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Identification, Isolation and Characterization of HCN4‐Positive Pacemaking Cells Derived from Murine Embryonic Stem Cells during Cardiac Differentiation
Author(s) -
MORIKAWA KUMI,
BAHRUDIN UDIN,
MIAKE JUNICHIRO,
IGAWA OSAMU,
KURATA YASUTAKA,
NAKAYAMA YUJI,
SHIRAYOSHI YASUAKI,
HISATOME ICHIRO
Publication year - 2010
Publication title -
pacing and clinical electrophysiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.686
H-Index - 101
eISSN - 1540-8159
pISSN - 0147-8389
DOI - 10.1111/j.1540-8159.2009.02614.x
Subject(s) - embryonic stem cell , transfection , immunostaining , microbiology and biotechnology , green fluorescent protein , stem cell , cellular differentiation , gene , embryoid body , biology , medicine , adult stem cell , genetics , immunology , immunohistochemistry
Background: Development of biological pacemaker is a potential treatment for bradyarrhythmias. Pacemaker cells could be extracted from differentiated embryonic stem (ES) cells based on their specific cell marker hyperpolarization‐activated cyclic nucleotide‐gated (HCN)4. The goal of this study was to develop a method of identification, isolation, and characterization of pacemaking cells derived from differentiated ES cells with GFP driven by HCN4 promoter.Methods and Results: Polymerase chain reaction (PCR) screening and southern blot analysis revealed that HCN4p‐EGFP trans‐gene was stably integrated into the chromosome of mouse AB1 ES cells. RT‐PCR and immunostaining results showed similar expression of the specific cardiac pacemaker markers of the HCN4p‐EGFP ES cells and its parental AB1 ES cell lines. Although HCN4p‐EGFP trans‐gene may have slight effect on the general mesodermal differentiation, it had no effect on the pluripotency of ES cells, on the transcription of cardiac specific factors and cardiac contractile proteins, and on the capability of ES cells to differentiate into pacemaker cells. Electrophysiological study indicated that HCN4p‐GFP‐positive cells revealed the spontaneous action potential, which was slowed by the treatment with 2 mM Cs + , and expressed the hyperpolarization‐activeted cation current I f encoded by HCN4 gene.Conclusion: By the approach of using stable transfectant of HCN4p‐EGFP gene, the identification, isolation, and characterization of ES cell‐derived pacemaking cells could be carried out. PACE 2010; 33:290–303)