Induction of Cardiogenesis in Embryonic Stem Cells via Downregulation of Notch1 Signaling

Abstract

Abstract-Embryonic stem cells represent an attractive source of cardiomyocytes for cell-replacement therapies. However, before embryonic stem cells can be successfully used for the treatment of cardiac diseases, the precise molecular mechanisms that underlie their cardiogenic differentiation must be identified. A network of intrinsic and extrinsic factors regulates embryonic stem cell self-renewal and differentiation into a variety of different cell lineages. Here, we show that Notch signaling takes place in some but not all embryonic stem cells and that the Notch pathway is shut down during the course of differentiation concomitantly with downregulation of Notch receptor and ligand expression. Moreover, gain-and loss-of-function experiments for Notch signaling components show that this pathway is a crucial regulator of cardiomyocyte differentiation within ES cells. Differentiation of ES cells into cardiomyocytes is favored by inactivation of the Notch1 receptor, whereas endogenous Notch signaling promotes differentiation of ES cells into the neuronal lineage. We conclude that Notch signaling influences the cell fate decision between mesodermal and the neuroectodermal cell fates during embryonic stem cell differentiation. These findings should help to optimize the production of specific cell types via modulation of the Notch pathways and, in particular, to improve the production of embryonic stem cell-derived cardiomyocytes. (Circ Res. 2006;98:1471-1478.)Key Words: Notch Ⅲ embryonic stem cells Ⅲ cardiomyogenesis Ⅲ differentiation Ⅲ gene targeting H eart failure has become the leading cause of death in developed countries. Hundreds of thousands of new cases are diagnosed each year, and despite a large battery of pharmacological agents, heart transplant remains the ultimate therapy for patients with end-stage heart failure. However, the request for organs far exceeds the number of potential donors. As an alternative approach, the regeneration of the myocardium via controlled differentiation of cardiomyocyte progenitors is receiving much attention. Embryonic stem (ES) cells demonstrate several characteristics that suggest that they might serve as a source of cells for the therapeutic regeneration of the heart. First, ES cells can be readily isolated from the inner cell mass of the blastocyst 1,2 and subsequently maintained indefinitely in vitro. [3][4][5] Second, ES cells are totipotent and can be induced to differentiate into a variety of cell types including cardiac myocytes. 6 Third, the recent generation of human ES cell lines has brought further support to the concept of regenerative medicine based on the controlled differentiation of ES cells to replace lost cells in damaged organs. 7 Nevertheless, because of their totipotency, ES cells could paradoxically represent a possible risk of producing teratomas following transfer in vivo. Therefore, knowledge of the precise control of the necessary differentiation processes will be required before ES cells can be used in therapy.The most commonly used method to indu...

Notch1 signaling regulates chondrogenic lineage determination through Sox9 activation

“…26 Vice versa, blockage of Notch signaling in RBP-J-deficient ES cells results in enhanced mesodermal and cardiogenic specification 34 and expression of a dominant negative mutant of RBP-J, incapable of DNA binding, forces cardiogenesis. 29 Furthermore, in the presence of appropriate other signals that support neuronal differentiation, Notch signaling drives ESCs towards the neuro-ectodermal lineages. 35 Within the ectoderm, Notch signaling has a critical role in neural crest formation.…”

NOTCH activation interferes with cell fate specification in the gastrulating mouse embryo

“…Inducible activation of NICD in mESCs cultured under mesoderm differentiation conditions leads to a reduction of endothelial, haematopoietic and cardiac differentiation and favours the generation of smooth muscle cells (SMC) (Nemir et al, 2006;Schroeder et al, 2003Schroeder et al, , 2006. Activation of the NOTCH pathway in human mesenchymal or ESCs similarly induces SMC differentiation (Kurpinski et al, 2010).…”

“…NOTCH is dispensable for mESC and hESC self-renewal. Notch1 and Rbpj null mESCs can be established and maintained normally (Nemir et al, 2006;Schroeder et al, 2003) and blockade of the NOTCH pathway in hESCs by using a γ-SECRETASE inhibitor or a dominant negative form of MAML seems to enhance the growth of undifferentiated cells and to prevent their spontaneous differentiation (Noggle et al, 2006;Yu et al, 2008). However, canonical NOTCH signalling has been shown to regulate lineage commitment when ESCs are induced to differentiate.…”

“…Constitutive expression of NICD in mESCs or treatment of hESCs with DLL1-expressing feeders favours the acquisition of a neurectodermal fate (Das et al, 2010;Kurpinski et al, 2010;Lowell et al, 2006). By contrast, inactivation of the NOTCH pathway with a γ-SECRETASE inhibitor or via genetic deletion of Notch1 and Rbpj accelerates ESC differentiation towards mesoderm, and enhances their cardiogenic potential (Jang et al, 2008;Nemir et al, 2006;Schroeder et al, 2003). Further revealing the complexity of NOTCH signalling, ligand-dependent cell type specification was observed when mESCs were exposed to JAG1 or DLL4 (Ramasamy and Lenka, 2010).…”

Voie de signalisation Notch et développement précoce des mammifères

“…Les défauts les plus précoces observés chez ces embryons mutants concernent la mise en place de l'asymétrie droite-gauche suite à des anomalies d'expression du gène Nodal -une des cibles directes de la voie Notch -dans le noeud vers le 8 e jour de développement [6]. L'absence de défauts avant et/ou pendant la gastrulation chez ces mutants contraste avec des données récentes qui montrent que, dans différents modèles de différenciation in vitro de cellules souches embryonnaires murines et humaines (cellules ES qui dérivent de la MCI du blastocyste), la voie Notch joue un rôle essentiel dans le choix entre diffé-renciation neuronale et différenciation mésodermique [7,8]. Ainsi, l'absence de défauts importants de spécifica-tion des feuillets embryonnaires chez les embryons mutants montre que le rôle de la voie Notch dans ce processus est plus critique ex vivo qu'in vivo.…”