In vitro study on human umbilical cord mesenchymal stem cells transfected with lentivirus-mediated hNIS-EGFP dual reporter gene and co-labeled with superparamagnetic iron oxide.

Abstract

The aim of the present study was to establish a stem cell line for multi-mode imaging (in vivo fluorescence imaging, magnetic resonance imaging and 99mTc single-photon emission computed tomography) and to study the biological activity, stemness, proliferative activity and differentiation ability of superparamagnetic iron oxide (SPIO), human sodium/iodide symporter (hNIS) and enhanced green fluorescent protein (EGFP) co-labeled human umbilical cord mesenchymal stem cells (hUCMSCs). The EGFP reporter gene was selected to indirectly reflect the expression of target gene hNIS, and hUCMSCs were re-transfected with the successfully constructed recombinant plasmid pCMV-NIS-EF1-GFP-PGK-puro. When a stem cell line stably expressing hNIS and EGFP was obtained, the cells were incubated with 30 µg/ml SPIO to obtain hNIS, EGFP and SPIO co-labeled stem cells. The protein expressions of hNIS and EGFP were identified using western blot analysis, and the protein function of hNIS was identified by 125I influx and 125I efflux experiments. hNIS-EGFP-hUCMSCs were labeled with SPIO under the mediation of poly-L-lysine, and SPIO, hNIS and EGFP co-labeled hUCMSCs were established successfully. Staining with Prussian blue confirmed that 98% of cells were successfully labeled with SPIO. Western blotting results demonstrated positive hNIS and EGFP protein expression levels, and 125I influx and 125I efflux experiments confirmed that the protein function of hUCMSCs after expressing hNIS was normal. The uptake of 125I was higher in cell lines hNIS-EGFP-hUCMSCs than in control hUCMSCs (fold change: 16.43±2.30 times; P<0.05). The stemness of hNIS-EGFP-hUCMSCs was found to be slightly decreased but not statistically significant; the overall characteristics of stem cells remained unchanged. The assessments of adipogenic and osteogenic differentiation suggest that hNIS-EGFP-hUCMSCs have no significantly different characteristics compared with primary hUCMSCs.