- Email: [email protected]
Fully differentiated cardiomyocites from human amniotic fluid-derived stem cells
Abstracts
at peak exercise, representing a safer option to treat obstruction compared to Dis. (3) By shortening AP duration, all drugs may exert a significant antiarrhythmic effect in HCM patients. doi:10.1016/j.vph.2015.11.019
Mek1 inhibition in vivo mitigates progressive cardiac concentric hypertrophy promoted by activated Met receptor V. Salaa,b,, S. Galloa, S. Gattia, E. Medicoa,c, E. Vignaa,c, D. Cantarellac, L. Fontanic, M. Nataled, M. Morellob, A. Ponzettob, T. Crepaldia a Department of Oncology, University of Turin, Turin, Italy b Department of Medical Sciences, University of Turin, Turin, Italy c FPO-IRCCS, Candiolo (TO), Italy d Politecnico di Torino, Turin, Italy Objectives: The Hepatocyte Growth Factor and its tyrosine-kinase receptor c-Met are involved in many physio-pathological processes, including heart development, repair. However, few information exists on the role of Met in heart disease. Cardiac hypertrophy is a major risk factor for heart failure. Hence, its attenuation represents an important clinical goal. c-Met is a strong inducer of Ras-RafMekErk1,2 pathway. Erk1,2 signalling is pivotal in the cardiac response to stress, suggesting that its inhibition may be a good strategy to revert heart hypertrophy. Materials and methods: To investigate the effects of a sustained activation of Met signalling, we generated a transgenic mouse model with tetracycline-suppressible expression of Tpr-Met, the constitutively active form of Met, specifically to cardiac muscle by means of αMHC promoter. Results: Three weeks after the induction of activated Met, the heart of Tpr-Met mice presented a remarkable concentric hypertrophy, with no signs of congestive failure and preserved contractility. Cardiac enlargement was accompanied by upregulation of growthregulating transcription factors, immediate early genes, natriuretic peptides and cytoskeletal proteins, and by remodelling of Extracellular Matrix. Within one week, hypertrophy progressed into congestive heart failure, accompanied by cardiac cachexia. Prevention trial by suppressing activated Met showed that cardiac hypertrophy was reversible, and progression to heart failure and cachexia were prevented. Notably, i.p. treatment of Tpr-Met mice with Pimasertib, Mek1 inhibitor, from P21 to P23, mitigated the cardiac hypertrophic phenotype. Conclusions: Our results suggest that modulation of Erk1,2 signalling may constitute a new therapeutic approach for treating cardiac hypertrophies. doi:10.1016/j.vph.2015.11.020
Fully differentiated cardiomyocites from human amniotic fluid-derived stem cells M.A. D'Amicoa,, B. Ghinassia, P. Izzicupoa, D. Sirabellab, M.A. Mariggiòc, S. Guarnieric, L. Stuppiad, A. Di Baldassarreb a Department of Medicine and Aging Science, University of Chieti-Pescara, Chieti, Italy b Columbia University, New York, NY, USA c Department of Basic and Applied Medical Science, University of Chieti-Pescara, Chieti, Italy d DISPUTER, University of Chieti-Pescara, Chieti, Italy Objectives: Human amniotic fluid-derived stem cells (hAFSC) are considered a novel class of multipotent stem cells sharing characteristics of both embryonic and adult stem cells. In fact, hAFSC proliferate rapidly, are able to differentiate into cells of all the three
47
embryonic germ layers, but show low immunogenicity and do not form teratoma. It has been already reported that hAFSC can differentiate toward cardiac lineage through the embryoid body (EB) formation. However, the three-dimensional structure and high cellular heterogeneity of EB can lower cell differentiation efficiency and reproducibility, hinder direct cell differentiation control and make cell differentiation studies more difficult than in a monolayer format. The aim of this study was to fully differentiate the hAFSC in cardiomyocytes, in a specific monolayer culture. Materials and methods: hAFSC were obtained from normal amniocentesis. Cells cultured in monolayer were exposed sequentially to ascorbic acid, 5-azacytidine, BMP4, ActivinA, and VEGF. Differentiation was evaluated by monitoring the expression of CD90, as mesenchymal stem cell marker, and of Nkx2.5, Gata4, sarcomeric α-actinin (αSA), α cardiac myosin heavy chain (αMHC), cardiac T-troponin (TnT) and Connexin43 as cardiac markers. Physiological studies of intracellular calcium variation were also performed. Results: During the in vitro differentiation cells underwent toward a significant decrease of CD90 (99.3 ± 0.5% vs 4.5 ± 2.5% in hAFSC and cardiomyocytes like cells, respectively), a significant increase in Gata4 and Nkx2.5 expression (Gata4 +: 25.1 ± 5.6% vs 95.7 ± 