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Abstract
The rat pancreatic AR42J-B13 (B-13) cell line differentiates into non-replicative hepatocyte-like (B-13/H) cells in response to glucocorticoid. Since this response is dependent on an induction of serine/threonine protein kinase 1 (SGK1), this may suggest that a general pivotal role for SGK1 in hepatocyte maturation. To test this hypothesis, the effects of expressing adenoviral-encoded flag tagged human SGK1F (AdV-SGK1F) was examined at 3 stages of human induced pluripotent stem cell (iPSC) differentiation to hepatocytes. B-13 cells infected with AdV-SGK1F in the absence of glucocorticoid resulted in expression of flag tagged SGK1F protein; increases in β-catenin phosphorylation; decreases in Tcf/Lef transcriptional activity; expression of hepatocyte marker genes and conversion of B-13 cells to a cell phenotype near-similar to B-13/H cells. Given this demonstration of functionality, iPSCs directed to differentiate towards hepatocyte-like cells using a standard protocol of chemical inhibitors and mixtures of growth factors were additionally infected with AdV-SGK1F, either at an early time point during differentiation to endoderm; during endoderm differentiation to anterior definitive endoderm and hepatoblasts and once converted to hepatocyte-like cells. SGK1F expression had no effect on differentiation to endoderm, likely due to low levels of expression. However, expression of SGK1F in both iPSCs-derived endoderm and hepatocyte-like cells both resulted in promotion of cells to an hepatoblast phenotype. These data demonstrate that SGK1 expression promotes an hepatoblast phenotype rather than maturation of human iPSC towards a mature hepatocyte phenotype and suggest a transient role for Sgk1 in promoting an hepatoblast state in B-13 trans-differentiation to B-13/H cells.
Highlights
A common difficulty encountered with stem cell-derived differentiated cells in vitro, is their maturation into fully differentiated phenotypes that quantitatively reflect the function of cells in vivo or directly after isolation from tissues [1,2,3]
We demonstrate that an AdV-encoded SGK1F capable of efficiently promoting an hepatocytelike phenotype in B-13 cells promoted hepatoblast formation when SGK1F was expressed in both induced pluripotent stem cell (iPSC)-derived endoderm and hepatocyte-like cells
Adenoviral-mediated expression of SGK1F alone induces the conversion of B-13 cells into B-13/H cells
Summary
A common difficulty encountered with stem cell-derived differentiated cells in vitro, is their maturation into fully differentiated phenotypes that quantitatively reflect the function of cells in vivo or directly after isolation from tissues [1,2,3]. Stem cell-derived hepatocyte-like cells resist functioning as hepatocytes in vitro likely because of several drivers These include sub-optimal differentiation protocols; a sub-optimal in vitro environment (e.g. extracellular matrix, appropriate cell-cell contacts, cell density) and aberrant levels of regulating factors (e.g. hormones controlling gene expression). These in combination, promote a de-differentiation process, a response encountered when hepatocytes are isolated from intact organs and placed under similar conditions in vitro [4]. These issues have resulted in extensive efforts to manipulate the in vitro environment to generate and/or preserve hepatic functionality (e.g. co-culture systems [5], 3D culture systems [6], flow cultures [7] etc.)
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