Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.

• Klíčová slova
• 2D-monolayer, cardiac spheroids, contractile properties, embryoid bodies, hPSC-derived cardiomyocytes, intracellular calcium handling, mitochondrial oxygen consumption, oxygen exposure,
• MeSH
• biologické markery MeSH
• buněčná diferenciace * MeSH
• buněčné kultury MeSH
• buněčné sféroidy MeSH
• exprese genu MeSH
• kardiomyocyty cytologie   metabolismus   MeSH
• kyslík metabolismus   MeSH
• lidé MeSH
• pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• sarkoplazmatické retikulum metabolismus   MeSH
• srdeční mitochondrie metabolismus   MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické markery MeSH
• kyslík MeSH
• vápník MeSH

Recent data on Duchenne muscular dystrophy (DMD) show myocyte progenitor's involvement in the disease pathology often leading to the DMD patient's death. The molecular mechanism underlying stem cell impairment in DMD has not been described. We created dystrophin-deficient human pluripotent stem cell (hPSC) lines by reprogramming cells from two DMD patients, and also by introducing dystrophin mutation into human embryonic stem cells via CRISPR/Cas9. While dystrophin is expressed in healthy hPSC, its deficiency in DMD hPSC lines induces the release of reactive oxygen species (ROS) through dysregulated activity of all three isoforms of nitric oxide synthase (further abrev. as, NOS). NOS-induced ROS release leads to DNA damage and genomic instability in DMD hPSC. We were able to reduce both the ROS release as well as DNA damage to the level of wild-type hPSC by inhibiting NOS activity.

• Klíčová slova
• DMD, NO synthases, ROS, dystrophin, genome stability, pluripotent stem cells,
• MeSH
• buněčné linie MeSH
• Duchennova muskulární dystrofie genetika   MeSH
• dystrofin nedostatek   genetika   MeSH
• indukované pluripotentní kmenové buňky metabolismus   patologie   MeSH
• lidé MeSH
• nestabilita genomu * MeSH
• oxidační stres MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• synthasa oxidu dusnatého, typ I metabolismus   MeSH
• synthasa oxidu dusnatého, typ II metabolismus   MeSH
• synthasa oxidu dusnatého, typ III metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dystrofin MeSH
• NOS1 protein, human MeSH Prohlížeč
• NOS2 protein, human MeSH Prohlížeč
• NOS3 protein, human MeSH Prohlížeč
• reaktivní formy kyslíku MeSH
• synthasa oxidu dusnatého, typ I MeSH
• synthasa oxidu dusnatého, typ II MeSH
• synthasa oxidu dusnatého, typ III MeSH