Background: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have tremendous promise for application in cardiac regeneration, but their translational potential is limited by an immature phenotype. We hypothesized that large-scale manufacturing of mature hPSC-CMs could be achieved via culture on polydimethylsiloxane (PDMS) lined roller bottles and that the transplantation of these cells would mediate better structural and functional outcomes than with conventional immature hPSC-CM populations. Methods: We comprehensively phenotyped hPSC-CMs after in vitro maturation for 20 and 40 days on either PDMS or standard tissue culture plastic (TCP) substrates. All hPSC-CMs were generated using a transgenic hPSC line that stably expressed a voltage-sensitive fluorescent reporter to facilitate in vitro and in vivo electrophysiological studies, and cardiomyocyte populations were also analyzed in vitro by immunocytochemistry, ultrastructure and fluorescent calcium imaging, as well as bulk and single-cell transcriptomics. We next compared outcomes after the transplantation of these populations into a guinea pig model of myocardial infarction (MI) using endpoints including histology, optical mapping of graft- and host-derived action potentials, echocardiography, and telemetric electrocardiographic (ECG) monitoring. Results: We demonstrated the economic generation of >1x10 8 mature hPSC-CMs per PDMS-lined roller bottle. Compared to their counterparts generated on TCP substrates, PDMS-matured hPSC-CMs exhibited increased cardiac gene expression and more mature structural and functional properties in vitro. More importantly, intra-cardiac grafts formed with PDMS-matured myocytes showed greatly enhanced structure and alignment, better host-graft electromechanical integration, less pro-arrhythmic behavior, and greater beneficial effects on contractile function. Conclusions: In summary, we describe practical methods for the scaled generation of mature hPSC-CMs and provide the first evidence that the transplantation of more mature cardiomyocytes yields better outcomes in vivo.

BackgroundHigh titers of lentiviral vectors are required for the efficient transduction of a gene of interest. During preparation of lentiviral the vectors, the protein of interest is inevitably expressed in the viral vector-producing cells. This expression may affect the production of the lentiviral vector.MethodsWe prepared lentiviral vectors expressing inwardly rectifying potassium channel (Lv-Kir2.1), its dominant-negative form (Lv-Kir-DN), and other K+ channels, using the ubiquitously active β-actin and neuron-specific synapsin I promoters.ResultsThe titer of Lv-Kir-DN was higher than that of Lv-Kir2.1, suggesting a negative effect of induced K+ currents on viral titer. We then blocked Kir2.1 currents with the selective blocker Ba2+ during Lv-Kir2.1 production, and obtained about a 5-fold increase in the titer. Higher extracellular K+ concentrations increased the titer of Lv-Kir2.1 about 9-fold. With a synapsin I promoter Ba2+ increased the titer because of the moderate expression of Kir2.1 channel. Channel blockade also increased the titers of the lentivirus expressing Kv1.4 and TREK channels, but not HERG. The increase in titer correlated with the K+ currents generated by the channels expressed.ConclusionIn the production of lentivirus expressing K+ channels, titers are increased by blocking K+ currents in the virus-producing cells. This identifies a crucial issue in the production of viruses expressing membrane channels, and should facilitate basic and gene therapeutic research on channelopathies.

