Name: detection of residual donor erythroid progenitor cells after hematopoietic stem cell transplantation for patients with hemoglobinopathies
Uploaded: 2017-09-06T13:00:00
Description: 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

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?...

<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. 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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?...

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。

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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,...

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 ...

Research

JoVE Journal

Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

科研

发育生物学

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

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 ...

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

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 ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

定量造血干和祖细胞 (HSPCs) 在斑马鱼胚胎发育中的体外试验研究

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Single-cell Photoconversion in Living Intact Zebrafish

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 ...

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

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 ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

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

人诱导多能干细胞体外生成体细胞衍生物的研究

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

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

人类诱导多能干细胞衍生神经细胞治疗移植小鼠脑损伤控制皮质影响模型

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...