Abstract 15783: Lineage Reprogramming of Mouse Fibroblasts to Proliferative and Multipotent Induced Cardiac Progenitor Cells by Defined Factors

Several studies have reported reprogramming of fibroblasts to induced cardiomyocytes (CMs). 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. We cloned a library of 22 genes, including cardiac-related transcription factors and chromatin remodeling agents, into a doxycycline inducible lentivirus vector. Fibroblasts used for reprogramming were derived from an Nkx 2.5-EYFP cardiac progenitor reporter mouse model, which when uninfected yielded no EYFP+ cells. However, adult cardiac fibroblasts infected with all 22 factors yielded some EYFP expressing cells only after doxycycline treatment. The factor pool was subsequently reduced to 11, and eventually to 5 factors. After infection with cardiac factors, EYFP+ cells were first observed 4-6 days after doxycycline treatment. By day 20, EYFP+ cells developed into highly proliferative EYFP+ colonies of cells that, upon passaging, exhibited a high nuclear-to-cytoplasmic ratio. Transcriptome analysis and immunocytochemistry of EYFP+ cells revealed up-regulation of CPC markers including Mesp1, Mef2c, Irx4, Gata6, Tbx5, Tbx 20 accompanied by downregulation of fibroblast-specific genes such as Fsp1 and Thy1. Based on lineage specific marker expression, the EYFP+ cells could be differentiated into various cardiac lineages, including cardiomyocytes (α-MHC), smooth muscle (SM-MHC) and endothelium (CD31). Even after doxycycline withdrawal cells remained EYFP+, expressed CPC markers, and retained multipotency for over 30 passages, all indicative of stable reprogramming to an induced CPC (iCPC) state. iCPCs co-cultured with mESC-derived CMs formed intercellular gap junctions and showed synchronous beating accompanied by spontaneous calcium transients. When injected into the cardiac crescent of E8.5 mouse embryos, iCPCs migrated to the developing heart tube and differentiated into MLC2v and cardiac actin expressing CMs. Similar iCPC reprogramming was achieved using adult lung- and tail tip-derived fibroblasts. These results demonstrate the ability of inducible defined factors to reprogram mouse fibroblasts into proliferative and multipotent iCPCs.