MicroRNA-27a Modulates HCV Infection in Differentiated Hepatocyte-Like Cells from Adipose Tissue-Derived Mesenchymal Stem Cells (original) (raw)
Figure 1
Successful differentiation of AT-hMSCs into DHCs in conditioned medium.
AT-hMSCs were differentiated over 3 weeks using a two-step protocol. (A) The morphology of the cells changed markedly from a fibroblast-like morphology to a polygonal shape during differentiation. (a) Pre-treatment. (b) After treatment with one-step medium. (c) After treatment with two-step medium. To determine whether the cells exhibited increased in the expression of hepatic markers, we assessed the expression of hepatic markers at the RNA (B) and protein levels (C). (B) RT-PCR analysis for the detection of Alb, CK7, and CK19 expression in Huh7 cells, AT-hMSCs, and DHCs. β-Actin served as a loading control; Huh7 was the positive control. (C) Alb protein expression was detected in AT-hMSC and DHCs by ICC. These cells were stained with anti-albumin (red) and DAPI (blue), and image merging revealed strong cytoplasmic localization of Alb (indicated by the arrows) in DHCs. (D) To verify whether the DHCs exhibited decreased expression of AT-hMSC markers during differentiation, AT-hMSCs and DHCs were stained with FITC-conjugated CD90 or CD44. The FITC-positive cells were then examined by FACS analysis. To certify the hepatic function of the DHCs, PAS staining (E) and urea assays (F) were carried out using AT-hMSCs and DHCs. (E) Stored glycogen is represented by pink staining; arrows show cells with high levels of stored glycogen. (F) Urea production was measured in AT-hMSCs or DHCs. Scale bars are 50 µm. All data are representative of at least three independent experiments. *p<0.05 and **p<0.001 compared with the control.