Human embryonic stem (hES) cells represent a promising source for cell transplantation because of their high degree of self-renewal and their unique ability to give rise to most somatic cell lineages. Despite the promise of obtaining various lineages, the ultimate goal of expanding and differentiating hESCs in three-dimensional environments remains challenging. We report on the idea that three-dimensional substrates can be designed for specific geometry and molecular matrix presentation to maintain the stemness of hESCs or induce the directed differentiation of isolated hES cells into strategic lineages (e.g. neural) without the use of aggregates, embryoid bodies or neurospheres.

To this end, our study offers a three-pronged strategy to direct the differentiation of hES cells into neural lineage on 2-D substrates and in 3-D scaffold cultures. The first relies on the display of adhesion ligands that can control directed differentiation vs. pluripotency. As a surface molecular engineering approach, we have incorporated 2-D and 3-D substrates with fragments of cadherin, fibronectin or L1 molecules that can be used to more precisely control pluripotency versus lineage commitment fates of hESCs in situ. Second, we have designed 3-D substrates for hES cells fabricated from a subset of combinatorial library of biodegradable polymers, tyrosine-derived polycarbonates. Using electrospinning, various fibrous scaffolds with varying microgeometries and surface composition were compared. Such scaffolds can yield controlled adhesion and outgrowth of individual hESCs, which can be used as another basis to regulate cell-cell vs. cell-matrix interactions and thereby induce expansion while limiting spontaneous differentiation. The third novel approach of the paper will demonstrate the possibility of integrating nanoscale adhesion ligands within the polymeric scaffolds, so as to switch cell fates to specific neuronal differentiation pathways in situ within three-dimensional microenvironments.