The CMV early enhancer/chicken beta actin (CAG) promoter can be used to drive transgene expression during the differentiation of murine embryonic stem cells into vascular progenitors

Abstract

Background: Mouse embryonic stem cells cultured in vitro have the ability to differentiate into cells of the three germ layers as well as germ cells. The differentiation mimics early developmental events, including vasculogenesis and early angiogenesis and several differentiation systems are being used to identify factors that are important during the formation of the vascular system. Embryonic stem cells are difficult to transfect, while downregulation of promoter activity upon selection of stable transfectants has been reported, rendering the study of proteins by overexpression difficult.

Results: CCE mouse embryonic stem cells were differentiated on collagen type IV for 4-5 days, Flk1+ mesodermal cells were sorted and replated either on collagen type IV in the presence of VEGFA to give rise to endothelial cells and smooth muscle cells or in collagen type I gels for the formation of vascular tubes. The activity of the CMV and beta-actin promoters was downregulated during selection of stable transfectants and during differentiation to the Flk1 stage, while the CMV immediate enhancer/beta-actin promoter in the pCAGIPuro-GFP vector led to 100% of stably transfected undifferentiated and differentiated cells expressing GFP. To further test this system we expressed syndecan-2 and -4 in these cells and demonstrated high levels of transgene expression in both undifferentiated cells and cells differentiated to the Flk1 stage.

Conclusion: Vectors containing the CAG promoter offer a valuable tool for the long term expression of transgenes during stem cell differentiation towards mesoderm, while the CMV and beta-actin promoters lead to very poor transgene expression during this process.

Figure 1

ES cell differentiation into Flk1⁺ mesodermal, endothelial and smooth muscle cells. A. Schematic showing the protocol for differentiation of CCE cells to mesodermal lineages. B. Expression of cell surface markers at day 0 and days 3–5 of differentiation; cells were analysed by FACS for the markers shown. C-D. Flk1⁺ cells were plated on collagen type IV in the presence of VEGFA. Cells were double immunostained for VE-cadherin (green) and αSMA (red) (C) or PECAM-1 (green) and αSMA (red) (D). EC sheets were found surrounded by SMCs. E-F. Phase contrast micrographs of Flk1⁺ cells after FACS sorting and seeding into collagen type I gels in the presence of VEGFA for 6 days. Vascular tube structures are arrowed. G-H. Collagen I gels were sectioned and stained for Pecam-1 (green) and αSMA (red) and showed Pecam-1⁺ tubular structures (arrows) surrounded by smooth muscle cells.

Figure 2

The CMV promoter is down regulated in mouse embryonic CCE cells and during differentiation into mesoderm. A-D. CCE cells were transfected with pEGFP-N1 and subjected to G418 selection 48 hours later. Cells expressing GFP are obvious 2 days after transfection (A, B) while few GFP positive cells remain 7 days post transfection (C, D). E-F. CCE cells were transfected with pEGFP-N1 and differentiation was initiated 2 days post transfection. No GFP⁺ cells were observed on day 5 of differentiation to the Flk1 stage (pool of containing both Flk1+ and Flk1- cells) when transient transfectants were used. G-I. CCE cells were transfected with pEGFP-N1 and placed under G418 selection 2 days post transfection. Undifferentiated cells (G, I) and cells differentiated to the Flk1 stage (H, I) were analysed by FACS for GFP expression 14–19 days post transfection. The data suggest that less than 50% of the undifferentiated cells express GFP, while this percentage declines during differentiation to the Flk1 stage. Similar levels of GFP expression were obtained during differentiation of cells transfected with pLK444-GFP.

Figure 3

The CMV and chicken β-actin promoters cannot drive gene expression in bicistronic vectors transfected into CCE cells. CCE cells (A) or rat embryo fibroblasts (B) were transfected with the bicistronic vectors pIRES2-EGFP and pIRES-EGFP(β-actin) in which the CMV promoter had been substituted with the chicken β-actin promoter. GFP expression was not evident in CCE cells transfected with these vectors; GFP⁺ cells were only obtained when cells were transfected with pEGFP-N1. In contrast GFP expression was apparent in fibroblasts transfected with all three constructs (B). Micrographs were obtained 24 hours after transfection.

Figure 4

Sustained GFP expression in CCE cells during differentiation into mesoderm is obtained using the CAG promoter. CCE cells were transfected with pCAGIPuro-GFP (A, B) or pCAGGSneo-GFP (C, D) and selected for puromycin or G418 resistance, respectively. FACS analysis was performed on undifferentiated CCE cells (A, C) and cells differentiated to the Flk1 stage (B, D)14–18 days posts transfection and GFP expression was compared in vectors expressing GFP (red) and empty vector (blue) transfected controls.

Figure 5

Ectopic expression of Syndecan-2 and -4 through differentiation of CCE cells to mesodermal lineages. Undifferentiated CCE cells (A) as well as Flk1⁺ (B) and Flk1^- (C) cells FACS sorted on day 4 of differentiation were analysed by RT-PCR for syndecan expression. D. CCE cells were transfected with either pCAGIPuro-sdc2 or pCAGIPuro-sdc4 and were selected for puromycin resistance. FACS analysis was performed on undifferentiated and cells differentiated to the Flk1 stage as indicated using antibodies against syndecan-2 and -4. In each analysis, the dotted black line denotes the relevant secondary antibody control, the blue line corresponds to cells transfected with pCAGIPuro empty vector and the red line are cells transfected with constructs containing syndecan-2 or-4 cDNA. E. Levels of Flk1 were measured on day 5 of differentiation in cells transfected with pCAGIPuro-sdc2, pCAGIPuro-sdc4 or empty vector control. Data from 5 independent experiments are shown, where a total of 9 flasks were analysed for each construct. The bars represent the standard deviation, and the results were analysed with repeated-measures ANNOVA, using Prism Graphpad 4.0, assuming Gaussian distribution. No statistical significance was observed for the difference in FLK1 expression in cells transfected with the different constructs.