231 USE OF SOFT SUBSTRATES TO PROMOTE AND MAINTAIN OCT4 EXPRESSION IN EPIGENETICALLY ERASED FIBROBLASTS

Abstract

Pluripotency and commitment are driven by transcription factors that act as molecular switches to activate or repress specific gene expression programs. Among these, the POU transcription factor OCT4 (encoded by POU class 5 homeobox 1, POU5F1) is known to be a regulator in pluripotent cells and it is essential for the maintenance of a high plasticity state in mammalian cells. In recent years, particular attention has been given to the importance of physical extracellular matrix properties in regulating pluripotent cell maintenance and differentiation. In particular, recent studies demonstrated that high plasticity cells are intrinsically soft and respond optimally to physical forces when cultured on a “low stiffness” substrate that matches their intrinsic softness. To investigate these aspects, here we used a protocol where terminally differentiated cells are driven into a high plasticity/OCT4-positive state through the use of the epigenetic eraser, 5-aza-cytidine (5-aza-CR) and we observed the effect of substrate stiffness on the maintenance of OCT4 expression. To this purpose, human skin fibroblasts were plated either on standard plastic dishes (group A) or on polyacrylamide gels (PAA) of low stiffness (1 kPa; group B). Cells were treated with 1 μM 5-aza-CR for 18 h and were subsequently maintained and cultured in embryonic stem cell medium for 14 days. Assessment of OCT4 was carried out both by real-time PCR and immunocytochemical analysis, at different time points: untreated fibroblasts (T0), after 5-aza-CR treatment, and on Days 2, 4, 6, 8, 10, and 14 of culture. The results obtained indicate that untreated fibroblasts (T0) were negative for OCT4, whereas, after 5-aza-CR exposure, cells actively expressed the pluripotency marker, in both the A and B groups. However, whereas group A cells progressively down-regulated OCT4 expression and eliminated its positivity by Day 6, group B cells steadily transcribed and were homogeneously immunopositive for the pluripotency factor until Day 14, when cultures were arrested. Overall, the data obtained suggest that a soft substrate, matching the intrinsic stiffness distinctive of high plasticity cells, may help in promoting their maintenance, via a biophysical mechanism of low-traction/low-stiffness-dependent manner.

Study was supported by Carraresi Foundation.