Connexin 32-mediated cell-cell communication is essential for hepatic differentiation from human embryonic stem cells

Abstract

Gap junction-mediated cell-cell interactions are highly conserved and play essential roles in cell survival, proliferation, differentiation and patterning. We report that Connexin 32 (Cx32)-mediated gap junctional intercellular communication (GJIC) is necessary for human embryonic stem cell-derived hepatocytes (hESC-Heps) during step-wise hepatic lineage restriction and maturation. Vitamin K2, previously shown to promote Cx32 expression in mature hepatocytes, up-regulated Cx32 expression and GJIC activation during hepatic differentiation and maturation, resulting in significant increases of hepatic markers expression and hepatocyte functions. In contrast, negative Cx32 regulator 2-aminoethoxydiphenyl borate blocked hESC-to-hepatocyte maturation and muted hepatocyte functions through disruption of GJIC activities. Dynamic gap junction organization and internalization are phosphorylation-dependent and the p38 mitogen-activated protein kinases pathway (MAPK) can negatively regulate Cxs through phosphorylation-dependent degradation of Cxs. We found that p38 MAPK inhibitor SB203580 improved maturation of hESC-Heps correlating with up-regulation of Cx32; by contrast, the p38 MAPK activator, anisomycin, blocked hESC-Heps maturation correlating with down-regulation of Cx32. These results suggested that Cx32 is essential for cell-cell interactions that facilitate driving hESCs through hepatic-lineage maturation. Regulators of both Cx32 and other members of its pathways maybe used as a promising approach on regulating hepatic lineage restriction of pluripotent stem cells and optimizing their functional maturation.

Human embryonic stem cell–derived hepatoblasts are an optimal lineage stage for hepatitis C virus infection

“…Immunofluorescent and Immunohistochemistry analyses were carried out as previously described . Details and dilutions about antibodies were listed in http://onlinelibrary.wiley.com/doi/10.1002/hep.29134/suppinfo.…”

“…Immunoprecipitation and western blotting were carried out as previously described . Briefly, cells were harvested in lysis buffer and precleared with Protein G–Sepharose (GE Healthcare, Uppsala, Sweden) precoated with anti‐OCLN at 4°C for 90 minutes.…”

“…Immunoprecipitation and western blotting were carried out as previously described. (16) Briefly, cells were harvested in lysis buffer and precleared with Protein G-Sepharose (GE Healthcare, Uppsala, Sweden) precoated with anti-OCLN at 48C for 90 minutes. The beads were collected by centrifugation, washed thoroughly in lysis buffer, and the precipitated proteins eluted with Laemmli buffer.…”

Establishment of a feeder and serum-free culture system for human embryonic stem cells

“…Human embryonic stem cells (hESCs) are pluripotent cells isolated from early blastocysts or primordial germ cells with the ability to proliferate and differentiate into any type of cell in the human body (Pera et al, 2000;Lior, 2002). hESCs have become a unique tool in regenerative medicine and tissue engineering, with broad prospects for tissue repair, organ transplantation, and drug screening and development (Qin et al, 2016). Accordingly, the induction of hESCs differentiation has been a major research topic since their initial isolation and establishment from human embryos in 1998 (Cowan et al, 2004).…”

Mind the Gaps in Tumor Immunity: Impact of Connexin-Mediated Intercellular Connections

“…Gap junction-mediated intercellular communications (GJICs) are critical for several physiological processes, including: electric current propagation in the heart and neurons ( 4 , 5 ); embryonic development ( 6 ); cell differentiation ( 7 ); tissue homeostasis ( 8 ); autophagosome biogenesis ( 9 ); cell survival, proliferation, and cell death ( 10 , 11 ); and the immune response ( 12 ). As GJs are involved in countless cellular and physiological processes, the cells need to establish delicate regulatory mechanisms of GJICs, which occurs at different levels, such as that of Cx gene expression, the life cycle of Cx protein level, or GJ assembly and permeability.…”