Full length articleElectrospun polystyrene scaffolds as a synthetic substrate for xeno-free expansion and differentiation of human induced pluripotent stem cells

The use of human induced pluripotent stem cells (hiPSCs) for clinical tissue engineering applications requires expansion and differentiation of the cells using defined, xeno-free substrates. The screening and selection of suitable synthetic substrates however, is tedious, as their performance relies on the inherent material properties. In the present work, we demonstrate an alternative concept for xeno-free expansion and differentiation of hiPSCs using synthetic substrates, which hinges on the structure-function relationship between electrospun polystyrene scaffolds (ESPS) and pluripotent stem cell growth. ESPS of differential porosity was obtained by fusing the fibers at different temperatures. The more porous, loosely fused scaffolds were found to efficiently trap the cells, leading to a large number of three-dimensional (3D) aggregates which were shown to be pluripotent colonies. Immunostaining, PCR analyses, in vitro differentiation and in vivo teratoma formation studies demonstrated that these hiPSC aggregates could be cultured for up to 10 consecutive passages (P10) with maintenance of pluripotency. Flow cytometry showed that more than 80% of the cell population stained positive for the pluripotent marker OCT4 at P1, P5 and P10. P10 cells could be differentiated to neuronal-like cells and cultured within the ESPS for up to 18 months. Our results suggest the usefulness of a generic class of synthetic substrates, exemplified by ESPS, for 'trapped aggregate culture' of hiPSCs.Statement of SignificanceTo realize the potential of human induced pluripotent stem cells (hiPSCs) in clinical medicine, robust, xeno-free substrates for expansion and differentiation of iPSCs are required. In the existing literature, synthetic materials have been reported that meet the requirement for non-xenogeneic substrates. However, the self-renewal and differentiation characteristics of hiPSCs are affected differently by the biocompatibility and physico-chemical properties of individual substrates. Although some rules based on chemical structure and substrate rigidity have been developed, most of these efforts are still empirical, and most synthetic substrates must still be rigorously screened for suitability.In this paper, we demonstrate an alternative concept for xeno-free expansion and differentiation of hiPSCs using synthetic substrates, which hinges on the structure-function relationship between electrospun polystyrene scaffolds (ESPS) and pluripotent stem cell growth. ESPS of differential porosity was obtained by fusing the fibers at different temperatures. The more porous, loosely fused scaffold was found to efficiently trap the cells, leading to a large number of three-dimensional (3D) aggregates. In the form of these trapped aggregates, we showed that hiPSCs could be cultured for up to 10 consecutive passages (P10) with maintenance of pluripotency, following which they could be differentiated to a chosen lineage. We believe that this novel, generic class of synthetic substrates that employs 'trapped aggregate culture' for expansion and differentiation of hiPSCs is an important conceptual advance, and would be of high interest to the readership of Acta Biomaterialia.

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