Intercellular communication via gap junctions is thought to play an important role in embryonic cell survival and differentiation. Classical studies demonstrated both dye and electrical coupling of cells in the inner cell mass of mouse embryos, as well as the development of restrictions against coupling between cells of the inner cell mass and surrounding trophectoderm. Here we demonstrate extensive gap junctional communication between human embryonic stem (ES) cells, the pluripotent cells isolated from the inner cell mass of preimplantation blastocysts. Human ES cells maintained in vitro expressed RNA for 18 of the 20 known connexins; only connexin 40.1 (Cx40.1) and Cx50 were not detected by reverse transcription-polymerase chain reaction. Cx40, Cx43, and Cx45 were visualized by immunofluorescence at points of contact between adjacent cells. Electron microscopy confirmed that neighboring cells formed zones of tight membrane apposition characteristic of gap junctions. Fluorescent dye injections demonstrated extensive coupling within human ES cell colonies growing on mouse embryonic fibroblast (MEF) feeder cells, whereas dye coupling between human ES cells and adjacent MEFs was extremely rare. Physiological recordings demonstrated electrical and dye coupling between human ES cells in feeder-free monolayers and between isolated human ES cell pairs. Octanol, 18-α-glycyrrhetinic acid, and arylaminobenzoates inhibited transjunctional currents. Dye uptake studies on human ES cell monolayers and recordings from solitary human ES cells gave evidence for the surface expression of connexon hemichannels. Human ES cells provide a unique system for the study of human connexin proteins and their potential functions in cellular differentiation and the maintenance of pluripotency.
- Dye coupling
- Electron microscopy
- Patch clamp
- Reverse transcription-polymerase chain reaction