Connexin 43 Functions as a Positive Regulator of Stem Cell Differentiation into Definitive Endoderm and Pancreatic Progenitors

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe identification of conditions that allows for the in vitro differentiation of embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) toward desired cell lineages have emerged as revolutionary new strategies for the development of cell-based replacement therapies. However, despite significant progress over the past few years, protocols for the induction of these pluripotent stem cells to differentiate into rare cell types, such as the pancreatic islet cells producing hormones like insulin and glucagon, remain relatively inefficient, often leading to heterogeneous cell preparations comprising unwanted cell types that may pose risks of teratoma development following transplantation (, , ).To date, the majority of protocols for the in vitro-directed differentiation of stem cells toward the islet cell lineage have focused on the administration of select growth factors and signaling molecules at defined time points that elicit the activation or inhibition of signaling pathways originally discovered to regulate islet cell development in animal models (). In these efforts, one aspect that remains relatively unexplored at the molecular level is the possible role of direct cell-to-cell communication, a mechanism known to regulate cell fate commitment and tissue morphogenesis during development (, , , , , ). Among proteins that have been shown to participate in these processes of cell communication, connexins (Cxs) are of special interest as they represent the building blocks of gap junction (GJ) channels, mediating the intercellular exchange of signaling molecules such as microRNAs, cations and anions, cyclic nucleotides, as well as small peptides and interfering RNAs (, ; , , , href="#bib30" rid="bib30" class=" bibr popnode">Lim et al., 2011, href="#bib22" rid="bib22" class=" bibr popnode">Kanaporis et al., 2008, href="#bib23" rid="bib23" class=" bibr popnode">Kanaporis et al., 2011). These channels have been shown to be indispensable for the proper growth, differentiation, and functional maturation of many cell types, both during embryonic development and in postnatal life (href="#bib29" rid="bib29" class=" bibr popnode">Levin, 2007). Among Cxs known to participate to the biology of pancreatic cell lineages, Cx43 is of particular interest as it is expressed in the developing pancreas where, together with Cx36, it gets progressively restricted to the endocrine cell lineage (href="#bib38" rid="bib38" class=" bibr popnode">Serre-Beinier et al., 2009), and is required for the control of secretory function and survival (href="#bib39" rid="bib39" class=" bibr popnode">Serre-Beinier et al., 2002, href="#bib26" rid="bib26" class=" bibr popnode">Klee et al., 2011, href="#bib1" rid="bib1" class=" bibr popnode">Carvalho et al., 2010, href="#bib2" rid="bib2" class=" bibr popnode">Carvalho et al., 2012, href="#bib33" rid="bib33" class=" bibr popnode">Nlend et al., 2006, href="#bib28" rid="bib28" class=" bibr popnode">Le Gurun et al., 2003). Interestingly, Cx43 has also been found to be involved in the maintenance of stem cell pluripotency (href="#bib8" rid="bib8" class=" bibr popnode">Dyce et al., 2014), and in the regulation of the cell cycle during tissue development and regeneration (href="#bib18" rid="bib18" class=" bibr popnode">Hoptak-Solga et al., 2008). Of further interest are studies demonstrating that interference with Cxs' expression or function results in significant alterations of cell fate development, survival, and differentiated functions (href="#bib40" rid="bib40" class=" bibr popnode">Scherer et al., 2005, href="#bib33" rid="bib33" class=" bibr popnode">Nlend et al., 2006, href="#bib47" rid="bib47" class=" bibr popnode">Wang and Belousov, 2011, href="#bib10" rid="bib10" class=" bibr popnode">Evans et al., 2012).In this study, building on the notion that the function of GJ channels is dependent on their gating status, we tested a simple gain-of-function approach that promotes the activation or opening of GJ channels composed of Cx43. The approach consisted in treating ESCs undergoing controlled differentiation toward pancreatic cell lineages with the AAP10-activating peptide, reported to promote Cx43 GJ channel opening (href="#bib48" rid="bib48" class=" bibr popnode">Weng et al., 2002, href="#bib6" rid="bib6" class=" bibr popnode">Dhein et al., 2001, href="#bib21" rid="bib21" class=" bibr popnode">Jozwiak and Dhein, 2008, href="#bib10" rid="bib10" class=" bibr popnode">Evans et al., 2012). The results of these experiments demonstrate that activation of Cx43 GJ channels in ESCs significantly enhances the expression of definitive endoderm (DE) markers FoxA2 and Sox17, which in turn results in a more efficient derivation of DE and primitive gut tube (PGT) cells, as well as more prominent numbers of posterior foregut (PF), pancreatic progenitors (PP), and pancreatic endocrine progenitors (EP). Collectively, these results provide evidence for the functional involvement of GJ channels in the differentiation of ESCs into pancreatic cell lineages.