Abstract

Enhanced Differentiation to Mesendodermal and Ectodermal Lineages using Nanofibrous Scaffolds

Maricela Maldonado, Student, University of California-Riverside

The differentiation of induced pluripotent stem cells (iPSCs) to target cell types holds a promising potential for applications in regenerative medicine and tissue engineering. Traditionally, the formation of embryoid bodies has been used to enhance differentiation of stem cells, but the efficiency remains a major limitation. Considering significant effects of microenvironments on stem cell differentiation, a tightly controllable scaffolding may provide a means to maximize the differentiation efficiency while minimizing heterogeneous differentiation. In this study, electrospun nanofibrous scaffolds with varied mechanical properties, which were characterized by nanoindentation, were used to differentiate human iPSCs to mesendodermal or ectodermal lineages using a series of growth factor/cytokine stimulation. During pre-culture of the iPSCs using mTESR media, a flat, two-dimensional colony morphology was observed on stiffer scaffolds accompanied by enhanced proliferation. In contrast, a round, three-dimensional colony morphology was observed for iPSCs cultured on softer scaffolds with retarded self-renewal. After pre-culture, cells were subjected to a defined differentiation media containing specific growth factors for either mesendodermal or ectodermal lineages. Following differentiation, gene expression was analyzed using real time PCR and protein expression using immunocytochemistry for mesendodermal or ectodermal lineage markers. For mesendodermal differentiation, the expression of mesendodermal markers was positively correlated to increasing stiffness. In contrast, the expression of ectodermal markers was inversely correlated to increasing stiffness. These results offer promising solutions to enhance the efficiency of iPSC differentiation towards mesendodermal and ectodermal lineages, for further differentiation towards downstream target cell types.