Poster Presentation 6th Australian Health and Medical Research Congress 2012

Spontaneous Calcium Oscillations from Embryonic Stem Cell Derived Cardiomyocytes – Sorting Out the Cardiac Conduction System.  (#387)

Benjamin A Finnin 1 , Ebba Lagerqvist 2 , Colin W Pouton 1 , John M Haynes 1
  1. Monash Universtiy, Parkville, Vic, Australia
  2. Institut De Génomique Fonctionelle, Montpellier, France

Background:  The cardiac conduction system is comprised of cells that possess region specific phenotypic and rhythmic properties.  The differentiation of embryonic stem cells (ESCs) into functional cardiac tissue generates spontaneously active clusters of beating cardiomyocytes, but there is little evidence that these beating aggregates contain more than a single phenotype of spontaneously active ‘pacemaker’ cell. 

Aims:  To establish whether a simple ESC embryoid body differentiation procedure is capable of generating more than one spontaneously active pacemaker cell.

Methods: A Nkx2.5-eGFP transgenic cell line was used to identify all cells of the cardiac lineage. Differentiation of mouse ESCs was induced via hanging drop.  Differentiating cultures were digested and FACS sorted at early (day 9) and late (day 18) stages. Cells were replated at 15k/cm2 on collagen-coated plates. 3 days after sorting cells were loaded with the calcium indicator Fluo4 and imaged at 15-120 frames per second. Spontaneous activity was recorded before and after addition of pharmacological agents (Ryanodine, 2-APB, ZD7288, Thapsigargin, Nifedipine, Nickel chloride).

Results:  Fluorescence activated cell sorting of a pan-cardiac reporter line followed by high speed confocal imaging reveals the presence of 6 common waveforms with unique properties.   These waveforms had distinct rates of spontaneous activity; from 139±6 to 14±1 oscillations per minute, which correlated with cell size (µm2, r2=0.93). Faster cells were less sensitive to the If channel blocker ZD7288 (10 µM), but more sensitive to the blockade of intracellular store-mediated Ca2+ release by ryanodine (10 µM).  All cells were sensitive to blockade by the SERCA inhibitor thapsigargin (1 µM) and the L-type voltage operated Ca2+ channel inhibitor, nifedipine (1 µM).

Conclusions: This study shows that hanging drop differentiation generates distinctly identifiable populations of spontaneously active Nkx2.5-eGFP+ positive cells.  These cells should prove an invaluable aid to investigations of the mechanisms and pathophysiology of cardiac pacemaker cells.