Abstract
Pluripotent mouse embryonic stem cells (mESC) are cell lines derived from the inner cell mass of blastocyst-stage early mammalian embryos. Since ion channel modulation has been reported to interfere with both growth and differentiation process in mouse and human ESC it is important to characterize the electrophysiological properties of newly generated mESC and compare them to other lines. In this work, we studied the intercellular communication by way of gap junctions in a Brazilian derived mESC (USP-1, generated by Dr. Lygia Pereira's group) and characterized its electrophysiological properties. We used immunofluorescence and RT-PCR to reveal the presence of connexin 43 (Cx43), pluripotency markers and ion channels. Using a co-culture of neonatal mouse cardiomyocytes with mESC, where the heart cells expressed the enhanced Green Fluorescent Protein, we performed dye injections to assess functional coupling between the two cell types observing dye diffusion. The patch-clamp study showed outward currents identified as two types of potassium currents, transient outward potassium current (Ito) and delayed rectifier outward potassium current (Iks), by use of specific drug blockage. Calcium or sodium currents in undifferentiated mESC were not identified. We conclude that USP-1 mESC has functional Cx43 channels establishing intercellular communication among themselves and with cardiomyocytes and has a similar electrophysiological profile compared to other mESC cell lines.
Highlights
Pluripotent mouse embryonic stem cells are cell lines derived from the inner cell mass of blastocyst-stage early mammalian embryos
We investigated the presence of transcripts related to pluripotency and ion channels subunits in the USP-1 cells
The same transcripts were analyzed in extracts of heart, as positive controls, and in pure cultures of mouse embryonic fibroblasts (MEF), since these cells are used as feeder layers for the mouse embryonic stem cells (mESC) and could contaminate the PCR results
Summary
Pluripotent mouse embryonic stem cells (mESC) are cell lines derived from the inner cell mass of blastocyst-stage early mammalian embryos These unique cells can self-renew while maintaining their pluripotency and differentiate into cellular derivates of all three primary germ layers in vivo or under appropriate cultivation in vitro Sartiani et al (2007) described the presence of both potassium and calcium currents in human ESC (hESC), but Jiang et al (2010) could only find potassium currents and not calcium currents in hiPSC These differences highlight the importance of characterizing the electrophysiological properties of pluripotent stem cells before differentiation and usage in in vitro or in vivo experimental models
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