Date Published: June 26, 2019
Publisher: Public Library of Science
Author(s): Fouzeyyah Alsaeedi, Rachel Wilson, Charlotte Candlish, Ibrahim Ibrahim, Alistair C. Leitch, Tarek M. Abdelghany, Colin Wilson, Lyle Armstrong, Matthew C. Wright, Yun-Wen Zheng.
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.
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 [1–3]. The main functional cell of the liver–hepatocytes–is an exemplar of this problem . Hepatocytes in vivo are highly metabolically active and perform a diverse range of functions (many of which are specific to this cell type). 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 also encountered when hepatocytes are isolated from intact organs and placed under similar conditions in vitro . 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 , 3D culture systems , flow cultures  etc.).
The rat Sgk1 gene is currently known to encode 3 validated mRNA transcripts and likely 1 further transcript [NCBI database, see also Table 2]. All 4 transcripts encode an identical core amino acid sequence and differ only in the their N terminal amino acid sequences. It is not known whether the Sgk1 isoforms have different functions, however, only the rat Sgk1c transcript appears to be irreversibly induced in B-13 cells after exposure to glucocorticoid . This suggests that endogenous Sgk1c may be responsible for its differentiating effects in B-13 cells. This is supported by the high expression of the murine orthologue in pancreatic tissue from mice with high circulating endogenous glucocorticoids (these mice experience a conversion of the pancreatic exocrine tissue into hepatocyte-like cells ). Since expression of plasmid-encoded human-specific SGK1F protein promoted B-13 cell differentiation to B-13/H cells , the cDNA for this transcript was cloned into a replication-deficient adenoviral genome. The rationale for focusing on SGK1F was also driven by the knowledge that the pancreas from a patient treated for many decades with systemic glucocorticoid experienced a degree of hepatic differentiation and also contained high levels of SGK1F mRNA transcripts . The data in this paper demonstrate that adenoviral-mediated SGK1F expression in B-13 cells induces their differentiation into B-13/H cells, similarly to their response to exposure to glucocorticoid. These data therefore support our proposed glucocorticoid-dependent mechanism for converting B-13 cells into B-13/H cells, as schematically outlined in Fig 8, involving glucocorticoid receptor activation, Sgk1 induction, phosphorylation of β-catenin and a suppression of endogenously high Wnt signaling activity in B-13 cells.
This paper reports for the first time that SGK1F expression in iPSCs promotes an hepatoblast phenotype and by implication, that this hepatoblast promotion is likely the major role played by glucocorticoid-induced Sgk1c expression in B-13 cells. These insights could be exploited to modulate expression to i) enhance the early stages of iPSC differentiation into hepatoblasts and ii) enhance B-13 conversion into B-13/H cells to promote hepatocyte phenotype.