Heart disease often leads to cardiomyocyte death and pathologic remodelling. Heart transplantation is the usual solution, although limited by donors number and restrictive inclusion criteria. These limits prompted research into stem cell-based alternatives, restricted however by a scarce source of adult stem cells and their relatively inefficient contribution to heart regeneration. Embryonic stem cells (ESCs) retain great promise as unlimited source of pluripotent progenitors for myocardial regeneration, but their therapeutics use is still impaired by ethical concerns and incomplete understanding of factors governing cardiomyocytes differentiation. We aimed at creating ESC-derived cardiomyocytes for experimental cell transplantation therapies in mice. We treated murine ESCs (mESCs) with Triiodothyronine (T3) and/or anacardic acid (AA), a natural epigenetic drug inhibiting histone acetylases, and investigated whether and how efficiently cardiac cell differentiation occurred. For easier identification of differentiated cells, engineered mESCs expressing red fluorescent protein (RFP) under the NCX1 gene promoter, an early cardiac differentiation marker, were used. The hanging-drop embryoid body (EB) technique was adopted to reproduce in vitro an embryo-like architecture. mESC-derived RFP-positive cardiomyocytes were collected and analysed by RT-PCR, western blot, electrophysiology and FACS. Both AA and T3 promoted cardiac differentiation, anticipating EBs beating and increasing beating areas but with effects apparently mediated by non-overlapping pathways. While T3 did not affect acetylation, AA decreased lysine acetylation of histonic and non histonic proteins. RT-PCR showed decreased stemness genes expression in EBs and only AA increased expression of Nkx2.5, a cardiac differentiation key gene.Gene profiling showed that AA upregulated only genes for early cardiomyocites differentiation, generating immature cardiac cells, whereas T3 induced genes for terminal cardiac differentiation, facilitating formation of mature cardiomyocytes. These results were confirmed by electrophysiology showing increased spontaneous firing, in support of T3 inducing peacemaker-cell-like differentiation. These findings reveal a master role of multiple epigenetically controlled signals mediated by T3 and AA, and provide an alternative for potentiating production of mature and functional cardiac cells for therapeutic intervention.
Role of thyroid hormones and epigenetic drugs in cardiac differentiation of mouse embryonic stem cells
A Aiello;A Farsetti
2013
Abstract
Heart disease often leads to cardiomyocyte death and pathologic remodelling. Heart transplantation is the usual solution, although limited by donors number and restrictive inclusion criteria. These limits prompted research into stem cell-based alternatives, restricted however by a scarce source of adult stem cells and their relatively inefficient contribution to heart regeneration. Embryonic stem cells (ESCs) retain great promise as unlimited source of pluripotent progenitors for myocardial regeneration, but their therapeutics use is still impaired by ethical concerns and incomplete understanding of factors governing cardiomyocytes differentiation. We aimed at creating ESC-derived cardiomyocytes for experimental cell transplantation therapies in mice. We treated murine ESCs (mESCs) with Triiodothyronine (T3) and/or anacardic acid (AA), a natural epigenetic drug inhibiting histone acetylases, and investigated whether and how efficiently cardiac cell differentiation occurred. For easier identification of differentiated cells, engineered mESCs expressing red fluorescent protein (RFP) under the NCX1 gene promoter, an early cardiac differentiation marker, were used. The hanging-drop embryoid body (EB) technique was adopted to reproduce in vitro an embryo-like architecture. mESC-derived RFP-positive cardiomyocytes were collected and analysed by RT-PCR, western blot, electrophysiology and FACS. Both AA and T3 promoted cardiac differentiation, anticipating EBs beating and increasing beating areas but with effects apparently mediated by non-overlapping pathways. While T3 did not affect acetylation, AA decreased lysine acetylation of histonic and non histonic proteins. RT-PCR showed decreased stemness genes expression in EBs and only AA increased expression of Nkx2.5, a cardiac differentiation key gene.Gene profiling showed that AA upregulated only genes for early cardiomyocites differentiation, generating immature cardiac cells, whereas T3 induced genes for terminal cardiac differentiation, facilitating formation of mature cardiomyocytes. These results were confirmed by electrophysiology showing increased spontaneous firing, in support of T3 inducing peacemaker-cell-like differentiation. These findings reveal a master role of multiple epigenetically controlled signals mediated by T3 and AA, and provide an alternative for potentiating production of mature and functional cardiac cells for therapeutic intervention.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