3.1%; Nkx2.5+: 37.45 + 1.29% vs 97.26 + 1.01% in hAFSC and cardiomyocytes like cells, respectively) together with their significant nuclear translocation (Gata4 + nuclei: 9.1 ± 0.9% vs 88.5 ± 5.8%; Nkx2.5 + nuclei: 11.06 + 2.71% vs 50.7 + 3.62%, in hAFSC and cardiomyocytes like cells, respectively). After 15 days of culture in differentiation condition we obtained a homogeneous population of cadiomyocyte like cells positive for αMHC (98.7 ± 1.1%), αSA (89.5 ± 5.4%) and cTnT (95.4 ± 3.1%) and Connexin 43 (95.5 ± 4.2%). Moreover, differentiated cells showed spontaneous calcium waves and responded to KCl and, albeit sporadically, also to caffeine stimulation. Some small beating foci (about 8–10% of the plate) were also observed. Conclusions: We demonstrate that hAFSC can fully differentiate into myocytes giving rise to a homogenous population with cardiacspecific molecular and functional properties. doi:10.1016/j.vph.2015.11.021
GPER activation mitigates cardiotoxicity induced by the anticancer agent doxorubicin E.M. De Francescoa,, C. Roccab, F. Scavellob, D. Ameliob, D.C. Rigiraccioloa, A. Scarpellia, G. Mesianib, N. Amodioc, M.C. Cerrab,d, M. Maggiolinia, T. Angeloneb,d a Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende (CS), Italy b Department of Biology, Ecology and E.S., University of Calabria, Arcavacata di Rende (CS), Italy c Department of Experimental Clinical Medicine, University of Catanzaro Magna Græcia, Catanzaro, Italy d National Institute of Cardiovascular Research, Bologna, Italy Objectives: The highly effective anticancer agent doxorubicin (Dox) is a frontline drug to treat a number of cancers. However, its clinical use is often complicated by dose-limiting cardiotoxicity that causes congestive heart failure in approximately 20% of patients. At the cellular level, Doxinduced cardiomyopathy has long been associated with the generation of mitochondrial ROS and with cell death. The G-protein estrogen receptor GPER has been shown to mediate the biological actions exerted by estrogenic signaling in diverse patho-physiological conditions. GPER is widely distributed among a variety of mammalian tissues including the cardiovascular system, where it may elicit beneficial actions. In particular, GPER activation by the selective agonist G-1 was shown to reduce infarct size in isolated rat and mouse hearts subjected to ischemia/reperfusion injury. On the basis of these findings,
at peak exercise, representing a safer option to treat obstruction compared to Dis. (3) By shortening AP duration, all drugs may exert a significant antiarrhythmic effect in HCM patients. doi:10.1016/j.vph.2015.11.019
Mek1 inhibition in vivo mitigates progressive cardiac concentric hypertrophy promoted by activated Met receptor V. Salaa,b,, S. Galloa, S. Gattia, E. Medicoa,c, E. Vignaa,c, D. Cantarellac, L. Fontanic, M. Nataled, M. Morellob, A. Ponzettob, T. Crepaldia a Department of Oncology, University of Turin, Turin, Italy b Department of Medical Sciences, University of Turin, Turin, Italy c FPO-IRCCS, Candiolo (TO), Italy d Politecnico di Torino, Turin, Italy Objectives: The Hepatocyte Growth Factor and its tyrosine-kinase receptor c-Met are involved in many physio-pathological processes, including heart development, repair. However, few information exists on the role of Met in heart disease. Cardiac hypertrophy is a major risk factor for heart failure. Hence, its attenuation represents an important clinical goal. c-Met is a strong inducer of Ras-RafMekErk1,2 pathway. Erk1,2 signalling is pivotal in the cardiac response to stress, suggesting that its inhibition may be a good strategy to revert heart hypertrophy. Materials and methods: To investigate the effects of a sustained activation of Met signalling, we generated a transgenic mouse model with tetracycline-suppressible expression of Tpr-Met, the constitutively active form of Met, specifically to cardiac muscle by means of αMHC promoter. Results: Three weeks after the induction of activated Met, the heart of Tpr-Met mice presented a remarkable concentric hypertrophy, with no signs of congestive failure and preserved contractility. Cardiac enlargement was accompanied by upregulation of growthregulating transcription factors, immediate early genes, natriuretic peptides and cytoskeletal proteins, and by remodelling of Extracellular Matrix. Within one week, hypertrophy progressed into congestive heart failure, accompanied by cardiac cachexia. Prevention trial by suppressing activated Met showed that cardiac hypertrophy was reversible, and progression to heart failure and cachexia were prevented. Notably, i.p. treatment of Tpr-Met mice with Pimasertib, Mek1 inhibitor, from P21 to P23, mitigated the cardiac hypertrophic phenotype. Conclusions: Our results suggest that modulation of Erk1,2 signalling may constitute a new therapeutic approach for treating cardiac hypertrophies. doi:10.1016/j.vph.2015.11.020
