Engineering Stem Cell Microenvironments From the Inside-out

Pluripotent embryonic stem cells (ESCs) capable of differentiating into an array of different cell types represent a robust cell source for the development of various regenerative cellular therapies and novel cell diagnostic platforms. Despite the clear potential of pluripotent stem cells, the inability to precisely control the presentation of morphogenic factors to ESCs in a manner that efficiently directs cell differentiation is a critical limitation. Microspheres fabricated from different biomaterials can be engineered to control the presentation of bioactive molecules, thereby simultaneously providing spatial, temporal and dose-dependent control of biomolecular delivery to cells. The objective of our work is to integrate engineered microspheres into stem cell microenvironments for controlled, local delivery of morphogenic factors to stem cells in order to more efficiently direct differentiation. Microspheres fabricated from poly(lactic-co-glycolic acid) (PLGA), gelatin or agarose were incorporated within ESC aggregates using rotary orbital suspension culture or forced aggregation within microwell substrates. The incorporation of fluorescently labeled microspheres was controlled in a dose-dependent manner as a function of the microsphere-to-cell ratios and the relative adhesivity of the materials. Introducing microspheres of different materials, but similar sizes (~5 µm) and extents of incorporation, did not significantly affect embryoid body (EB) formation or initial differentiation. However, EBs with gelatin microspheres expressed higher levels of AFP and lower levels of NKX 2.5 after 7 and 10 days of differentiation compared to the PLGA, agarose or untreated EBs. Overall, these studies demonstrate the ability to control the extracellular composition within differentiating ESC microenvironments via novel biomaterials-based delivery methods. This approach represents a fundamentally new route to direct the differentiation of stem cells through controlled, local presentation of molecules, a broadly applicable principle that can be integrated into the development of stem cell technologies.