Varma N, Janic B, Ali M, Iskander Asm, Arbab A
Cellular and Molecular Imaging Laboratory, Department of Radiology, Henry Ford Hospital , Detroit, MI 48202, USA.
J Stem Cells Regen Med. 2011 Apr 1;7(1):41-53. doi: 10.46582/jsrm.0701005. eCollection 2011.
Human hematopoietic stem cells (hHSCs) have enormous potential for clinical use in cell-based therapies, especially as a gene delivery system. Moreover, lentiviral transduction in stem cells is very often associated with low transduction efficiency and low levels of foreign gene expression. Therefore, it is important to analyze vector and promoter systems that can generate robust foreign gene expression in these cells. In this study, we evaluated and compared the ability of different commercially available promoters to drive the expression of exogenous reporter genes in hHSCs and evaluated the effect of different doses of stem cell growth factors on the expression of transgenes. We used lentivirus based vector system carrying the following promoters: 1) Human cytomegalovirus (CMV) promoter, 2) Simian virus 40 (SV40) promoter, 3) mammalian Ubiquitin C (UBC) promoter and 4) cellular polypeptide chain elongation factor 1 alpha (EF1) promoter. EF1 and CMV promoters robustly drove the expression of green fluorescence protein (GFP) reporter gene, while SV40 and UBC promoters induced very low level of GFP expression. Lentivectors containing EF1 and CMV promoters showed high-level stable GFP expression in human cord blood stem cells for 6 weeks period after post transduction. CD133+ hHSCs stimulated with higher concentration of growth factors exhibited enhancement of transduction rate. Cord blood derived CD133+ hHSCs could be effectively transduced with lentivectors under CMV or EF-1 promoters for the expression of foreign gene.
人类造血干细胞（hHSCs）在基于细胞的治疗中具有巨大的临床应用潜力，尤其是作为一种基因传递系统。此外，干细胞中的慢病毒转导常常与低转导效率和低水平的外源基因表达相关。因此，分析能够在这些细胞中产生强大外源基因表达的载体和启动子系统非常重要。在本研究中，我们评估并比较了不同市售启动子驱动hHSCs中外源报告基因表达的能力，并评估了不同剂量的干细胞生长因子对转基因表达的影响。我们使用携带以下启动子的慢病毒载体系统：1）人巨细胞病毒（CMV）启动子，2）猿猴病毒40（SV40）启动子，3）哺乳动物泛素C（UBC）启动子和4）细胞多肽链延伸因子1α（EF1）启动子。EF1和CMV启动子有力地驱动了绿色荧光蛋白（GFP）报告基因的表达，而SV40和UBC启动子诱导的GFP表达水平非常低。含有EF1和CMV启动子的慢病毒载体在转导后6周内在人脐血干细胞中显示出高水平的稳定GFP表达。用更高浓度的生长因子刺激的CD133 + hHSCs表现出转导率的提高。在CMV或EF-1启动子下，慢病毒载体可有效转导脐带血来源的CD133 + hHSCs以表达外源基因。
