Induction of highly functional hepatocytes from human umbilical cord mesenchymal stem cells by HNF4α transduction.

To investigate the differentiation potential of human umbilical mesenchymal stem cells (HuMSCs) and the key factors that facilitate hepatic differentiation.HuMSCs were induced to become hepatocyte-like cells according to a previously published protocol. The differentiation status of the hepatocyte-like cells was examined by observing the morphological changes under an inverted microscope and by immunofluorescence analysis. Hepatocyte nuclear factor 4 alpha (HNF4α) overexpression was achieved by plasmid transfection of the hepatocyte-like cells. The expression of proteins and genes of interest was then examined by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) or real-time RT-PCR methods.Our results demonstrated that HuMSCs can easily be induced into hepatocyte-like cells using a published differentiation protocol. The overexpression of HNF4α in the induced HuMSCs significantly enhanced the expression levels of hepatic-specific proteins and genes. HNF4α overexpression may be associated with liver-enriched transcription factor networks and the Wnt/β-Catenin pathway.The overexpression of HNF4α improves the hepatic differentiation of HuMSCs and is a simple way to improve cellular sources for clinical applications

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