Magnetic cell sorting purification of differentiated embryonic stem cells stably expressing truncated human CD4 as surface marker
Magnetic cell sorting purification of differentiated embryonic stem cells stably expressing truncated human CD4 as surface marker
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DOI：
10.1634/stemcells.2004-0177
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发表时间：
2005-04-01
期刊：
影响因子：
5.2
通讯作者：
Franz, WM
中科院分区：
文献类型：
--
作者：
David, R;Groebner, M;Franz, WM
胚胎干细胞在再生医学和组织工程方面具有巨大的潜力。由于缺乏温和、高效地分离特定细胞类型以供移植而不表达免疫原性标志物的方案，临床应用仍然受到阻碍。我们描述了在用于磁性细胞分选(MACs)的磷酸甘油酸激酶启动子(MACs)的控制下，稳定表达缺乏胞内结构域的人CD4分子的ES细胞的标记。为了跟踪标记的ES细胞，我们将Delta CD4融合到细胞内增强型绿色荧光蛋白结构域(Delta CD4EGFP)。我们展示了膜结合的荧光融合蛋白的功能及其对Mac的适用性，导致纯度超过97%。同样，Delta CD4的表达产生了高达98.5%的阳性细胞，与其分化状态无关。纯度不受初始Delta CD4(+)细胞百分比的限制，范围为0.6%-16%。MACS选择的细胞通过发育所有三个胚层的类胚体的重新聚集和从头形成来证明其活性。因此，在分化的ES细胞中表达Delta CD4可能使组织工程和移植研究中所需的细胞类型的快速、高产量纯化成为可能。
Embryonic stem (ES) cells offer great potential in regenerative medicine and tissue engineering. Clinical applications are still hampered by the lack of protocols for gentle, high-yield isolation of specific cell types for transplantation expressing no immunogenic markers. We describe labeling of stably transfected ES cells expressing a human CD4 molecule lacking its intracellular domain (Delta CD4) under control of the phosphoglycerate kinase promoter for magnetic cell sorting (MACS). To track the labeled ES cells, we fused Delta CD4 to an intracellular enhanced green fluorescent protein domain (Delta CD4EGFP). We showed functionality of the membrane-bound fluorescent fusion protein and its suitability for MACS leading to purities greater than 97%. Likewise, expression of Delta CD4 yielded up to 98.5% positive cells independently of their differentiation state. Purities were not limited by the initial percentage of Delta CD4(+) cells, ranging from 0.6%-16%. The viability of MACS-selected cells was demonstrated by reaggregation and de novo formation of embryoid bodies developing all three germ layers. Thus, expression of Delta CD4 in differentiated ES cells may enable rapid, high-yield purification of a desired cell type for tissue engineering and transplantation studies.