Induced Expression of Endogenous CXCR4 in iPSCs by Targeted CpG Demethylation Enhances Cell Migration Toward the Ligand CXCL12
Poor homing of cells after transplantation is an unresolved common issue in cardiac cell therapies. To enhance stem cell homing, the ligand CXC motif chemokine 12 (CXCL12) and its specific receptor CXC receptor type 4 (CXCR4) have been employed as a system in this study to show that induced expression of the endogenous CXCR4 gene in mouse-induced pluripotent stem cells (iPSCs) improved the cell migration. Loci-specific epigenome editing in the form of CpG demethylation at CXCR4 promoter region of the mouse iPSCs was accomplished with CXCR4b-TAL-Tet1c, chimeric fusion proteins of the catalytic domain of ten-eleven translocation 1 (TET1) to the C-terminal end of the DNA binding domains of predesigned synthetic transcription activator-like effectors (TALEs) that recognize specific DNA sequences within the mouse CXCR4 promoter region. Infection of the mouse iPSCs with the engineered CXCR4b-TAL-Tet1c in the form of lentiviral particles induced the loci-specific CpG demethylation and subsequent activation of CXCR4 expression in mouse iPSCs. As expected, the CXCR4-overexpressing iPSCs exhibited 3.9-fold greater migration than the control iPSCs did without alteration of the stemness and activated phosphorylation of AKT significantly. These results set a sound foundation for subsequent in vivo iPSCs transplantation studies in rodent models of acute myocardial infarction and heart failure. We show that TALEs can enhance the expression of CXCR4 by CpG methylation, and may retain the stemness. Migration of iPSCs activated by CXCL12 is associated with significant phosphorylation of AKT, not ERK1/2.
KEY WORDSIPSCs (induced pluripotent stem cells) TALEs (transcription activator-like effectors) CXCR4 CpG methylation epigenome editing
CJ conducted the most of the experiments and wrote the paper. JG conceived the idea for the paper, conducted experiments of real-time PCR and cell viability, and wrote the paper with CJ; and HYC conducted experiments on the CXCR4 reporter construction and in vitro CpG methylation. YHF revised the paper critically for important intellectual content, analyzed the experiments of Figs. 1 and 2, final approvaled of the version to be published.
This work was supported in part by the National Natural Science Foundation of China [81673635 and 81100078], Scientific research project of Guangdong Provincial Administration of traditional Chinese Medicine , and Science Research Foundation of Jinan university .
Compliance with Ethical Standards
The authors declare that they have no competing interests.
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