Abstract 19007: Lineage Reprogramming of Mouse Fibroblasts to Induced Cardiac Progenitor Cells by Defined Factors

Abstract

Several studies have reported reprogramming of mouse fibroblasts to induced cardiomyocytes (iCMs). However, reprogramming to cardiac progenitor cells (CPCs), which may be more favorable for cell therapy because of their proliferative properties and multipotency, remains to be accomplished. This study aims to reprogram mouse fibroblasts to induced CPCs (iCPCs) by transcription factor (TF)-mediated reprogramming. We have cloned a library of 25 genes including cardiac-related TFs and chromatin remodeling agents into a novel, inducible piggyBac transposon vector. Fibroblasts were derived from a Nkx 2.5- YFP cardiac progenitor reporter mouse model. We first confirmed efficient transcription/translation of our piggyBac TF library. Next, as a control experiment neonatal cardiac fibroblasts were transfected with Gata4, Mef2c, Tbx5 or Gata4, Mef2c, Tbx5, Hand2 (published factors) and monitored for appearance of beating cells. Spontaneously beating, YFP- iCMs were observed only after application of doxycycline (25 days) for both combinations of factors, demonstrating a robust reprogramming system. We then transfected neonatal cardiac fibroblasts with entire library of 25 factors and observed YFP+ cells on doxycycline treatment. The TF pool was subsequently narrowed down to 11 essential factors. On transfection with 11 factors, YFP+ cells were first observed 4 days after doxycycline treatment. By day 20 these YFP+ cells developed into two-dimensional, highly proliferative YFP+ colonies of cells that lost their parental fibroblast morphology and exhibited a high nucleus to cytoplasmic ratio. Similar reprogramming was later accomplished using adult cardiac and adult lung fibroblasts. Untransfected/iPSC factors transfected control fibroblasts yielded no beating or YFP+ cells. qPCR analysis of these YFP+ colonies revealed an upregulation of CPC markers including Mesp1, Mef2c, Irx4 accompanied with down regulation of fibroblast specific genes such as Fsp1, consistent with reprogramming towards an iCPC state. These results support the ability of inducible piggBac-based TFs to reprogram fibroblasts to iCPCs. Current experiments are focused on optimizing culture conditions to maintain iCPCs in a proliferative state and defining their potency.