Interestingly, we mentioned that the rate of recurrence of Sox9+Pdx1+ (Numbers 5A and 5D) and Pdx1+Nkx6.1+ progenitors (Numbers 5B and 5E) was also significantly increased by AAP10 treatment. alternative 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 generating hormones like insulin and glucagon, remain relatively inefficient, often leading to heterogeneous cell preparations comprising undesirable cell types that may present risks of teratoma development following transplantation (Tang et?al., 2013, Kushner et?al., 2014, Espes et?al., 2017). To day, 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 found out to regulate islet cell development in animal models (Sneddon et?al., 2018). In these attempts, one aspect that remains relatively unexplored in the molecular level is the possible role of direct cell-to-cell communication, a mechanism known to regulate cell fate commitment and cells morphogenesis during development (Constantin and Cronier, 2000, Wei et?al., 2004, Levin, 2007, Hatler et?al., 2009, Sozen et?al., 2014, Yamada et?al., 2016). Among proteins that have been shown to participate in these processes of cell communication, connexins (Cxs) are of unique interest as they represent the building blocks of space 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 (Goodenough et?al., 1996, S?hl and Willecke, 2004; Willecke et?al., 2002, Evans et?al., 2006, Charpantier et?al., 2007, Lim et?al., 2011, Kanaporis et?al., 2008, Kanaporis et?al., 2011). These channels have been shown to be CACNB4 indispensable for the proper growth, differentiation, and practical maturation of many cell types, MC-Sq-Cit-PAB-Gefitinib both during embryonic development and in postnatal existence (Levin, 2007). Among Cxs known to participate MC-Sq-Cit-PAB-Gefitinib to the biology of pancreatic cell lineages, Cx43 is definitely of particular interest as it is MC-Sq-Cit-PAB-Gefitinib definitely indicated in the developing pancreas where, together with Cx36, it gets gradually restricted to the endocrine cell lineage (Serre-Beinier et?al., 2009), and is required for the control of secretory function and survival (Serre-Beinier et?al., 2002, Klee et?al., 2011, Carvalho MC-Sq-Cit-PAB-Gefitinib et?al., 2010, Carvalho et?al., 2012, Nlend et?al., 2006, Le Gurun et?al., 2003). Interestingly, Cx43 has also been found to be involved in the maintenance of stem cell pluripotency (Dyce et?al., 2014), and in the rules of the cell cycle during tissue development and regeneration (Hoptak-Solga et?al., 2008). Of further interest are studies demonstrating that interference with Cxs’ manifestation or function results in significant alterations of cell fate development, survival, and differentiated functions (Scherer et?al., 2005, Nlend et?al., 2006, Wang and Belousov, 2011, Evans et?al., 2012). In this study, building on the notion 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 (Weng et?al., 2002, Dhein et?al., 2001, Jozwiak and Dhein, 2008, Evans et?al., 2012). The results of these experiments demonstrate that activation of Cx43 GJ channels in ESCs significantly enhances the manifestation of definitive endoderm (DE) markers FoxA2 and Sox17, which in turn results MC-Sq-Cit-PAB-Gefitinib 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 practical involvement of GJ channels in the differentiation of ESCs into pancreatic cell lineages. Results In a first series of experiments, to determine the manifestation profile of Cx43 in ESCs under conditions that favor spontaneous differentiation, we adopted a protocol of suspension tradition to generate embryoid body (EBs). EB formation recapitulates.