Lentiviral Based Gene Transduction and Promoter Studies in Human Hematopoietic Stem Cells (hHSCs)

Abstract

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.

Keywords: Cord blood stem cells (AC133+); Lentiviral vectors; Promoters and Green fluorescent protein (GFP).

Figure 1

Schematic representation of promoter regions controlling GFP expression in retroviral vectors.

Figure 2

Viral dose effect on transudation: hHSCs transduced with lentivirus at cell to virus ratio of 1:1000, 1:2000; 1:5000. Highest level of GFP expression was found in 1:5000 cell to virus ratio and also higher level of cell death was found at this dose. 1:2000 ratio was the optimal dose for good level of transgene expression as well as low level of cell death (see Figure 3).

Figure 3

Viral dose effect on cell viability: Cells were transduced with lenti virus at 1:1000, 1:2000, 1:5000 cell to virus ratio. Viral dose effect on cell viability was analyzed by Trypan blue assay. 1:5000 cell to viral ratio gave the highest level of GFP expression (46%), however cell death was also high, at 31-33%. On other hand, 1:2000 cell to virus ratio gave good level of GFP expression (42%) as well as low level of cell death (15-20%). * = significant differences from other viral doses and control. a = significant differences from control.

Figure 4

GFP expression in HSCs at day 10 of post transduction: (A1, A2) bright field and florescence images of stem cells infected with EF1-GFP, (B1, B2) bright field and fluorescence image of stem cells infected with CMV-GFP; (C1, C2) bright ield and florescence images of stem cells infected with SV40-GFP, (D1, D2) bright field and fluorescence images of stem cells infected with UBC-GFP.

Figure 5

Promoter functionality studies for 6 weeks period using flowcytometer: Flow cytometric analysis shows the expression of transgenes (GFP) over extended period. Note the loss of expression of GFP on day 38 when CMV was used as promoter.

Figure 6

Promoter functionality studies for 6 weeks period using florescence microscope: Same cell cultures were also photomicrographed using green fluorescent filter. Note the expression of GFP on extended culture conditions.

Figure 7

Improvement of transduction: hHSCs were stimulated with double dose of growth factors (GF) for 10 hours before the transduction. Cell to virus ratio of 1:2000 was used to for transduction. Cell stimulated with double dose of growth factors showed 33% of improved transduction.

Figure 8

Effect of growth factor on cell viability: Lentivirus-GFP vectors were transduced with pre-stimulated stem cells with different concentrations of growth factors (GF). * = significant differences compared to other culture conditions.