ABSTRACT
Objective To investigate the effects of oncostatin M(OSM) on differentiation of murine induced pluri-potent stem cells(miPSCs) into cardiomyocytes in vitro. Methods Using the embryoid bodies differentiation meth-od,OSM was added during early-phase and midphase(day 0-6) of miPSCs differentiation for 15 days.Semi-quan-titative RT-PCR,Western blot and immunofluorescence staining were used to detect the expression levels of myo-cardial cells-specific markers. Results The best concentration of OSM to induce the cardiac differentiation of miP-SCs was 20 pmol/L and cardiac troponin-T(cTnT) was expressed 7-fold higher in OSM-treated compared with vehi-cle control-treated embryoid bodies at day 15 post-differentiation. Cardiac-specific gene and protein expression of cTnT and Gata4 demonstrated that the OSM-treated embryoid bodies expressed cardiac-specific gene(Tnnt2, Nkx2.5,MLC2a,MLC2v,GATA4 and GATA6) and protein (cTnI,Gata4) at levels that were higher than those of the vehicle-control group after induced for 15 days. Furthermore, Immunostaining of the cardiac-specific protein markers cTnT showed that the miPSCs expressed cTnT across all groups, and exhibited a markedly increased of cTnT-positive cells in OSM-treated cells. Conclusions OSM promotes the differentiation of murine induced pluripo-tent stem cells into cardiomyocytes, and it could serve as a valuable strategy for myocardiac differentiation of miPSCs in vitro.
ABSTRACT
Objective To investigate the effects of oncostatin M(OSM) on differentiation of murine induced pluri-potent stem cells(miPSCs) into cardiomyocytes in vitro. Methods Using the embryoid bodies differentiation meth-od,OSM was added during early-phase and midphase(day 0-6) of miPSCs differentiation for 15 days.Semi-quan-titative RT-PCR,Western blot and immunofluorescence staining were used to detect the expression levels of myo-cardial cells-specific markers. Results The best concentration of OSM to induce the cardiac differentiation of miP-SCs was 20 pmol/L and cardiac troponin-T(cTnT) was expressed 7-fold higher in OSM-treated compared with vehi-cle control-treated embryoid bodies at day 15 post-differentiation. Cardiac-specific gene and protein expression of cTnT and Gata4 demonstrated that the OSM-treated embryoid bodies expressed cardiac-specific gene(Tnnt2, Nkx2.5,MLC2a,MLC2v,GATA4 and GATA6) and protein (cTnI,Gata4) at levels that were higher than those of the vehicle-control group after induced for 15 days. Furthermore, Immunostaining of the cardiac-specific protein markers cTnT showed that the miPSCs expressed cTnT across all groups, and exhibited a markedly increased of cTnT-positive cells in OSM-treated cells. Conclusions OSM promotes the differentiation of murine induced pluripo-tent stem cells into cardiomyocytes, and it could serve as a valuable strategy for myocardiac differentiation of miPSCs in vitro.
ABSTRACT
The effects of Zn2+ on spontaneous glutamate and GABA release were tested in mechanically dissociated rat CA3 pyramidal neurons which retained functional presynaptic nerve terminals. The spontaneous miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs, respectively) were pharmacologically isolated and recorded using whole-cell patch clamp technique under voltage-clamp conditions. Zn2+ at a lower concentration (30 micrometer) increased GABAergic mIPSC frequency without affecting mIPSC amplitude, but it decreased both mIPSC frequency and amplitude at higher concentrations (> or =300 micrometer). In contrast, Zn2+ (3 to 100 micrometer) did not affect glutamatergic mEPSCs, although it slightly decreased both mIPSC frequency and amplitude at 300 micrometer concentration. Facilitatory effect of Zn2+ on GABAergic mIPSC frequency was occluded either in Ca2+ -free external solution or in the presence of 100 micrometer 4-aminopyridine, a non-selective K+ channel blocker. The results suggest that Zn2+ at lower concentrations depolarizes GABAergic nerve terminals by blocking K+ channels and increases the probability of spontaneous GABA release. This Zn2+ -mediated modulation of spontaneous GABAergic transmission is likely to play an important role in the regulation of neuronal excitability within the hippocampal CA3 area.
Subject(s)
Animals , Rats , 4-Aminopyridine , gamma-Aminobutyric Acid , Glutamic Acid , Hippocampus , Inhibitory Postsynaptic Potentials , Neurons , ZincABSTRACT
Cholinergic modulation of GABAergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) by the activation of muscarine receptors was investigated in mechanically dissociated rat nucleus basalis of the Meynert neurons using the conventional whole-cell patch recording configuration. Muscarine (10microM) reversibly and concentration-dependently decreased mIPSC frequency without affecting the current amplitude distribution. Muscarine action on GABAergic mIPSCs was completely blocked by 1microM methoctramine, a selective M2 receptor antagonist, but not by 1microM pirenzepine, a selective M1 receptor antagonist. NEM (10microM), a G-protein uncoupler, attenuated the inhibitory action of muscarine on GABAergic mIPSC frequency. Muscarine still could decrease GABAergic mIPSC frequency even in the Ca2+-free external solution. However, the inhibitory action of muscarine on GABAergic mIPSCs was completely occluded in the presence of forskolin. The results suggest that muscarine acts presynaptically and reduces the probability of spontaneous GABA release, and that such muscarine-induced inhibitory action seems to be mediated by G-protein-coupled M2 receptors, via the reduction of cAMP production. Accordingly, M2 receptor-mediated disinhibition of nBM neurons might play one of important roles in the regulation of cholinergic outputs from nBM neurons as well as the excitability of nBM neurons themselves.