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

Objective: To construct a adenoviral vector containing a mouse, rat, and human general albumin promoter sequence and evaluate its albumin detecting in hepatic cells, non-hepatic cells and stem cells-derived hepatocyte-like cells, for real-time monitoring of the differentiation status of functional hepatocytes from stem cells in mouse, rat, and human. Methods: ?. Cells: Human hepatoma cells(Hep G2) and mouse liver hepatoma cells(Hepa1-6) were grown in DMEM supplemented with 10% fetal bovine serum. Rat primary hepatocytes were isolated from male Sprague-Dawley rats by a two-step collagenase perfusion method and cultured in serum-free medium with collagen-Matrigel sandwich extracellular matrix configuration. SD rat bone marrow mesenchymal stem cells(BM-MSCs) from passage 6–8 were cultured in Sprague-Dawley rat MSC growth medium. Mouse BM-MSCs of passage 4–6 were cultured in C57BL/6 mouse MSC growth medium. All cells were kept at 37°C and 5% CO2 in a humidified incubator. Cells in the exponential phase of growth were counted, and after 24 h, cells at 50–70% confluence were transfected with the recombinant plasmids or adenovirus. ?. Construction of the p DRIVE-SV40-Albp-Lac Z recombinant plasmids and plasmids transfection: Three human albumin promoter fragments were obtained from a Gene Bank BLAST search based on ALB promoter bp site, the payload size of a gene delivery vector and the potential cis-acting element binding sites. The target sequences were subcloned into digested expression vector to generate p DRIVE-SV40-Albp-Lac Z recombinant plasmids by Hanbio. Escherichia coli transformed by p DRIVE-SV40-Albp-Lac Z recombinant plasmids were amplified by using the E. coli Fast-Media Zeo kit. Plasmid extraction and measurement plasmid DNA concentration under spectrophotometer. Hepa1-6 cells were transfected with the three different p DRIVE-SV40-Albp-Lac Z constructs using Lipo FiterTM Liposomal Transfection Reagent. Then blue staining was evaluated under a microscope after X-gal interaction. ALB promoter transcriptional levels were assessed by manual counting, which was aided by using Image-pro Plus 6.0. ?. Construction of SV40-Albp-Zs Green adenoviral vectors and Adenovirus transduction: The finial ALB promoter fragment sequence-173/+36(210bp, Fragment 3) was chosen based on the results of the plasmid transfection experiment. And we can also come out that the SV40-Albp sequence can induce efficient Lac Z gene expression. Thus, we use SV40-Albp sequence to construct adenoviral vectors. SV40-Albp sequence is supported by Hanbio and Zs Green sequence is achieved from Gene Bank. Designed primer of SV40-Albp sequence and Zs Green reporter gene. The target sequences were amplified by PCR and subcloned into multiple clone site Xba I/Mlu I of p HBAd-U6-CMV vector to generate the recombinant plasmid p HBAd-SV40-Albp-Zs Green. Then plasmid was transfected into E. coli DH5?, the positive clone was identified by endonuclease digestion and further conformed by DNA sequencing. Recombinant adenoviruses were produced by transfecting 293 T cells with the adenoviral expression plasmid p HBAd-SV40-Albp-Zs Green and the framework plasmid p HBAd-BHGlox. After amplification and infectious titer determination, SV40-Albp-Zs Green adenoviral vectors transduct hepatic cells and non- hepatic cells, a fluorescence microscope was used to detect Zs Green fluorescence in the transduced cells. CMV-GFP adenoviral vectors were served as positive control. Albumin expression in cells was assessed by immunofluorescence staining. ?. SV40-Albp-Zs Green adenoviral vectors transduct stem cells-derived hepatocyte-like cells: To induce hepatocyte differentiation, the two step induction protocol was applied. The induction protocol was as follows:(1) differentiation medium consisting of IMDM medium supplemented with 20 ng/m L HGF, 10 ng/m L b FGF, and 0.61 g/L NAM for 7 days;(2) maturation medium, which consisted of IMDM supplemented with 20 ng/m L OSM, 1mmol/L DXM, and 50 mg/m L ITS premix for 2 weeks. Then cells were transuded by SV40-Albp-Zs Green adenoviral vectors, a fluorescence microscope was used to detect Zs Green fluorescence in the transduced cells. CMV-GFP adenoviral vectors were served as positive control. Albumin expression in cells was assessed by immunofluorescence staining. Results: ?. Construction of the p DRIVE-SV40-Albp-Lac Z recombinant plasmid and screening of highest transcriptional ALB promoter fragment: The ALB promoter fragment sequences are-247/+36(284bp, Fragment 1),-173/-23 (151bp, Fragment 2) and-173/+36(210bp, Fragment 3). The recombinant p DRIVE-SV40-Albp-Lac Z plasmids were successfully constructed. Hepa1-6 cells transfected with the three recombinant p DRIVE-SV40-Albp-Lac Z plasmids displayed ?-gal activity. Fragment 3 showed the highest Lac Z gene expression among the three plasmid constructs. Therefore, we used Fragment 3 in subsequent experiments. ?. Construction of the recombinant SV40-Albp-Zs Green reporter adenovirus and detection of albumin expression in transduced cells: The recombinant p HBAd-SV40-Albp-Zs Green plasmids containing Fragment 3 were successfully constructed. The authenticity of the SV40-Albp-Zs Green fragment was confirmed by endonuclease digestion and sequencing. The SV40-Albp-Zs Green-tagged positive rate as determined by TCID50 was 2×1010 PFU/m L. Zs Green green fluorescence was observed in hepatic cells, but not in non-hepatic cells, which was consistent with the ALB immunofluorescence staining results. Meanwhile, an overall higher intensity of GFP fluorescence in all cells was noted for the CMV promoter positive control group. ?. Detection of albumin expression in mouse BM-MSCs-derived hepatocyte-like cells cells: Zs Green green fluorescence was observed in cells, which was consistent with the ALB immunofluorescence staining results. And GFP fluorescence was noted for the CMV promoter positive control group. Conclusions: ?.-173/+36(210bp) sequence shows the highest transcriptional activity in mouse, rat, and human albumin promoter general sequence. ?. A recombinant SV40-Albp-Zs Green reporter adenovirus containing a mouse, rat, and human general albumin promoter sequence was successfully constructed. ?. The SV40-Albp-Zs Green reporter adenovirus is useful for real-time monitoring of the differentiation status of functional hepatocytes from stem cells in mouse, rat, and human.