Maximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed... Show moreMaximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to iden- tify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC) model of hypertrophic cardiomyop- athy (HCM). A simple screen revealed the collabora- tive effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the elec- trophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condi- tion, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of hap- loinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM Show less
BACKGROUND Pluripotent stem cells (PSCs) offer a new paradigm for modeling genetic cardiac diseases, but it is unclear whether mouse and human PSCs can truly model both gain- and loss-of-function... Show moreBACKGROUND Pluripotent stem cells (PSCs) offer a new paradigm for modeling genetic cardiac diseases, but it is unclear whether mouse and human PSCs can truly model both gain- and loss-of-function genetic disorders affecting the Na(+) current (I(Na)) because of the immaturity of the PSC-derived cardiomyocytes. To address this issue, we generated multiple PSC lines containing a Na(+) channel mutation causing a cardiac Na(+) channel overlap syndrome. METHOD AND RESULTS Induced PSC (iPSC) lines were generated from mice carrying the Scn5a(1798insD/+) (Scn5a-het) mutation. These mouse iPSCs, along with wild-type mouse iPSCs, were compared with the targeted mouse embryonic stem cell line used to generate the mutant mice and with the wild-type mouse embryonic stem cell line. Patch-clamp experiments showed that the Scn5a-het cardiomyocytes had a significant decrease in I(Na) density and a larger persistent I(Na) compared with Scn5a-wt cardiomyocytes. Action potential measurements showed a reduced upstroke velocity and longer action potential duration in Scn5a-het myocytes. These characteristics recapitulated findings from primary cardiomyocytes isolated directly from adult Scn5a-het mice. Finally, iPSCs were generated from a patient with the equivalent SCN5A(1795insD/+) mutation. Patch-clamp measurements on the derivative cardiomyocytes revealed changes similar to those in the mouse PSC-derived cardiomyocytes. CONCLUSION Here, we demonstrate that both embryonic stem cell- and iPSC-derived cardiomyocytes can recapitulate the characteristics of a combined gain- and loss-of-function Na(+) channel mutation and that the electrophysiological immaturity of PSC-derived cardiomyocytes does not preclude their use as an accurate model for cardiac Na(+) channel disease. Show less
More than 10 years after their first isolation, human embryonic stem cells are finally 'coming of age' in research and biotechnology applications as protocols for their differentiation and... Show moreMore than 10 years after their first isolation, human embryonic stem cells are finally 'coming of age' in research and biotechnology applications as protocols for their differentiation and undifferentiated expansion in culture become robust and scalable, and validated commercial reagents become available. Production of human cardiomyocytes is now feasible on a daily basis for many laboratories with tissue culture expertise. An additional recent surge of interest resulting from the first production of human iPSCs (induced pluripotent stem cells) from somatic cells of patients now makes these technologies of even greater importance since it is likely that (genetic) cardiac disease phenotypes can be captured in the cardiac derivatives of these cells. Although cell therapy based on replacing cardiomyocytes lost or dysfunctional owing to cardiac disease are probably as far away as ever, biotechnology and pharmaceutical applications in safety pharmacology and drug discovery will probably impact this clinical area in the very near future. In the present paper, we review the cutting edge of this exciting area of translational research. Show less
More than 10 years after their first isolation, human embryonic stem cells are finally 'coming of age' in research and biotechnology applications as protocols for their differentiation and... Show moreMore than 10 years after their first isolation, human embryonic stem cells are finally 'coming of age' in research and biotechnology applications as protocols for their differentiation and undifferentiated expansion in culture become robust and scalable, and validated commercial reagents become available. Production of human cardiomyocytes is now feasible on a daily basis for many laboratories with tissue culture expertise. An additional recent surge of interest resulting from the first production of human iPSCs (induced pluripotent stem cells) from somatic cells of patients now makes these technologies of even greater importance since it is likely that (genetic) cardiac disease phenotypes can be captured in the cardiac derivatives of these cells. Although cell therapy based on replacing cardiomyocytes lost or dysfunctional owing to cardiac disease are probably as far away as ever, biotechnology and pharmaceutical applications in safety pharmacology and drug discovery will probably impact this clinical area in the very near future. In the present paper, we review the cutting edge of this exciting area of translational research. Show less
Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. Previously we reported that maggot excretions/secretions (ES) break down Staphylococcus aureus biofilms but do... Show moreMaggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. Previously we reported that maggot excretions/secretions (ES) break down Staphylococcus aureus biofilms but do not kill the bacteria. As many antibiotics are not effective against biofilms we assessed the effect of combinations of ES and antibiotics on S. aureus biofilms and on the survival of the bacteria released from the biofilms. Effects of ES, antibiotics (vancomycin, daptomycin or clindamycin) and combinations thereof on S. aureus ATCC 29 213 biofilms and bacterial viability were determined using microtitre plates and in vitro killing assays. Vancomycin and daptomycin dose-dependently enhanced biofilm formation, whereas clindamycin reduced S. aureus biofilm size. Adding ES to antibiotic incubations caused a complete biofilm breakdown. After a lag time the bacteria derived from biofilms became susceptible to vancomycin and clindamycin, provided that the medium was refreshed. Daptomycin dose-dependently eliminated the biofilm-derived bacteria immediately. Furthermore, it was significantly more effective against bacteria derived from ES-exposed biofilms than those from control biofilms. ES did not affect the activity of the antibiotics against log-phase S. aureus. Combinations of maggot ES and antibiotics eliminate S. aureus biofilms and the bacteria derived therefrom. Show less
