(453g) Characterization of Acellular Matrix Derived From ESC Aggregates As Bioactive Scaffolds

In recent years, embryonic stem cells (ESCs) have emerged as a powerful tool for the potential treatment of various incurable diseases. For therapeutic perspectives, mimicking stem cell niche or microenvironment, including ad equate cell-cell interactions and combination of extracellular matrix (ECM) molecules and biochemical factors, is essential for efficient pluripotent stem cell (PSC) expansion and differentiation.  Acellular matrices derived from ESC aggregates, undifferentiated or embryoid bodies (EBs), can serve as potential scaffolds providing a more controlled bioactive three-dimensional microenvironment.

In this study, different chemical acellularization methods (i.e. Triton X-100, ammonia solution, SDS, and their combinations) are compared in association with DNAse treatment.  The optimized protocol efficiently preserves ECM expression and improves DNA removal of ESC aggregates. These results highlight that the various defined structures generated from ESC aggregates could be efficiently acellularized and used as intact ECM-derived scaffolds.  Next, ECM (fibronectin, laminin, collagen type IV, and vitronectin) expression is kinetically assessed for ESC monolayer, undifferentiated aggregates, and differentiated EBs with or without acellularization. Confocal microscopy and in situ ELISA are used to characterize ECM expression qualitatively and quantitatively.  It is observed that ECM expression pattern is dynamically remodeled during expansion and differentiation. These results indicate that various culture conditions enable to generate different ESC-derived structures composed of specific ECM molecules composition.  Finally, to prove the effectiveness of such scaffolds for ESC expansion and differentiation, undifferentiated ESCs are seeded on these ESC-ECM derived scaffolds and the markers of pluripotency and lineage differentiation are kinetically monitored to measure the degree of stemness and commitment of the newly seeded cells.

In summary, these results open the way towards the development of characterized bioactive scaffolds for more rapid and homogeneous ESC expansion and specific lineage differentiation.