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

Abstract

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.

CCBE1 in Cardiac Development and Disease

“…In agreement with a possible role during cardiogenesis in mice, disruption of normal CCBE1 activity by shRNA knockdown (KD) or by a blocking antibody in differentiating mouse embryonic stem cells (mESCs) in vitro, results in a strong decrease in the expression of several cardiac lineage markers without affecting the pan-mesoderm marker BRACHYURY (Bover et al, 2018). Moreover, this in vitro model for cardiac differentiation demonstrated that, as occurs during mouse and chick cardiac development, high Ccbe1 expression correlates with the onset of cardiac specification, as observed in SHF and PE cardiac progenitors (Facucho-Oliveira et al, 2011;Furtado et al, 2014;Bover et al, 2018). In addition, Ccbe1 KD resulted in an impairment of embryoid body (EB) growth caused by reduced cell proliferation and a relative increase in cell death, especially from day 4 onwards (Bover et al, 2018).…”

“…Moreover, this in vitro model for cardiac differentiation demonstrated that, as occurs during mouse and chick cardiac development, high Ccbe1 expression correlates with the onset of cardiac specification, as observed in SHF and PE cardiac progenitors (Facucho-Oliveira et al, 2011;Furtado et al, 2014;Bover et al, 2018). In addition, Ccbe1 KD resulted in an impairment of embryoid body (EB) growth caused by reduced cell proliferation and a relative increase in cell death, especially from day 4 onwards (Bover et al, 2018). This cardiac fate during mESCs differentiation is EB size, growth factor signaling and ECM proteins niche dependent, but in particular, how mESCs interact with this developmental niche (Czyz and Wobus, 2001;Bratt-leal et al, 2009;Goh et al, 2013;Higuchi et al, 2013;Taylor-Weiner et al, 2013;Zeng et al, 2013).…”

“…This cardiac fate during mESCs differentiation is EB size, growth factor signaling and ECM proteins niche dependent, but in particular, how mESCs interact with this developmental niche ( Czyz and Wobus, 2001 ; Bratt-leal et al, 2009 ; Goh et al, 2013 ; Higuchi et al, 2013 ; Taylor-Weiner et al, 2013 ; Zeng et al, 2013 ). However, after blocking CCBE1 activity using anti-CCBE1 antibodies, the expression of Mesp1 was also affected even before the difference in size could be detected, indicating that the normal cardiac mesoderm commitment is dependent on normal levels of CCBE1 and not a consequence of the reduced size of the EBs ( Bover et al, 2018 ). This showed that CCBE1 is important and specific for the proper cardiac specification in vitro .…”

“…Mutations in CCBE1 were identified in human patients with Hennekam syndrome (HS), a rare autosomal recessive disorder of lymphatic development that presents a wide variety of symptoms including primary lymphedema, and heart defects ( Van Balkom et al, 2002 ; Alders et al, 2009 ; Connell et al, 2010 ; Connell et al, 2012 ; Alders et al, 2013 ; Shah et al, 2013 ). Most recently, CCBE1 has been reported as an important protein in cardiovascular development ( Furtado et al, 2014 ; Bonet et al, 2018 ; Bover et al, 2018 ). In this review, we focus on the current knowledge of the role of CCBE1 in the context of heart development and the future perspectives regarding its implications for translational/regenerative medicine.…”

DAND5 Inactivation Enhances Cardiac Differentiation in Mouse Embryonic Stem Cells

“…Undifferentiated Dand5 KO and the respective E14 WT control mESC lines were used to test cells pluripotency potential and spontaneous differentiation by the hanging droplet method (Bover et al, 2018). Briefly, cells were dissociated into a single cell suspension and resuspended in fresh mESC medium without LIF.…”