P-selectin-coated microtube for enrichment of CD34+ hematopoietic stem and progenitor cells from human bone marrow Hematology
P-selectin-coated microtube for enrichment of CD34+ hematopoietic stem and progenitor cells from human bone marrow Hematology
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DOI：
10.1373/clinchem.2007.089896
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发表时间：
2008-01-01
影响因子：
9.3
通讯作者：
King, Michael R.
中科院分区：
文献类型：
--
作者：
Narasipura, Srinivas D.;Wojciechowski, Joel C.;King, Michael R.
背景：造血干细胞和祖细胞（HSPC）的富集和纯化对于血液疾病的移植治疗和基础干细胞研究非常重要。原始CD34(+) HSPCs 对选择素的滚动粘附力比成熟CD34(-) 单核细胞(MNCs) 更强。我们利用这种不同的滚动行为，通过选择素涂层的微管灌注 MNC，从骨髓中捕获和纯化 HSPC。方法：通过涂有粘附分子的细胞捕获微管灌注骨髓 MNC。我们清洗了装置内腔，并通过视频显微镜对捕获的细胞进行可视化和估计。通过高剪切、无钙缓冲液和空气栓塞洗脱贴壁细胞。我们使用免疫荧光染色和流式细胞术来分析 CD34(+) HSPC。 结果：粘附分子涂层装置中捕获的 CD34(+) HSPC 纯度显着高于骨髓 MNC 中发现的 CD34(+) 细胞比例 [平均 (SE) 2.5% (0.8%)]。 P-选择素包被的表面产生了16%至20%的CD34(+)细胞纯度，而抗体包被的表面产生了12%至18%。尽管选择素和抗体表面之间的 CD34(+) 细胞纯度相当，但 P-选择素装置中捕获的 CD34(+) HSPC 总数（约 5.7 X 10(4) 至 7.1 X 10(4)）明显高于抗体装置（约 1.74 X 10(4) 至 2.61 X 10(4)）。结论： P-选择素可用于紧凑型流动装置中以捕获 HSPC。选择素介导的 CD34(+) HSPC 捕获导致富集度比骨髓 MNC 中的 CD34(+) 细胞群高约 8 倍。这项研究支持以下假设：基于流的粘附分子介导的捕获可能是捕获和纯化 HSPC 的可行替代方法。 (c) 2007 年美国临床化学协会。
BACKGROUND: Enrichment and purification of hematopoietic stem and progenitor cells (HSPCs) is important in transplantation therapies for hematologic disorders and in basic stem cell research. Primitive CD34(+) HSPCs have demonstrated stronger rolling adhesion on selectins than mature CD34(-) mononuclear cells (MNCs). We have exploited this differential rolling behavior to capture and purify HSPCs from bone marrow by perfusing MNCs through selectin-coated microtubes.METHODS: Bone marrow MNCs were perfused through the cell-capture microtubes coated with adhesion molecules. We washed the device lumen and visualized and estimated captured cells by video microscopy. Adherent cells were eluted by high shear, calcium-free buffer, and air embolism. We used immunofluorescence staining followed by flow cytometry to analyze CD34(+) HSPCs.RESULTS: CD34(+) HSPC purity of cells captured in adhesion molecule-coated devices was significantly higher than the fraction of CD34(+) cells found in bone marrow MNCs [mean (SE) 2.5% (0.8%)]. P-selectin-coated surfaces yielded 16% to 20% CD34(+) cell purity, whereas antibody-coated surfaces yielded 12% to 18%. Although CD34(+) cell purity was comparable between selectin and antibody surfaces, the total number of CD34(+) HSPCs captured was significantly higher in P-selectin devices (approximately 5.7 X 10(4) to 7.1 X 10(4)) than antibody devices (approximately 1.74 X 10(4) to 2.61 X 10(4)).CONCLUSIONS: P-selectin can be used in a compact flow device to capture HSPCs. Selectin-mediated capture of CD34(+) HSPCs resulted in enrichment approximately 8-fold higher than the CD34(+) cell population from bone marrow MNCs. This study supports the hypoth esis that flow-based, adhesion molecule-mediated capture maybe a viable alternative approach to the capture and purification of HSPCs. (c) 2007 American Association for Clinical Chemistry.