Le Tracking Analysis (NTA) and dot blot. Final results: In 2D culture, only DPPSC cultured within the default HS medium proliferated and showed the anticipated morphology. In 3D culture, DPPSC in SR1 medium formed spheroids of related morphology and size to that of HS medium. Drastically smaller sized spheroids were formed by DPPSC in ED-HS medium, even though DPPSC barely formed spheroids in SR2 medium. qPCR analysis showed that while expression of Oct4A gene in DPPSC cells from 2D and 3D culture (each in HS and SR1 media) was related, expression of Nanog in DPPSC spheroids in SR1 medium was significantlyhigher than the spheroids in HS medium and also the cells from 2D culture. Vesicles isolated from DPPSC spheroid in SR1 conditioned medium from Day 12 and Day 134 of culture showed sizes that fall inside the exosomal size variety, and are positive for the exosomal markers CD81, CD9 and CD63. Vesicle yield for Day 134 was higher than that of Day 12, but a bigger percentage of particles from the latter had been good for the three exosomal markers. Summary/Conclusion: 3D spheroid culture of DPPSC in SR1 medium showed improvement in pluripotency, and allows to get a serum-free culture for exosome production.PT10.Enhanced exosome secretion is essential for myeloma stem cells to survive in hypoxic situation Sayaka Nakayama, Yuki Toda, Shigekuni Hosogi and Eishi Ashihara Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto-shi, JapanIntroduction: Cancer stem cells (CSCs) with the extremely tumorigenic cell population are critically linked together with the poor prognosis of sufferers in numerous forms of cancer. In our prior study, the multiple myeloma (MM) cells which were chronically cultured in a hypoxic condition (over six months, 1 oxygen) exhibited stem cell characteristics. It suggests that MM stem cells are capable of adapting to hypoxic tension even though the adaptation mechanism remains unclear. We focused on the excessive secretion of exosomes from hypoxia-adapted MM cells (HA-MM cells). Exosomes are thought of as a garbage bin to take away unnecessary molecules from the cytoplasm to sustain cellular homeostasis, as well as a novel intercellular DNAM-1/CD226 Proteins Storage & Stability communication tool. Methods: GW4869, an inhibitor on the ceramidemediated inward budding with the multivesicular bodies for exosome biogenesis, was applied to analyse the response to a deficiency of exosome secretion from their decreased production in HA-MM cells. Results: GW4869 enhanced the rate of Annexin V constructive (apoptotic) cells and induced the expression of fragmented PARP in HA-MM cells, but not inISEV2019 ABSTRACT CD49c/Integrin alpha-3 Proteins Recombinant Proteins BOOKparental cells cultured in a normoxic situation (20 oxygen). Together with the addition of HA-MM-derived exosomes, GW4869-induced apoptosis was not attenuated. From these results, HA-MM cells are most likely to release exosomes to retain the intracellular atmosphere in a state of homeostasis, but to not receive them for autocrine signal. Hexokinase two (HK2) generates glucose-6-phosphate, that is additional metabolized by each the glycolytic pathway as well as the pentose phosphate pathway (PPP). PPP plays a major part in supplying NADPH for detoxification of intracellular reactive oxygen species (ROS). The upregulated HK2 protein expression in HA-MM cells was diminished by GW4869. With dichlorodihydrofluorescein staining assay, GW4869 elevated intracellular ROS production in HA-MM cells. Thus, the failure of exosome secretion could possibly alter the energy metabolism top to ROSassociated apoptosis.