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are synthesized from dietary carbohydrates by colonic bacterial fermentation. These SCFAs supply energy, suppress cancer, and affect ion transport. However, their roles in ion transport and regulation in the intracellular environment remain unknown. In order to elucidate the roles of SCFAs, we measured short-circuit currents (ISC) and performed RT-PCR and immunohistochemical analyses of ion transporters in rat rectal colon. The application of 30 mM butyrate shifted ISC in a negative direction, but did not attenuate the activity of epithelial Na+ channels (ENaC). The application of bumetanide, a Na+-K+-2Cl− cotransporter inhibitor, to the basolateral side reduced the negative ISC shift induced by butyrate. The application of XE991, a KCNQ-type K+ channel inhibitor, to the apical side decreased the ISC shift induced by butyrate in a dose-dependent manner. The ISC shift was independent of HCO3− and insensitive to ibuprofen, an SMCT1 inhibitor. The mucosa from rat rectal colon expressed mRNAs of H+-coupled monocarboxylate transporters (MCT1, MCT4, and MCT5, also referred to as SLC16A1, SLC16A3, and SLC16A4, respectively). RT-PCR and immunofluorescence analyses demonstrated that KCNQ2 and KCNQ4 localized to the apical membrane of surface cells in rat rectal colon. These results indicate that butyrate, which may be transported by H+-coupled monocarboxylate transporters, activates K+ secretion through KCNQ-type K+ channels on the apical membrane in rat rectal colon. KCNQ-type K+ channels may play a role in intestinal secretion and defense mechanisms in the gastrointestinal tract.

The TREK-1 channel, the TWIK-1-related potassium (K+) channel, is a member of a family of 2-pore-domain K+ (K2P) channels, through which background or leak K+ currents occur. An interesting feature of the TREK-1 channel is the run-up of current: i.e. the current through TREK-1 channels spontaneously increases within several minutes of the formation of the whole-cell configuration. To investigate whether intracellular transport is involved in the run-up, we established 293T cell lines stably expressing the TREK-1c channel (K2P2.1) and examined the effects of inhibitors of membrane protein transport, N-methylmaleimide (NEM), brefeldin-A, and an endocytosis inhibitor, pitstop2, on the run-up. The results showing that NEM and brefeldin-A inhibited and pitstop2 facilitated the run-up suggest the involvement of intracellular protein transport. Correspondingly, in cells stably expressing the mCherry-TREK-1 fusion protein, NEM decreased and pitstop2 increased the cell surface localization of the fusion protein. Furthermore, the run-up was inhibited by the intracellular application of a peptide of the C-terminal fragment TREK335–360, corresponding to the interaction site with microtubule-associated protein 2 (Mtap2). This peptide also inhibited the co-immunoprecipitation of Mtap2 with anti-mCherry antibody. The extracellular application of an ezrin inhibitor (NSC668394) also suppressed the run-up and surface localization of the fusion protein. The co-application of these inhibitors abolished the TREK-1c current, suggesting that the additive effects of ezrin and Mtap2 enhance the surface expression of TREK-1c channels and the run-up. These findings clearly showed the involvement of intracellular transport in TREK-1c current run-up and its mechanism.

The pancreatic duct epithelium secretes the HCO3−-rich pancreatic juice. The HCO3− transport across the luminal membrane has been proposed to be mediated by SLC26A Cl−–HCO3− exchangers. To examine the electrophysiological properties of Cl−–HCO3− exchangers, we directly measured HCO3− conductance in the luminal membrane of the interlobular pancreatic duct cells from guinea pigs using an inside-out patch-clamp technique. Intracellular HCO3− increased the HCO3− conductance with a half-maximal effective concentration value of approximately 30 mM. The selectivity sequence based on permeability ratios was SCN− (1.4) > Cl− (1.2) = gluconate (1.1) = I− (1.1) = HCO3− (1.0) > methanesulfonate (0.6). The sequence of the relative conductance was HCO3− (1.0) > SCN− (0.7) = I− (0.7) > Cl− (0.5) = gluconate (0.4) > methanesulfonate (0.2). The current dependent on intracellular HCO3− was reduced by replacement of extracellular Cl− with gluconate or by H2DIDS, an inhibitor of Cl−–HCO3− exchangers. RT-PCR analysis revealed that the interlobular and main ducts expressed all SLC26A family members except Slc26a5 and Slc26a8. SLC26A1, SLC26A4, SLC26A6, and SLC26A10 were found to be localized to the luminal membrane of the guinea pig pancreatic duct by immunohistochemistry. These results demonstrate that these SLC26A Cl−–HCO3− exchangers may mediate the electrogenic HCO3− transport through the luminal membrane and may be involved in pancreatic secretion in guinea pig ducts.