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.

The pea aphid, Acyrthosiphon pisum , is an important agricultural pest and an ideal model organism for various studies. Chitin synthase (CHS) catalyses chitin synthesis, a critical structural component of insect exoskeletons. Here, we identified a CHS gene from A . pisum , ApisCHS . The ApisCHS expression profiles showed that ApisCHS was expressed in various developmental stages and in all tested tissues of A . pisum , including the epidermis, embryo, gut and haemolymph. Notably, ApisCHS exhibited peak expression in the middle of each nymphal period and was extremely highly expressed in the epidermis and embryo. RNA interference (RNAi) showed that ~600 ng of dsRNA is an effective dose for gene silencing by injection for dsRNA delivery; moreover, 1200 ng·μL −1 dsRNA induced CHS gene silencing by a plant-mediated feeding approach. A 44.7% mortality rate and a 51.3% moulting rate were observed 72 h after injection of ds ApisCHS into fourth-instar nymphs, compared with the levels in the control (injected with ds GFP ). Moreover, a longer period was required for nymph development and a 44.2% deformity rate among newborn nymphs was obtained upon ingestion of ds ApisCHS . These results suggest that ApisCHS plays a critical role in nymphal growth and embryonic development in pea aphids, and is a potential target for RNAi-based aphid pest control.

The pea aphid, Acyrthosiphon pisum, is an important agricultural pest and biological model organism, and RNA interference (RNAi) is an important tool for functional genomics and for insect pest management. However, the efficiency of RNAi in pea aphids is variable, limiting its application in aphids. In this study, we present optimized conditions for inducing and increasing the gene silencing efficiency of RNAi in pea aphids. The optimal gene silencing of the target Aphunchback gene was achieved by injecting 600 ng double-stranded (ds) RNA, and the highest mRNA depletion rate (74%) was detected at 36 h after injection. Moreover, the same gene silencing conditions were used to achieve transcript silencing for nine different genes in the pea aphid, although the silencing efficiencies for the different genes varied. Furthermore, the pre-exposure of aphids to dsRNA (600 ng dsGFP) led to significant hunchback silencing following a secondary exposure to 60 ng of dshunchback, a dose which did not lead to gene silencing when independently injected. The information presented here can be exploited to develop more efficient RNAi bioassays for pea aphids, both as gene functional study tools and an insect pest control strategy.

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