A novel approach for the endothelialization of xenogeneic decellularized vascular tissues by human cells utilizing surface modification and dynamic culture.

Decellularized xenogeneic vascular grafts can be used in revascularization surgeries. We have developed decellularization methods using high hydrostatic pressure (HHP), which preserves the extracellular structure. Here, we attempted ex vivo endothelialization of HHP-decellularized xenogeneic tissues using human endothelial cells (ECs) to prevent clot formation against human blood. Slices of porcine aortic endothelium were decellularized using HHP and coated with gelatin. Human umbilical vein ECs were directly seeded and cultured under dynamic flow or static conditions for 14 days. Dynamic flow cultures tend to demonstrate higher cell coverage. We then coated the tissues with the E8 fragment of human laminin-411 (hL411), which has high affinity for ECs, and found that Dynamic/hL411showed high area coverage, almost reaching 100% (Dynamic/Gelatin vs Dynamic/hL411; 58.7 ± 11.4 vs 97.5 ± 1.9%, P = 0.0017). Immunostaining revealed sufficient endothelial cell coverage as a single cell layer in Dynamic/hL411. A clot formation assay using human whole blood showed low clot formation in Dynamic/hL411, almost similar to that in the negative control, polytetrafluoroethylene. Surface modification of HHP-decellularized xenogeneic endothelial tissues combined with dynamic culture achieved sufficient ex vivo endothelialization along with prevention of clot formation, indicating their potential for clinical use as vascular grafts in the future.

Novel device prototyping for endoscopic cell sheet transplantation using a three-dimensional printed simulator.

INTRODUCTION: Considering higher risks of candidates for cardiac regenerative therapy with compromised cardiac function, it is anticipated to develop less invasive surgical procedures. In the present study, we aimed to develop a prototype of totally endoscopic cell sheet delivery device and evaluate the surgical technique for epicardial cell sheet placement using three-dimensional (3D) printed simulators based on human computed tomography data. METHODS: We designed an endoscopic cell sheet delivery device with outer and inner frame with self-expandable applicator which can be opened in thoracic cavity. We launched spout line to provide liquids on the applicator surface and tension line to gently bend the applicator dorsally. We prepared human mesenchymal stem cell (MSC) sheets and compared wet/dry conditions of 3D printed heart/porcine heart and applicator to identify suitable conditions for cell sheet transplantation. Finally we validated the feasibility of endoscopic transplantation to anterior and lateral wall of left ventricle using 3D printed simulators. RESULTS: Moist condition of both 3D printed heart/porcine heart surface and applicator at transplantation yielded highest successful rate (100%, p = 0.0197). For both endoscopic transplantation sites, MSC sheets were successfully deployed. The procedure duration was 157 ± 23 s for anterior wall and 123 ± 13 s for the lateral wall in average, respectively. CONCLUSIONS: We developed a novel prototype of endoscopic cell sheet delivery device for minimally-invasive cardiac regenerative therapy utilizing a 3D printed simulator. The commercialization of the prototype may provide a safe minimally-invasive method to deliver potential cardiac regenerative therapy in the future.

Tumor Mesenchymal Stromal Cells Regulate Cell Migration of Atypical Teratoid Rhabdoid Tumor through Exosome-Mediated miR155/SMARCA4 Pathway.

Atypical teratoid/rhabdoid tumor (ATRT) is a rare pediatric brain tumor with extremely high aggressiveness and poor prognosis. The tumor microenvironment is regulated by a complex interaction among distinct cell types, yet the crosstalk between tumor-associated mesenchymal stem cells (tMSCs) and naïve ATRT cells are unclear. In this study, we sought to identify the secretory factor(s) that is responsible for the tMSC-mediated regulation of ATRT migration. Comparing with ATRT cell alone, co-culture of tMSCs or addition of its conditioned medium (tMSC-CM) promoted the migration of ATRT, and this effect could be abrogated by exosome release inhibitor GW4869. The exosomes in tMSC-CM were detected by transmission electron microscope and flow cytometry. ATRT naïve cell-derived conditioned media (ATRT-CM) also enhanced the exosome secretion from tMSCs, indicating the interplay between ATRT cells and tMSCs. Microarray analysis revealed that, compared with that in bone marrow-derived MSCs, microRNA155 is the most upregulated microRNA in the tMSC-CM. Tracing the PK67-labeled exosomes secreted from tMSCs confirmed their incorporation into naïve ATRT cells. After entering ATRT cells, miR155 promoted ATRT cell migration by directly targeting SMARCA4. Knockdown of SMARCA4 mimicked the miR155-driven ATRT cell migration, whereas SMARCA4 overexpression or the delivery of exosomes with miR155 knockdown suppressed the migration. Furthermore, abrogation of exosome release with GW4869 reduced the tumorigenesis of the xenograft containing naïve ATRT cells and tMSCs in immunocompromised recipients. In conclusion, our data have demonstrated that tMSCs secreted miR155-enriched exosomes, and the exosome incorporation and miR155 delivery further promoted migration in ATRT cells via a SMARCA4-dependent mechanism.