Construction of a general albumin promoter reporter system for real-time monitoring of the differentiation status of functional hepatocytes from stem cells in mouse, rat and human

Abstract

Genetic constructs with promoters fused to reporter genes for simultaneous monitoring of cellular events have been the focus of attention in recent years. Adenoviral vectors, which have distinctive characteristics, have been used to monitor the differentiation of stem cells in vitro. In the present study, a modified adenoviral vector was constructed, containing a mouse, rat, and human general albumin promoter sequence fused to a ZsGreen reporter gene, and evaluated its efficiency in different cell types. Two hepatocyte cell lines (Hepa1-6 and HepG2), rat primary hepatocytes, rat bone marrow mesenchymal stem cells (BM-MSCs) and rat BM-MSCs-derived hepatocyte-like cells were transduced with this vector, and the transfection efficiency and functional capabilities of the promoter were evaluated by fluorescent microscopy. The results demonstrated efficient expression of ZsGreen in Hepa1-6 cells, HepG2 cells, rat primary hepatocytes, and rat BM-MSCs-derived hepatocyte-like cells, but not in rat BM-MSCs. In conclusion, the current study demonstrates a simple, high-efficiency, general tool for real-time monitoring of the differentiation status of hepatocytes from stem cells in mice, rats, and humans. This tool may be useful for evaluating different protocols to generate functional hepatocytes from stem cells in multiple species.

Construction of bioengineered hepatic tissue derived from human umbilical cord mesenchymal stem cells via aggregation culture in porcine decellularized liver scaffolds

“…To detect the completion of hepatic differentiation for the 2D, 3D, 2D‐DLS, and 3D‐DLS groups, a human albumin promoter‐ZsGreen reporter adenovirus was designed and constructed as described previously (for promoter sequences, Figure S1). 2D, 3D, 2D‐DLS, and 3D‐DLS hepatocyte‐like cells were incubated with adenovirus (multiplicity of infection (MOI): 1:100) for 2 hours, and ZsGreen expressions were then examined using a Leica Microsystems DM40000B upright microscope after 48 hours.…”

Hepatic differentiation of mouse bone marrow-derived mesenchymal stem cells using a novel 3D culture system

“…To monitor the differentiation of hepatocytes from BM-MSCs, the Albp-ZsGreen adenoviral vector containing the ALB promoter was designed and constructed as previously described ( 24 ). 2D and 3D hepatocyte-like cells (1×10 6 cells/well) were incubated with the Albp-ZsGreen adenoviral vector (10 µl; 1×10 8 plaque formation units; multiplicity of infection, 100) in 6-well tissue culture plates for 2 h prior to examining ALB expression at 48 h using a Leica DM40000B microscope (Leica Microsystems GmbH).…”

Heterotopic vascularization and functionalization of implantable bio engineered hepatic tissue alleviates liver injury in rats

“…The albumin (ALB) promoter‐ZsGreen reporter adenovirus was designed and constructed as described previously to dynamically monitor hepatocyte spheroids . The hepatocyte spheroids were incubated with adenovirus (multiplicity of infection [MOI]: 1:100) for 2 hours before inoculation of the scaffold, and ZsGreen expression was examined using a Leica Microsystems DM40000B upright microscope 48 hours after infection (Leica).…”