Abstract
Substrate stiffness, biochemical composition, and matrix topography deeply influence cell behavior, guiding motility, proliferation, and differentiation responses. The aim of this work was to determine the effect that the stiffness and protein composition of the underlying substrate has on the differentiation of induced pluripotent stem (iPS) cells and the potential synergy with specific soluble cues. With that purpose, murine iPS-derived embryoid bodies (iPS-EBs) were seeded on fibronectin- or collagen I-coated polyacrylamide (pAA) gels of tunable stiffness (0.6, 14, and 50 kPa) in the presence of basal medium; tissue culture polystyrene plates were employed as control. Specification of iPS cells toward the three germ layers was analyzed, detecting an increase of tissue-specific gene markers in the pAA matrices. Interestingly, soft matrix (0.6 kPa) coated with fibronectin favored differentiation toward cardiac and neural lineages and, in the case of neural differentiation, the effect was potentiated by the addition of specific soluble factors. The generation of mature astrocytes, neural cells, and cardiomyocytes was further proven by immunofluorescence and transmission electron microscopy. In summary, this work emphasizes the importance of using biomimetic matrices to accomplish a more specific and mature differentiation of stem cells for future therapeutic applications.
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