Sequences of PCR and real-time PCR primers.

<p>Sequences of PCR and real-time PCR primers.</p

Mechanism of HNF4α-mediated improved hepatic differentiation.

<p><i>(</i><b><i>A</i></b><i>)</i> Detection of liver-enriched transcription factors in induced and HNF4α-transfected HuMSCs by RT-PCR. 0 d: HuMSCs before induction; DM 9 d and 21 d: Cells cultured in hepatic differentiation medium for nine days and 21 days, respectively; HNF4α 12 d and 21 d: Cells transfected with HNF4α nine days after induction (gene expression was determined three and 12 days later, respectively). <i>(</i><b><i>B</i></b><i>)</i> Wnt/β-catenin signaling-related genes were examined by real-time RT-PCR. All of the data are presented as the means ± SD (n = 3), and the fold induction for each gene by HNF4α overexpression was significant (P<0.05).</p

Hallmark function assays of mature hepatocytes.

<p><i>(</i><b><i>A</i></b><i>)</i> Periodic Acid-Schiff staining assay in hepatogenic differentiated cells. A1:HuMSCs; A2: HuMSCs were cultured in hepatocyte differentiation medium for 21 days; A3: HuMSCs were transfected with HNF4α plasmid and cultured until day 21. <i>(</i><b><i>B</i></b><i>)</i> Expression of MHC (HLA-DR and HLA-DQ) assay by flow cytometry analysis. B1,B4:HuMSCs;B2,B5: HuMSCs were cultured in hepatocyte differentiation medium for 21 days transfected with PCDNA3.1 alone;B3,B6: HuMSCs were transfected with HNF4α plasmid and cultured until day 21. <i>(</i><b><i>C, D</i></b><i>)</i> Albumin levels in hepatogenic transfected with HNF4α differentiated cells in comparison to cells cultured in differentiation medium for 21 days transfected with PCDNA3.1 alone by enzyme linked immunosorbent assay (P<0.05) and Flow cytometry analysis (D1: control group D2:HNF4α group).</p

In vitro hepatic differentiation of HuMSCs and HNF4α transfection.

<p><i>(</i><b><i>A</i></b><i>)</i> Morphological changes observed as undifferentiated HuMSCs differentiated into hepatocytes under hepatogenic conditions. A1: Undifferentiated HuMSCs; A2: 1 week postinduction. A3: 2 weeks postinduction. A4–A5: 3 weeks postinduction. Original magnification, ×100 (A1–A4), ×200 (A5). <i>(</i><b><i>B</i></b><i>)</i> Immunocytochemical analysis of hepatocyte-specific marker expression. HuMSCs were cultured in hepatocyte differentiation medium for 21 days (B1, B3) or in growth medium (negative controls; B2, B4). The cells were stained with mouse antibodies against the hepatocyte-specific markers ALB (B1, B2) and AFP (B3, B4) and incubated with Dylight594-conjugated anti-mouse IgG. The cell nuclei were counterstained with DAPI. Original magnification, ×400 (B1–B4). <i>(</i><b><i>C</i></b><i>)</i> Overexpression of HNF4α was detected by immunofluorescence two days after transfection. C1: Green light could be detected from the nuclei of the transfected HNF4α group; C2: Control group transfected with PCDNA3.1. Original magnification, ×400 (C1, C2) <i>(</i><b><i>D</i></b><i>)</i> Overexpression of HNF4α was detected by RT-PCR two days after transfection.</p