الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
 |  | from | 131 |  |  |
| | | from | 131 | | |
Abstract
Stem cells have unlimited capacity of replication and demonstrate the potential to generate any cell type such as functional CMs, which represent a promising source for regenerative therapy in various cardiovascular diseases associated with loss of cardiac cells. Several growth factors and signaling pathways such as Bone morphogenic protein (BMP), Transforming growth factor beta (TGFB/activin/Nodal), Wnt and Fibroblast growth factors (FGF), which act sequentially, individually or in synergy, are involved in early cardiac development. Therefore optimization of these signaling pathways should give rise to well-functioning CMs with proper electrophysiological response to beta adrenergic stimuli.
In the present study, various growth factors (DKK1, VEGF and bFGF) were used at particular points in time to drive the differentiation of rhesus embryonic stem cells (Lyon-ES1) into CMs. Furthermore, we evaluated the electrophysiological responses of these stem cell derived CMs to adrenergic and cholinergic stimulation. Functional characterization was carried out to validate the properties of these differentiated cells to the stimuli of the autonomic nervous system, and to ensure that the derived CMs will act alike the native myocardium when transplanted. In addition the beta adrenergic receptor expression was evaluated.
The cardiac transcription factor genes (GATA4 and Nkx2.5) and cardiac specific genes (cTNT and αMHC) showed a tendency of higher expression in the control group than in the treated one. Both groups showed well organized sarcomeric structures, which is a hallmark of functional myocytes, when immunostaining for the structural proteins, α-actinin and cTNT, was done at day 25, but the treated group showed less normalized positive percentage for α-actinin and cTNT.
After application of 1 µM isoproterenol (beta adrenergic agonist), the control group showed a positive chronotropic effect (increase in the field potential beating rate compared to its basal rate), which was significantly higher than the one observed in the treated group, when measuring the field potential of whole embryoid body and the action potential of individual CMs. In contrast, both groups showed negative chronotropic effect on application of 10µM carbachol, muscarinic agonist. These findings suggested that the beta adrenergic pathway is significantly affected in the treated group but the muscarinic pathway remains unaffected. Although there was no change in the chronotropic response of both the control group and the treated group after stimulation by forskolin and IBMX. These data suggested that the beta adrenergic signaling was intact in both groups beyond the beta adrenergic receptor.
The beta1 adrenergic receptor RNA expression of the control group was 5.14 fold more than that of the treated group, this was statistically significant. Similarly, beta 2 adrenergic receptor’s RNA expression of the control group was also higher than that of the treated one; with an approximate 0.83 fold increase, however without statistical significance. The results correlated with the expression of beta 1 adrenergic receptors immunostaining in the differentiated CMs of the control group was significantly higher than of the treated group, while there was no statistically significant difference between beta 2 adrenergic receptor expressions of the control and treated group.
To know which growth factor is responsible for these results, we performed differentiation of rhesus embryonic stem cells (Lyon-ES1) into CMs using only one growth factor (Dkk1 or VEGF) than that used in the control group, then we measured the action potential on adding 1 µM isoproterenol (beta adrenergic agonist) and there was no differences in the response of these 2 groups than the control group.
In conclusion, application of these distinct growth factors together in our specific differentiation protocol decreased both the chronotropic response of the stem cell derived CMs to the beta adrenergic stimulation, and their beta adrenergic receptor expression.