We have previously demonstrated that contactin2 (Cntn2) protein is enriched in Purkinje cells of the adult cardiac conduction system (CCS). Here we present a mouse embryonic stem cell (mESC) reporter line to identify Purkinje-like cardiomyocytes in vitro.
Methods and Results: Dual reporter Purkinje cell mESC were generated from Cntn2-eGFP blastocysts, transduced with αMHC-mCherry lentivirus. The mESC expressed markers of pluripotency (Oct3/4; Sox2, Nanog) and differentiated into all three germ layers (Nestin, a-fetoprotein, Brachyury). Cultures differentiated under serum-free, cardiogenic conditions were enriched in cardiomyocytes (n=5), consisting of single positive aMHC-mCherry [57.76±3.7%] and double positive αMHC-mCherry/Cntn2-eGFP cells [1.9±0.9%]. Compared with eGFP negative cardiomyocytes (n=15), double positive cells (n=11) revealed electrophysiological characteristics consistent with Purkinje-like derivatives, including increased peak sodium currents (180.3±20.5 pA/pF vs. =-118.0±21.2pA/pF), faster upstroke velocities (133.7±12.3 V/s vs.60.0±13.2V/s) and elongated action potential duration (APD90=170.2±17.5ms vs. APD90=120.6±17.3ms). Calcium imaging demonstrated chronotrop regulation and spontaneous Ca2+ oscillations in mCherry+eGFP+ cells. Immunofluorecence analysis demonstrated endogenous expression of Purkinje cell markers, including Cntn2, Cx40 and Troponin-T, in double positive cells. Quantitative real-time PCR analysis of double positive cells verified cardiomyocyte-specific transcript expression [Mlc2v: 3.8x106-fold (p<0.05); Nkx2.5: 178-fold (p<0.05) compared to double negative cells]. Moreover, double positive cells expressed significantly elevated levels of CCS-specific transcripts compared to mCherry-single positive cells [Cntn2: 31-fold (p<0.001); Cx40: 878 -fold (p<0.01); Cav3.1: 11-fold (p<0.05); HCN4: 7-fold (p<0.001)].
Conclusion: The dual reporter Purkinje cell mESC reporter line permits identification and enrichment of ventricular CCS derivatives. [Supported by NYSTEM contracts #C024327 and #C028115 to GIF].