(149b) Microenvironment Regulation of Tissue Development From Pluripotent Stem Cells Post-Cryopreservation

Neural progenitor cells (NPCs) are usually derived from pluripotent stem cells (PSCs) through the formation of embryonic bodies (EBs), a 3-D microtissue-like structure mimicking embryonic development. PSCs could also be expanded as microtissue-like aggregates in suspension culture, which enables the large scale production in stirred bioreactors. The cryo-banking of PSCs and committed EBs is required to meet the demand of large amount of functional neural populations in view of potential clinical application for the treatment of various neurological disorders.

In this study, it is hypothesized that the microenvironment (including biochemical factors and cell configuration) of undifferentiated PSC aggregates and EBs affects their recovery and neural differentiation post-cryopreservation. To assess the influence of PSC structural environment on cell recovery, intact microtissues from PSCs and EBs (I-P and I-E) were compared to dissociated single cells (D-P and D-E). The results showed that I-P and I-E recovered better after thawing compared to D-P and D-E respectively, due to the preserved extracellular matrix (ECM) and cell organization. Moreover, the F-actin organization was maintained in I-E group while it was depolymerized in D-E group. Reactive oxygen species (ROS) and caspase expression were also investigated under various conditions. Small aggregates generated significantly less ROS and caspases compared to large ones. In the same vein, mesenchymal stem cell secretome, known to bear broad spectrum of protective factors, enhance EB recovery through anti-oxidative and anti-apoptotic effects. Finally, I-P maintained the pluripotent marker expression, and the ability to differentiate into three-germ layers. I-E and D-E showed the enhanced neuronal differentiation and D-E further enhanced astrocyte differentiation.

In summary, modulation of structural and biochemical environment is essential to promote efficient recovery and minimize ROS-mediated apoptosis for PSC tissue development. The structural microenvironment also sustained PSC differentiation potential and regulated the neural commitment post-cryopreservation.