71/TER119流流式细胞仪检测小鼠红系祖细胞的识别和分析

慢病毒介导的击倒在体外红细胞

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