Crazzolara Roman, Kropshofer Gabriele, Steurer Michael, Sopper Sieghart, Schwinger Wolfgang
Department of Pediatrics, Medical University Innsbruck;
Department of Pediatrics, Medical University Innsbruck.
J Vis Exp. 2017 Sep 6(127):56002. doi: 10.3791/56002.
The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.
在接受骨髓移植治疗重型地中海贫血或镰状细胞病的大部分患者中，发现存在不完全嵌合现象。这一观察结果具有重大意义，因为后续的治疗性免疫调节策略可改善临床结局。传统上，基于聚合酶链反应的短串联重复序列分析用于识别供体来源血细胞中的嵌合现象。然而，该方法仅限于有核细胞，无法区分解离的单细胞谱系。我们将短串联重复序列分析应用于流式细胞术分选的造血祖细胞，并将其与从骨髓中收集的选定红系爆式集落形成单位获得的短串联重复序列分析进行比较。通过这种方法，我们能够证明供体细胞在红系区室中的不同增殖和分化情况。这项技术有资格完成当前干细胞移植环境中的嵌合监测，因此可能应用于未来的临床研究、干细胞研究以及基因治疗试验的设计。
