Maturation of Human iPSC-Derived Cardiac Microfiber with Electrical Stimulation Device.

Here an electrical stimulation system is described for maturing microfiber-shaped cardiac tissue (cardiac microfibers, CMFs). The system enables stable culturing of CMFs with electrical stimulation by placing the tissue between electrodes. The electrical stimulation device provides an electric field covering whole CMFs within the stimulation area and can control the beating of the cardiac microfibers. In addition, CMFs under electrical stimulation with different frequencies are examined to evaluate the maturation levels by their sarcomere lengths, electrophysiological characteristics, and gene expression. Sarcomere elongation (14% increase compared to control) is observed at day 10, and a significant upregulation of electrodynamic properties such as gap junction protein alpha 1 (GJA1) and potassium inwardly rectifying channel subfamily J member 2 (KCNJ2) (maximum fourfold increase compared to control) is observed at day 30. These results suggest that electrically stimulated cultures can accelerate the maturation of microfiber-shaped cardiac tissues compared to those without electrical stimulation. This model will contribute to the pathological research of unexplained cardiac diseases and pharmacologic testing by stably constructing matured CMFs.

Adeno-Associated Virus-Encapsulated Alginate Microspheres Loaded in Collagen Gel Carriers for Localized Gene Transfer.

This work reports localized in vivo gene transfer by biodegradation of the adeno-associated virus-encapsulating alginate microspheres (AAV-AMs) loaded in collagen gel carriers. AAV-AMs are centrifugally synthesized by ejecting a mixed pre-gel solution of alginate and AAV to CaCl2 solution to form an ionically cross-linked hydrogel microsphere immediately. The AAV-AMs are able to preserve the AAV without diffusing out even after spreading them on the cells, and the AAV is released and transfected by the degradation of the alginate microsphere. In addition, AAV-AMs can be stored by cryopreservation until use. By implanting this highly convenient AAV-encapsulated hydrogel, AAV-AMs can be loaded into collagen gel carriers to fix the position of the implanted AAV-AMs and achieve localized gene transfer in vivo. In vivo experiments show that the AAV-AMs loaded in collagen gel carriers are demonstrated to release the encapsulated AAV for gene transfer in the buttocks muscles of mice. While conventional injections caused gene transfer to the entire surrounding tissue, the biodegradation of AAV-AMs shows that gene transfer is achieved locally to the muscles. This means that the proposed AAV-loaded system is shown to be a superior method for selective gene transfer.

Spatiotemporal single-cell tracking analysis in 3D tissues to reveal heterogeneous cellular response to mechanical stimuli.

Mechanical stimuli have been recognized as important for tissue maturation, homeostasis and constructing engineered three-dimensional (3D) tissues. However, we know little about the cellular mechanical response in tissues that could be considerably heterogeneous and spatiotemporally dynamic due to the complex structure of tissues. Here, we report a spatiotemporal single-cell tracking analysis of in vitro 3D tissues under mechanical stretch, to reveal the heterogeneous cellular behavior by using a developed stretch and optical live imaging system. The system could affect the cellular orientation and directly measure the distance of cells in in vitro 3D myoblast tissues (3DMTs) at the single-cell level. Moreover, we observed the spatiotemporal heterogeneous cellular locomotion and shape changes under mechanical stretch in 3DMTs. This single-cell tracking analysis can become a principal method to investigate the heterogeneous cellular response in tissues and provide insights that conventional analyses have not yet offered.