Selective immunoaffinity-based enrichment of CD34(+) cells transduced with retroviral vectors containing an intracytoplasmatically truncated version of the human low-affinity nerve growth factor receptor (Delta LNGFR) gene
Selective immunoaffinity-based enrichment of CD34(+) cells transduced with retroviral vectors containing an intracytoplasmatically truncated version of the human low-affinity nerve growth factor receptor (Delta LNGFR) gene
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DOI：
10.1089/hum.1997.8.15-1815
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发表时间：
1997-10-10
影响因子：
4.2
通讯作者：
Stockschlader, M
中科院分区：
文献类型：
--
作者：
Fehse, B;Uhde, A;Stockschlader, M
人类造血干细胞仍然是治疗恶性和非恶性疾病的基因治疗方法中最有前途的靶细胞之一，为了快速表征转导细胞并将其与残留的未转导但生物学等效的细胞分离，我们使用了基于莫洛尼鼠白血病病毒（Mo-MuLV）的逆转录病毒载体，其中含有胞浆内截短的人类低亲和力神经生长因子受体（Delta LNGFR）cDNA作为标记基因，在纤连蛋白包被的组织培养瓶中转导CD34(+)细胞(平均纯度97%)的上清液，导致5.5-45%(平均26%)的转导细胞表达Delta LNGFR(LNGFR(+)细胞)，转导后，超过65%的转导细胞保留CD34(+)，与对照(模拟和未转导)CD34(+)细胞相比，转导没有改变不会降低 CD34(+) 细胞的克隆效率，用单克隆抗 LNGFR 抗体对转导细胞进行免疫磁选，产生 >90% LNGFR(+) 细胞。这些高度富集的 LNGFR(+) 细胞的进一步表型表征表明，大多数共表达 CD34 和 CD38 抗原。这些结果表明，表达异位细胞表面蛋白的转导细胞可以通过以下方法快速、方便地定量和表征：荧光激活细胞分选（FACS）分析以及使用磁珠通过免疫粘附快速有效地富集，细胞表面报告基因的使用应有助于优化将基因转移到更原始的造血祖细胞中的方法。
Human hematopoietic stem cells remain one of the most promising target cells for gene therapeutic approaches to treat malignant and nonmalignant diseases, To rapidly characterize transduced cells and to isolate these from residual nontransduced, but biologically equivalent, cells, we have used a Moloney murine leukemia virus (Mo-MuLV)-based retroviral vector containing the intracytoplasmatically truncated human low-affinity nerve growth factor receptor (Delta LNGFR) cDNA as a marker gene, Supernatant transduction of CD34(+) cells (mean purity 97%) in fibronectin-coated tissue culture flasks resulted in 5.5-45% (mean 26%) transduced cells expressing Delta LNGFR (LNGFR(+) cells), After transduction, more than 65% of the transduced cells remained CD34(+), Compared with control (mock-and nontransduced) CD34(+) cells, transduction did not decrease the cloning efficiency of CD34(+) cells, Immunomagnetic selection of the transduced cells with a monoclonal anti-LNGFR antibody resulted in >90% LNGFR(+) cells, Further phenotypic characterization of these highly enriched LNGFR(+) cells indicated that the majority co-expressed the CD34 and CD38 antigens, These results show that transduced cells expressing an ectopic cell-surface protein can be rapidly and conveniently quantitated and characterized by fluorescence-activated cell sorting (FACS) analysis and fast and efficiently enriched by immunoadhesion using magnetic beads, The use of cell-surface reporters should facilitate optimization of methods of gene transfer into more primitive hematopoietic progenitors.