Abstract 15792: Reprogramming of Fibroblasts Into Induced Cardiac Progenitor Cells With the CRISPR-dCas9 System
Background and Objective: Currently available regenerative clinical therapies for myocardial infarction could benefit from additional resources of cardiac lineage cells. Although the CRISPR-dCas9 system allows for precise manipulation of individual loci by customized guide RNAs (gRNAs), it has not been widely used in the generation of induced cardiac progenitor cells (iCPCs). This study was designed to determine the feasibility and effectiveness of reprogramming fibroblasts into iCPCs using the dCas9-based transcription activation system.
Methods: Tail-tip fibroblasts (TTFs) were isolated from Nkx2-5RFP reporter mice. A gRNA pool targeting 17 progenitor genes was synthesized and transduced with dCas9-VP64 lentivirus into TTFs (Fig. 1A). The phenotype of iCPCs was then characterized by expression of progenitor genes and proliferation markers. Finally, the cardiac-lineage differentiation potential of iCPC was determined under defined induction mediums.
Results: Progenitor genes (Such as Gata4, Mesp1, and Isl-1) were specifically activated in TTFs after transduction with the CRISPR-dCas9 system as compared to control TTFs (with Null virus) (Fig. 1B). Sphere clusters of RFP+ iCPCs were only generated in TTFs with the CRISPR-dCas9 system (Fig. 1C). Proliferation markers (such as Ki67) were expressed in iCPCs and the cell population was expanded after serial passages. The differentiation of iCPCs into cardiomyocytes, smooth muscle cells, and endothelial cells was identified by immunostaining (Fig. 1D).
Conclusion: The CRISPR-dCas9 system is an efficient and specific way to generate iCPCs, which could provide a novel source of cells for therapy after myocardial infarction.
Author Disclosures: L. Jiang: None. J. Liang: None. W. Huang: None. W. Cai: None. C. Paul: None. Y. Wang: None.
- © 2016 by American Heart Association, Inc.