Isolation of ventricular cardiomyocytes (vCMs) has been challenging due to the lack of specific surface markers. Here we show that vCMs can be purified from differentiating mouse embryonic stem cells (mESCs) using molecular beacons (MBs) targeting specific intracellular mRNAs. We designed MBs (IRX4 MBs) to target mRNA encoding Iroquois homeobox protein 4 (Irx4), a transcription factor specific for vCMs. To purify mESC vCMs, IRX4 MBs were delivered into cardiomyogenically differentiating mESCs, and IRX4 MBs-positive cells were FACS-sorted. We found that, of the cells isolated, ∼98% displayed vCM-like action potentials by electrophysiological analyses. These MB-purified vCMs continuously maintained their CM characteristics as verified by spontaneous beating, Ca2+ transient, and expression of vCM-specific proteins. Our study shows the feasibility of isolating pure vCMs via cell sorting without modifying host genes. The homogeneous and functional ventricular CMs generated via the MB-based method can be useful for disease investigation, drug discovery, and cell-based therapies.
Bibliographical noteFunding Information:
We gratefully acknowledge the Emory Children’s Pediatric Research Center flow cytometry core. This work was supported in part by the National Heart, Lung, and Blood Institute of the NIH as a Program of Excellence in Nanotechnology award (HHSN268201000043C to G.B. and Y.-S.Y.) and by grants from NIDDK (DP3DK094346 to Y.-S.Y.), NHLBI (R01HL127759 to Y.-S.Y. and R01HL088488 to M.W.), Faculty Research Assistance Program of Yonsei University College of Medicine 2015, and the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIP) (No 2015M3A9C6031514). K.B. is a recipient of an American Heart Association postdoctoral fellowship grant.
All Science Journal Classification (ASJC) codes
- Developmental Biology
- Cell Biology