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Rebuilding a Broken Heart with ESC-derived Purkinje cells

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Review of “Isolation and Characterization of ESC-Derived Cardiac Purkinje Cells” from Stem Cells by Stuart P. Atkinson

Purkinje cells (PCs) are a specialised component of the cardiac conduction system (CCS) which functions to control cardiac action potentials essential for a consistent heartbeat. Studies suggest that dysfunctional PCs are a potential source of arrhythmias in numerous syndromes, but the mechanisms underlying this behaviour have not been fully studied. In order to start this characterisation, the laboratory of Glenn I. Fishman (New York University School of Medicine, USA) have generated an engineered mouse embryonic stem cell (ESC) line which can generate vast numbers of PCs which normally represent a rare sub‐population of cardiomyocytes [1].

Based on previous expression studies [2, 3], the group utilised ESCs expressing a Cntn2-GFP reporter as a marker of cardiac progenitors differentiating into cells of the Purkinje fiber conduction network. As Cntn2 is also expressed in certain neuronal cell subtypes, specificity was ensured using another marker - a cardiomyocyte‐specific MHCα‐mCherry reporter construct. Following a previously published protocol [4], the researchers then differentiated these ESCs into PCs, observing a time course paralleling the activation of αMHC and Cntn2 in the developing murine heart. ESC-derived PCs made up around 2% of the cell population at 4 weeks, and presented with an elongated morphology and increased surface area compared to ESC‐derived cardiomyocytes which made up around 60% of the cell population. Isolated ESC-PCs expressed a range of proteins similar to that of endogenous PCs (Cntn2, Troponin T in a sarcomeric pattern, and the conduction‐system specific connexin40 gap junctional protein - See figure), and further analysis demonstrated the heightened expression of many genes associated with the cardiac conduction system (Nkx2‐5, Connexin40, HCN4, CACNA1G, Scn5a, and SCN10A). Finally, using patch-clamp techniques, the group confirmed that ESC-PCs had similar electrophysiological properties to that of endogenous PCs; a highly important characteristic.

In combination with ESC-derived sinoatrial cells [5], pacemaking cells [6]; and atrial‐like cardiomyocytes [7], the creation of PC cells in this study may represent an exciting step forward in cell therapy for the failing heart. The data here presented represents 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 hearts conduction system in a patient-tailored manner.

Discussion Points

  • How functional are mouse PC cells in an in vivo setting?
  • Can a similar strategy be used with hESCs to produce human PCs?
  • How far away are we from being able to reconstruct a mouse/human heart?

References

  1. 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.
  2. Pallante BA, Giovannone S, Fang-Yu L, et al. Contactin-2 expression in the cardiac Purkinje fiber network. Circ Arrhythm Electrophysiol 2010;3:186-194.
  3. Kim EE, Shekhar A, Lu J, et al. PCP4 regulates Purkinje cell excitability and cardiac rhythmicity. The Journal of clinical investigation 2014;124:5027-5036.
  4. Kattman SJ, Witty AD, Gagliardi M, et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 2011;8:228-240.
  5. Scavone A, Capilupo D, Mazzocchi N, et al. Embryonic stem cell-derived CD166+ precursors develop into fully functional sinoatrial-like cells. Circulation research 2013;113:389-398.
  6. Morikawa K, Bahrudin U, Miake J, et al. Identification, isolation and characterization of HCN4-positive pacemaking cells derived from murine embryonic stem cells during cardiac differentiation. Pacing Clin Electrophysiol 2010;33:290-303.
  7. Josowitz R, Lu J, Falce C, et al. Identification and purification of human induced pluripotent stem cell-derived atrial-like cardiomyocytes based on sarcolipin expression. PLoS One 2014;9:e101316.