Making Purkinje Cells in a Culture Dish


The beating heart is functionally divided into two levels; an upper set of chamber and a lower set of chambers.  The heart beat originates in upper chambers, but that signal to beat must be relayed to the lower chambers of the heart.  However does this signal get to the bottom of the heart?  The answer is that there is an extension cord that relays the signal to beat from the top of the heart to the lower chambers of the heart.  This extension cord comes in the form of a conduction system that consists of modified cells that do not contract, but conduct electrochemical signals from the upper chambers of the heart to the lower chambers.

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The beat originates in the upper part of the left atrium (upper chamber) in the so-called pacemaker or sinoatrial node.  The signal to beat spreads rapidly across the atrial tissue and the a transmission node called the atrioventricular node at the bottom of the heart.  Once the signal to beat gets to the atrioventricular node, that signal goes to the conductive tissues in the septum of the heart called the “bundle of His” or atrioventricular bundle.  From there, the signal splits into the left and right bundle branches that swing around bottom of the heart into the ventricle walls.  Tiny extensions of the conduction system called “purkinje fibers” move into the walls of the ventricles.  These purkinje fibers function to control cardiac action potentials essential for a consistent heartbeat.

Several studies suggest that dysfunctional purkinje fibers are a potential source of arrhythmias in several heart syndromes.  However, how purkinje fiber dysfunction is responsible for causing arrhythmias has not been fully studied.  In order to begin this studying the role of purkinje fibers in arrhythmias, the laboratory of Glenn I. Fishman (New York University School of Medicine, USA) has generated an engineered mouse embryonic stem cell (ESC) line which can generate huge numbers of purkinje fiber cells.  Normally, when embryonic stem cells are differentiated into heart muscle cells (cardiomyocytes), purkinje cells constitute a very minor, even rare cell sub‐population (see Maass K, Shekhar A, Lu J, et al. Isolation and characterization of embryonic stem cell-derived cardiac purkinje cells. Stem Cells 2015;33:1102-1112).

According to previous expression studies, Fishman and others utilized ESCs that expressed Green Fluorescent Protein (GFP) under the control of the Cntn2 promoter.  The Cntn2 gene encodes the Contacting-2 protein, which marks those cells that will differentiate into Purkinje fiber conduction network cells (Pallante BA, et al. Circ Arrhythm Electrophysiol 2010;3:186-194;Kim EE, et al. The Journal of clinical investigation 2014;124:5027-5036).  Cntn2, however is not a perfect marker because it is also expressed in certain neuronal cells.  Therefore an additional marker was used; “MHCα‐mCherry.”  MHCα‐mCherry expressed a very brightly colored protein under the control of the myosin heavy chain gene promoter.  Because the alpha-myosin heavy chain is a heart muscle-specific protein, this brightly-colored protein is only expressed in heart-specific cells. Any cells that express both the Cntn2-GFP and the MHCα‐mCherry are almost certainly purkinje fiber cells.

Fishman and his team differentiated mouse ESCs into purkinje fiber cells and characterized the parallel activation of αMHC and Cntn2 in the developing murine heart.  ESC-derived purkinje fibers made up around 2% of the cell population at 4 weeks, and appeared long and pointy.  They also expressed a range of proteins similar to that of endogenous purkinje fiber cells, such as Cntn2, Troponin T (in a sarcomeric pattern, no less), and the conduction‐system specific connexin40 gap junctional protein.  Further analysis demonstrated the heightened expression of many genes associated with the cardiac conduction system, such as Nkx2‐5, Connexin40, HCN4, CACNA1G, Scn5a, and SCN10A.  The use of patch-clamping showed that these cultured cells  had similar electrophysiological properties to that of endogenous PCs; a highly important characteristic.

In combination with ESC-derived sinoatrial cells (see Scavone A, et al. Circulation research 2013;113:389-398), pacemaker cells (see Morikawa K, et al. Pacing Clin Electrophysiol 2010;33:290-303), and atrial‐like cardiomyocytes (Josowitz R, et al. PLoS One 2014;9:e101316), the creation of PC cells in this study may represent an extremely exciting step towards cell therapy for the failing heart. These data represent a useful strategy for the production of a large amount of a useful cell type from a heterogeneous cardiac cell population, which may be used to inform on diverse study areas including developmental biology, disease pathogenesis and anti‐arrhythmic drug screening. The authors themselves hope that using patient-specific fibroblasts and a direct reprogramming process, PCs may be used to treat heritable, acquired and post‐surgical damage to the heart’s conduction system in a patient-tailored manner.

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mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).