Focal adhesion prevention on nanoparticle substrates upregulates stemness gene expression in primary melanocytes

Abstract

HypothesisCell focal adhesions are initiated by interactions of cell surface integrins with extracellular matrix (ECM) proteins, and cells which lack focal adhesions have been associated with an intermediate adhesive state, which is more labile towards cell reprogramming. We hypothesized that cells cultured under conditions where they exhibit reduced focal adhesions will assume an intermediate adhesive state with a more stem cell-like transcriptional profile. ExperimentsTo recreate culture conditions for cells to exhibit reduced focal adhesions, we synthesized PMMA nanoparticles by evaporation-induced self-assembly (EISA), employing varying conditions of surfactant concentrations, and allowed them to self-assemble into nanoparticle substrates. Following guidelines from previous work, we selected nanoparticle substrates of dimensions [~]70nm, suitable for the prevention of focal adhesion formation. Primary human melanocytes were cultured on these substrates to verify the reduction of their focal adhesions; following that the cultures were characterized in terms of their expression of distinct sets of genes related to stemness and differentiation. FindingsThe melanocytes showed reduced focal adhesions on both non-coated and fibronectin-coated nanoparticle substrates, when compared to melanocytes cultured on TCPs. Accompanying the reduction in focal adhesions, the cells exhibited an increase in the expression of general as well as melanocyte-specific stemness related genes such as OCT4, LIN28, NANOG and PAX6 and a concurrent downregulation of genes expressed by differentiated melanocytes, such as TYR, TRP1 and MITF. These observations support our hypothesis.