Reprogramming liver-stem WB cells into functional insulin-producing cells by persistent expression of Pdx1- and Pdx1-VP16 mediated by lentiviral vectors

Abstract

Adenovirus-mediated transient expression of the pancreatic duodenal homeobox transcription factor Pdx1 in mouse liver activates pancreatic endocrine and exocrine genes, the latter reportedly resulting in severe hepatitis. Expression of a super-active form of Pdx1 or Pdx1-VP16 selectively transdifferentiates hepatic WB cells into functional pancreatic beta-like insulin-producing cells, without evidence of exocrine differentiation. No study has systematically compared the transdifferentiation efficiency of Pdx1 and Pdx1-VP16 at the cellular and molecular level. Comparisons can be ambiguous when vectors harboring a transcription factor cDNA have differing extents and duration of gene expression. In view of the remarkable capacity of lentiviral vector (LV) for delivering and integrating transgene into both dividing and nondividing cells, we transduced rat hepatic stem cell-like WB cells with LV-Pdx1 or LV-Pdx1-VP16, and then used the limiting-dilution technique to clone singlecell-derived cell lines that stably express either Pdx1 or Pdx1-VP16. With these cell lines, we studied: (a) the expression of Pdx1 or Pdx1-VP16 protein by Western blotting and immunocytochemistry; (b) the repertoire of long-term expression of Pdx1-or Pdx1-VP16-induced pancreatic gene expression using RT-PCR methods; and (c) their capacity to serve as beta-cell surrogates in restoring euglycemia in streptozotocin-treated diabetic mice. We found that cell lines expressing either Pdx1 or Pdx1-VP16 long-term exhibited similar profiles for expression of genes related to pancreatic development and beta-cell function, and reversed hyperglycemia in diabetic mice. We also examined short-term expression of Pdx1 or Pdx1-VP16, and the results demonstrated that expression of Pdx1-VP16 is more efficient in initiating liver-to-endocrine pancreas transdifferentiation. Our findings demonstrate: (a) that the LV system is highly effective in producing persistent expression of Pdx1 or Pdx1-VP16 in WB hepatic cells; and (b) long-term, persistent expression of either Pdx1 or Pdx1-VP16 is similarly effective in converting hepatic stem cells into pancreatic endocrine precursor cells that, upon transplantation into diabetic mice, become functional insulin-producing cells and restore euglycemia. Laboratory Investigation (2006) 86, 83-93.

Liver stem cell-derived β-cell surrogates for treatment of type 1 diabetes

“…With these cell lines, we studied: the repertoire of both short-term and long-term expression of Pdx1-or Pdx1-VP16-induced pancreatic gene expression; and their capacity to serve as beta-cell surrogates in restoring euglycemia in streptozotocin-treated diabetic mice. We found that cell lines expressing either Pdx1 or Pdx1-VP16 long-term exhibited similar profiles for expression of genes related to pancreatic β-cell development and function; and similar capacity of reversal of hyperglycemia in diabetic mice [3]. However, short-term expression of Pdx1 or Pdx1-VP16 demonstrated that Pdx1-VP16 is much more efficient in initiating liver-to-pancreas transdifferentiation [3], this finding confirming the recent observations that adenovirus-mediated transient expression of Pdx1-VP16 in mouse liver is more effective than Pdx1 [22,23].…”

“…We found that cell lines expressing either Pdx1 or Pdx1-VP16 long-term exhibited similar profiles for expression of genes related to pancreatic β-cell development and function; and similar capacity of reversal of hyperglycemia in diabetic mice [3]. However, short-term expression of Pdx1 or Pdx1-VP16 demonstrated that Pdx1-VP16 is much more efficient in initiating liver-to-pancreas transdifferentiation [3], this finding confirming the recent observations that adenovirus-mediated transient expression of Pdx1-VP16 in mouse liver is more effective than Pdx1 [22,23]. In summary, long-term, persistent expression of either Pdx1 or Pdx1-VP16 is similarly effective in converting hepatic stem cells into pancreatic endocrine precursor cells that, upon transplantation into diabetic mice, can become functional IPCs and restore euglycemia.…”

“…Comparisons can be ambiguous, when adenovirus vectors harboring a transcription factor cDNA have differing extents and duration of gene expression. In view of the remarkable capacity of lentiviral vector (LV) for delivering and integrating transgene into both dividing and nondividing cells, we transduced rat hepatic stem-like WB cells with LV-Pdx1 or LV-Pdx1-VP16, and generated single-cell-derived cell lines that stably express either Pdx1 or Pdx1-VP16 [3]. With these cell lines, we studied: the repertoire of both short-term and long-term expression of Pdx1-or Pdx1-VP16-induced pancreatic gene expression; and their capacity to serve as beta-cell surrogates in restoring euglycemia in streptozotocin-treated diabetic mice.…”

“…Based on our published findings [2][3][4], the molecular events for Pdx1-VP16-induced transdifferentiation of hepatic stem-like WB cell into functional insulin-producing β-like cells can be summarized in Fig. 1.…”

“…Hepatocytes and pancreatic β-cells also possess built-in glucosesensing systems, allowing them to respond to changes in blood glucose concentrations. Recent studies demonstrate that conversion or transdifferentiation of hepatic stem cells [2][3][4], human fetal liver cells [5,6], human hepatoma cells [7] and human adult hepatocytes [8] into functional IPCs by genetic manipulation provides a promising option for cell replacement and gene therapy. Transdifferentiation/reprogramming of cells sharing a common embryonal origin can often be achieved by expressing tissue-specific/selective transcription factors (TFs) from one cell type into the other [9,10].…”