Enhanced hepatogenic transdifferentiation of human adipose tissue mesenchymal stem cells by gene engineering with Oct4 and Sox2.

Adipose tissue mesenchymal stem cells (ATMSCs) represent an attractive tool for the establishment of a successful stem cell-based therapy in the field of liver regeneration medicine. ATMSCs overexpressing Oct4 and Sox2 (Oct4/Sox2-ATMSCs) showed enhanced proliferation and multipotency. Hence, we hypothesized that Oct4 and Sox2 can increase "transdifferentiation" of ATMSCs into cells of the hepatic lineage. In this study, we generated Oct4- and Sox2-overexpressing human ATMSCs by liposomal transfection. We confirmed the expression of mesenchymal stem cell surface markers without morphological alterations in both red-fluorescent protein (RFP) (control)- and Oct4/Sox2-ATMSCs by flow cytometry. After induction of differentiation into hepatocyte-like cells, the morphology of ATMSCs changed and they began to appear as round or polygonal epithelioid cells. Hepatic markers were evaluated by reverse transcription-polymerase chain reaction and confirmed by immunofluorescence. The results showed that albumin was strongly expressed in hepatogenic differentiated Oct4/Sox2-ATMSCs, whereas the expression level of α-fetoprotein was lower than that of RFP-ATMSCs. The functionality of hepatocytes was evaluated by periodic acid-Schiff (PAS) staining and urea assays. The number of PAS-positive cells was significantly higher and urea production was significantly higher in Oct4/Sox2-ATMSCs compared to that in RFP-ATMSCs. Taken together, the hepatocyte-like cells derived from Oct4/Sox2-ATMSCs were mature hepatocytes, possibly functional hepatocytes with enhanced capacity to store glycogen and produce urea. In this study, we demonstrated the enhanced transdifferentiation of Oct4- and Sox2-overexpressing ATMSCs into hepatocyte-like cells that have enhanced hepatocyte-specific functions. Therefore, we expect that Oct4/Sox2-ATMSCs may become a very useful source for hepatocyte regeneration or liver cell transplantation

Correction: Enhanced Hepatogenic Transdifferentiation of Human Adipose Tissue Mesenchymal Stem Cells by Gene Engineering with Oct4 and Sox2.

[This corrects the article DOI: 10.1371/journal.pone.0108874.]

Production of urea in hepatocyte-like cells derived from RFP- and Oct4/Sox2-ATMSCs after 28 days hepatogenic differentiation.

<p>After hepatogenic induction, urea production was significantly increased in cultures of differentiated RFP- and Oct4/Sox2-ATMSCs. Thus, the amount of urea produced by hepatocyte-like cells derived from Oct4/Sox2-ATMSCs was significantly higher than that of RFP-ATMSCs. Undifferentiated ATMSC and HepG2 Cells were used as negative and positive control, respectively. The experiments were repeated at least three times and similar findings were observed. Data represent the mean ± SD of three independent experiments. Statistical analysis was performed by ANOVA followed by Tukey’s post hoc test of the means (significant, **<i>p</i> < 0.01).</p

Immunophenotyping of RFP- and Oct4/Sox2-transfected ATMSCs.

<p>RFP-transfected ATMSCs and Oct4/Sox2-transfected ATMSCs at passage 5 were immunophenotyped for CD29, CD31, CD34, CD44, CD45, CD73, CD90, and CD105 by flow cytometry. The expression of ATMSC surface markers characteristic of MSCs was maintained.</p

Period acid Schiff (PAS) staining of RFP- and Oct4/Sox2-ATMSCs after 28 days hepatogenic differentiation.

<p>(A) Detection of glycogen in the cytoplasm of MSCs subjected to the liver differentiation protocol was demonstrated by PAS staining. PAS-positive substances stain pink in the cytoplasm of cells. (B) The number of PAS-positive cells is expressed as percentage of the total number of counted cells and was significantly higher in Oct4/Sox2-ATMSCs than that of RFP-ATMSCs. The experiments were repeated at least three times and similar findings were observed. Data represent the mean ± SD of three independent experiments. Statistical analysis was performed by student <i>t</i>-test (significant, *<i>p</i> < 0.05).</p

PCR analysis and immunofluorescence of liver markers after 28 days hepatogenic differentiation.

<p>(A) The mRNA expression level of albumin (ALB) was strongly expressed in hepatogenically differentiated Oct4/Sox2-ATMSCs, whereas the expression level of α-fetoprotein (AFP) was lower than that of RFP-ATMSCs. The expression levels of transferrin were not significantly different in both cells. Undifferentiated ATMSCs and HepG2 were used as negative and positive controls, respectively. (B) Hepatocyte-like cells from RFP- and Oct4/Sox2-ATMSCs are confirmed by immunofluorescence staining for AFP and ALB. Nuclei were counterstained with Hoecst33342.</p

Analysis of the Oct4 and Sox2 expression in Oct4/Sox2-ATMSCs.

<p>(A) RT-PCR analysis revealed that the mRNA expression of Oct4 and Sox2 in Oct4/Sox2-ATMSCs were significantly higher than that in RFP-ATMSCs at 24 h post-transfection. (B) Western blot analysis showed high levels of Oct4 and Sox2 expression in Oct4/Sox2-ATMSCs. Data are representative of three independent experiments, with similar results. Statistical analysis was performed by student <i>t</i>-test (significant, **<i>p</i> < 0.01).</p

Antitumor effects of celecoxib in COX-2 expressing and non-expressing canine melanoma cell lines

Cyclooxygenase-2 (COX-2) is a potential target for chemoprevention and cancer therapy. Celecoxib, a selective COX-2 inhibitor, inhibits cell growth of various types of human cancer including malignant melanoma. In dogs, oral malignant melanoma represents the most common oral tumor and is often a fatal disease. Therefore, there is a desperate need to develop additional therapeutic strategies. The purpose of this study was to investigate the anticancer effects of celecoxib on canine malignant melanoma cell lines that express varying levels of COX-2. Celecoxib induced a significant anti-proliferative effect in both LMeC and CMeC-1 cells. In the CMeC cells, treatment of 50 µM celecoxib caused an increase in cells in the G0/G1 and a decreased proportion of cells in G-2 phase. In the LMeC cells, 50 µM of celecoxib led to an increase in the percentage of cells in the sub-G1 phase and a significant activation of caspase-3 when compared to CMeC-1 cells. In conclusion, these results demonstrate that celecoxib exhibits antitumor effects on canine melanoma LMeC and CMeC-1 cells by induction of G(1)-S cell cycle arrest and apoptosis. Our data suggest that celecoxib might be effective as a chemotherapeutic agent against canine malignant melanoma