Abstract 13281: In vivo Reprogramming of Mouse Fibroblasts Into Cardiac Progenitor Cells for Myocardial Infarction Treatment

Abstract

Introduction: Our previous study demonstrated that fibroblasts can be reprogrammed into induced cardiovascular progenitor cells (iCPCs) by a CRISPR activation approach. However, it is still unknown whether in vivo iCPC reprogramming can be achieved by direct activation of endogenous genes. Hypothesis: Direct activation of endogenous loci by using CRISPR tools targeting genomic DNA can fulfill in vivo iCPC reprogramming to repair infarcted hearts. Methods: A new fibroblast-specific CRISPRa model (Col1a2-cre/ERT;CAG-cas9) was generated. SgRNAs targeting the core promoter region of Gata4, Isl1, Nkx2-5, Baf60c, or Tbx5 were locally introduced through an AAV system in MI mice that were created by permanent LAD ligation. Heart function was measured by echocardiography and tissue sections were harvested for immunostaining. Cardiovascular cells derived from iCPCs were determined by immunostaining, ex vivo characterization, and electrophysiological assay. Results: The CRISPR system was activated in ~50% of fibroblasts after tamoxifen induction. The lineage conversion into iCPCs was identified by the Nkx2.5-eGFP reporter. eGFP+ cells were expressing proliferative markers (Ki67 and Pcna) and aggregated into spheres during suspension culture after sorting. Progenitor markers (Flk1, Ssea1, and Pdgf-α) were co-expressed in eGPF+ cells. The ejection fraction and scar formation of infarcted hearts were improved after injection of sgRNA-AAVs for 4 weeks. The engrafted iCPCs were identified by the lineage-tracing reporter and differentiated into cTnT+ cardiomyocytes, α-SMA+ smooth muscle cells, or CD31+ endothelial cells in infarcted hearts. Conclusions: Activation of endogenous loci enables in vivo iCPC reprogramming for cardiac regenerative medicine.