Loss of Ccbe1 affects cardiac-specification and cardiomyocyte differentiation in mouse embryonic stem cells

• DOI: 10.1371/journal.pone.0205108
• Publication Date: 2018-10-03T14:09:56Z
• Authors: Oriol Bover; Tiago Justo; Paulo N. G. Pereira; João Facucho-Oliv...; José M. Inácio; José S. Ramalho; Ibrahim J. Domian; José António Belo
• Keywords: 0301 basic medicine; Dysplasia; Science; LIM-Homeodomain Proteins; Progenitors; Mice, Transgenic; Induction; Mice; 03 medical and health sciences; Vegfc; Animals; Myocytes, Cardiac; Lymphangiogenesis; Lineages; RNA, Small Interfering; Cells, Cultured; Myocardium; Tumor Suppressor Proteins; Q; Calcium-Binding Proteins; R; Gene Expression Regulation, Developmental; Ccbe1; Cell Differentiation; Heart; Mouse Embryonic Stem Cells; 3. Good health; Gene Knockdown Techniques; Homeobox Protein Nkx-2.5; Medicine; Mutations; Research Article; Transcription Factors

Understanding the molecular pathways regulating cardiogenesis is crucial for the early diagnosis of heart diseases and improvement of cardiovascular disease. During normal mammalian cardiac development, collagen and calcium-binding EGF domain-1 (Ccbe1) is expressed in the first and second heart field progenitors as well as in the proepicardium, but its role in early cardiac commitment remains unknown. Here we demonstrate that during mouse embryonic stem cell (ESC) differentiation Ccbe1 is upregulated upon emergence of Isl1- and Nkx2.5- positive cardiac progenitors. Ccbe1 is markedly enriched in Isl1-positive cardiac progenitors isolated from ESCs differentiating in vitro or embryonic hearts developing in vivo. Disruption of Ccbe1 activity by shRNA knockdown or blockade with a neutralizing antibody results in impaired differentiation of embryonic stem cells along the cardiac mesoderm lineage resulting in a decreased expression of mature cardiomyocyte markers. In addition, knockdown of Ccbe1 leads to smaller embryoid bodies. Collectively, our results show that CCBE1 is essential for the commitment of cardiac mesoderm and consequently, for the formation of cardiac myocytes in differentiating mouse ESCs.