Direct genetic reprogramming of postnatal primary cells by

Direct genetic reprogramming of postnatal primary cells by forced expression of key developmental transcription factors has emerged as an alternative to in vitro differentiation of PSCs. This strategy has been used successfully to produce functional cells, including neurons, hepatocyte-like cells and cardiomyocytes from fibroblasts (Huang et al., 2011; Ieda et al., 2010; Vierbuchen et al., 2010). Similarly, in vivo reprogramming of hepatic cells by expressing different pancreatic transcription factors, including Neurog3 and Pdx1, was able to restore euglycemia in hyperglycemic mice (Ferber et al., 2000; Wang et al., 2007; Vijay Yechoor et al., 2009). Additionally, in vivo reprogramming of exocrine acinar cells into insulin-positive cells by expression of Neurog3, Pdx1 and MafA was also able to reverse hyperglycemia in mice (Zhou et al., 2008). Furthermore, overexpression of Neurog3 and Pdx1 has been shown to enhance pancreatic differentiation of embryonic stem cells (Kubo et al., 2011). Other cell types have also been tested for amenability to reprogramming towards the β-cell fate, including adipose tissue-derived stem cells, placenta-derived multipotent stem cells, hepatocytes, intrahepatic biliary epithelial cells and gall Caspase-1, human recombinant proteinase cells (Chandra et al., 2011; Chiou et al., 2011; Coad et al., 2009; Motoyama et al., 2009; Nagaya et al., 2009; Shigeru et al., 2007). The extrahepatic biliary tissue, including the gallbladder, is a particularly appealing source of cells for reprogramming to the pancreatic fate. The extrahepatobiliary system shares a common developmental origin with the ventral pancreas, from a cell termed the pancreatobiliary progenitor (Spence et al., 2009). Segregation of these distinct lineages is partly regulated by the Notch effector Hes1. Recently it was demonstrated that inhibition of Hes1 in cultured gall bladder cells (GBCs) was sufficient to induce some insulin expression (Coad et al., 2009). While this work highlighted the potential of GBCs as a source of transplantable β-cells, the full spectrum of β-cell expressed genes or the in vivo functionality of these cells was not determined. Moreover, the cells showed only limited proliferative potential under the culture conditions used. For GBCs to be a viable substrate of future β-cell replacement therapies, they would have to be robustly expandable (Yechoor and Chan, 2010). Therefore, the true utility of GBCs as a source of transplantable β-cells remains unknown. In this study, we investigated if mouse GBCs significantly expanded in vitro can still be reprogrammed towards the β-cell fate by using a combination of positive instructive signals as well as Notch inhibition. GBCs were transduced with adenoviruses expressing the transcription factors NEUROG3, Pdx1 and MafA and treated with retinoic acid and Notch inhibitors, resulting in their differentiation into islet-like cells. Reprogrammed cells had the ability to engraft, survive and remain insulin-positive up to 15weeks post-transplantation. However, there were also differences between the reprogrammed GBCs and true β-cells. Our findings confirm that the gall bladder represents a promising source of autologous reprogrammable cells for the treatment of type 1 diabetes mellitus.
Materials & methods
Results
Discussion The results presented here establish a combined genetic and small molecule approach to differentiate mouse GBCs towards the pancreatic β-cell lineage (summarized in Fig. S2). The expression of key transcription factors has been shown to be a powerful method to directly reprogram one cell type to another, without the need to go through a pluripotent intermediate (Huang et al., 2011; Ieda et al., 2010; Vierbuchen et al., 2010; Xie et al., 2004). It is interesting that the three transcription factors capable of reprogramming GBCs into islet-like cells are the same as those required for in vivo reprogramming of the exocrine pancreas into endocrine pancreas (Zhou et al., 2008), as well as in vitro reprogramming of pancreatic exocrine cells into β-like cells (Akinci et al., 2011). Therefore, together these results implicate Neurog3, Pdx1 and MafA as the key genetic reprogramming factors needed for the de novo generation of mouse β-cells. In addition to expressing these three transcription factors, the frequency of GFP+ rGBCs was augmented by the timed addition of RA and DBZ, a Notch signaling effector. Both these pathways are essential for normal pancreatic development (Apelqvist et al., 1999; Martin et al., 2005; Stafford and Prince, 2002). Manipulation of these pathways has also been used to differentiate pluripotent stem cells towards a differentiated β-cell fate, indicating manipulation of both RA-responsive genes and Notch signaling to also be critical for de novo β-cell differentiation (Kroon et al., 2008; Nostro et al., 2011).