Influence of scaffold physical properties and stromal cell coculture on hematopoietic differentiation of mouse embryonic stem cells

Recent studies have suggested that three-dimensional (3D) biomaterial-based scaffolds and dynamic culture conditions could provide significant enhancement in the differentiation efficiency of embryonic stem cells (ESCs). Here we report that scaffold physical properties, like pore size, polymer concentration and compression modulus as well as specific culture conditions, e.g. cell seeding density and coculture with stromal cells can significantly influence hematopoietic differentiation of ESCs. PLLA scaffolds of various polymer concentrations (7.5%, 10% and 20% w/v) and pore size distributions (<150 μm, 150–425 μm, >425 μm) were fabricated using a standard solvent casting-salt leaching method. Mouse R1 ESCs were allowed to differentiate on these scaffolds either alone or in coculture with OP9 cells, a bone-marrow derived murine stromal cell line. Following one week of culture, cells were detached and analyzed using flow cytometry to evaluate the frequency of hematopoietic progenitor cells (HPC). Our results indicate that decreasing scaffold pore size increases hematopoietic differentiation of ESCs. In addition, increasing polymer concentration which resulted in increased scaffold compression modulus also provided significantly enhanced hematopoiesis. Furthermore, higher cell seeding densities as well as coculture with marrow-derived stromal cells increased HPC generation. Collectively, these results indicate that physical and mechanical properties of the 3D microenvironment as well as cell–cell and cell–stromal interactions might play a significant role in ESC differentiation and therefore warrants further investigation to elucidate the molecular mechanisms.