Fully differentiated cardiomyocites from human amniotic fluid-derived stem cells M.A. D'Amicoa,, B. Ghinassia, P. Izzicupoa, D. Sirabellab, M.A. Mariggiòc, S. Guarnieric, L. Stuppiad, A. Di Baldassarreb a Department of Medicine and Aging Science, University of Chieti-Pescara, Chieti, Italy b Columbia University, New York, NY, USA c Department of Basic and Applied Medical Science, University of Chieti-Pescara, Chieti, Italy d DISPUTER, University of Chieti-Pescara, Chieti, Italy Objectives: Human amniotic fluid-derived stem cells (hAFSC) are considered a novel class of multipotent stem cells sharing characteristics of both embryonic and adult stem cells. In fact, hAFSC proliferate rapidly, are able to differentiate into cells of all the three
47
embryonic germ layers, but show low immunogenicity and do not form teratoma. It has been already reported that hAFSC can differentiate toward cardiac lineage through the embryoid body (EB) formation. However, the three-dimensional structure and high cellular heterogeneity of EB can lower cell differentiation efficiency and reproducibility, hinder direct cell differentiation control and make cell differentiation studies more difficult than in a monolayer format. The aim of this study was to fully differentiate the hAFSC in cardiomyocytes, in a specific monolayer culture. Materials and methods: hAFSC were obtained from normal amniocentesis. Cells cultured in monolayer were exposed sequentially to ascorbic acid, 5-azacytidine, BMP4, ActivinA, and VEGF. Differentiation was evaluated by monitoring the expression of CD90, as mesenchymal stem cell marker, and of Nkx2.5, Gata4, sarcomeric α-actinin (αSA), α cardiac myosin heavy chain (αMHC), cardiac T-troponin (TnT) and Connexin43 as cardiac markers. Physiological studies of intracellular calcium variation were also performed. Results: During the in vitro differentiation cells underwent toward a significant decrease of CD90 (99.3 ± 0.5% vs 4.5 ± 2.5% in hAFSC and cardiomyocytes like cells, respectively), a significant increase in Gata4 and Nkx2.5 expression (Gata4 +: 25.1 ± 5.6% vs 95.7 ± 3.1%; Nkx2.5+: 37.45 + 1.29% vs 97.26 + 1.01% in hAFSC and cardiomyocytes like cells, respectively) together with their significant nuclear translocation (Gata4 + nuclei: 9.1 ± 0.9% vs 88.5 ± 5.8%; Nkx2.5 + nuclei: 11.06 + 2.71% vs 50.7 + 3.62%, in hAFSC and cardiomyocytes like cells, respectively). After 15 days of culture in differentiation condition we obtained a homogeneous population of cadiomyocyte like cells positive for αMHC (98.7 ± 1.1%), αSA (89.5 ± 5.4%) and cTnT (95.4 ± 3.1%) and Connexin 43 (95.5 ± 4.2%). Moreover, differentiated cells showed spontaneous calcium waves and responded to KCl and, albeit sporadically, also to caffeine stimulation. Some small beating foci (about 8–10% of the plate) were also observed. Conclusions: We demonstrate that hAFSC can fully differentiate into myocytes giving rise to a homogenous population with cardiacspecific molecular and functional properties. doi:10.1016/j.vph.2015.11.021
GPER activation mitigates cardiotoxicity induced by the anticancer agent doxorubicin E.M. De Francescoa,, C. Roccab, F. Scavellob, D. Ameliob, D.C. Rigiraccioloa, A. Scarpellia, G. Mesianib, N. Amodioc, M.C. Cerrab,d, M. Maggiolinia, T. Angeloneb,d a Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende (CS), Italy b Department of Biology, Ecology and E.S., University of Calabria, Arcavacata di Rende (CS), Italy c Department of Experimental Clinical Medicine, University of Catanzaro Magna Græcia, Catanzaro, Italy d National Institute of Cardiovascular Research, Bologna, Italy Objectives: The highly effective anticancer agent doxorubicin (Dox) is a frontline drug to treat a number of cancers. However, its clinical use is often complicated by dose-limiting cardiotoxicity that causes congestive heart failure in approximately 20% of patients. At the cellular level, Doxinduced cardiomyopathy has long been associated with the generation of mitochondrial ROS and with cell death. The G-protein estrogen receptor GPER has been shown to mediate the biological actions exerted by estrogenic signaling in diverse patho-physiological conditions. GPER is widely distributed among a variety of mammalian tissues including the cardiovascular system, where it may elicit beneficial actions. In particular, GPER activation by the selective agonist G-1 was shown to reduce infarct size in isolated rat and mouse hearts subjected to ischemia/reperfusion injury. On the basis of these findings,