Characterization of the interface between adsorbed fibronectin and human embryonic stem cells

Abstract

The cell-substrate interface plays a key role in the regulation of cell behaviour. Defining the properties of this interface is particularly important for human embryonic stem (hES) cell culture, because changes in this environment can regulate hES cell differentiation. It has been established that fibronectincoated surfaces can promote the attachment, growth and maintenance of the undifferentiated phenotype of hES cells. We investigated the influence of the surface density of adsorbed fibronectin on hES cell behaviour in defined serum-free culture conditions and demonstrated that only 25 per cent surface saturation was required to maintain attachment, growth and maintenance of the undifferentiated phenotype. The influence of surface-adsorbed fibronectin fragments was compared with whole fibronectin, and it was demonstrated that the 120 kDa fragment central binding domain alone was able to sustain hES cells in an undifferentiated phenotype in a similar fashion to fibronectin. Furthermore, hES cell attachment to both fibronectin and the 120 kDa fragment was mediated by integrin a5b1. However, although a substrateattached synthetic arginine-glycine-aspartic acid (RGD) peptide alone was able to promote the attachment and spreading of fibroblasts, it was inactive for hES cells, indicating that stem cells have different requirements in order to attach and spread on the central fibronectin RGD-cell-binding domain. This study provides further information on the characteristics of the cellsubstrate interface required to control hES cell behaviour in clearly defined serum-free conditions, which are needed for the development of therapeutic applications of hES cells.

Combinatorial effect of substratum properties on mesenchymal stem cell sheet engineering and subsequent multi-lineage differentiation

“…Furthermore, different cell types were known to respond differently to the amount of surface-bound fibronectin. For instance, endothelial cell and embryonic stem cell were reported to adhere better on surfaces with higher fibronectin adsorption [55][56], while another study indicated that MG63 (human osteosarcoma) cells preferably adhered to surfaces with lower fibronectin adsorption [57]. To date, BMSC adhesion under varying fibronectin adsorption is still poorly understood.…”

Extracellular matrix‐immobilized nanotopographical substrates for enhanced myogenic differentiation

“…To quantify FN on different substrates, fluorescent intensity was measured modifying previously reported method by Kalaskar et al 12 In addition, presence of immobilized FN (10 mg mL 21 ) was confirmed with anti-FN antibody (Cat. EDC-NHS coupling of L-lysine (1 mg mL 21 , Sigma) was confirmed with FT-IR.…”

Making cardiomyocytes: How mechanical stimulation can influence differentiation of pluripotent stem cells

“…During the earliest stages of development, integrin subunits α5 and αv bind to laminin and fibronectin . These subunits, along with β1 and β5, are important in ESC self‐renewal and maintaining the pluripotent phenotype . As cells differentiate, the integrin profile changes, and consequently the cell's response to extracellular force changes.…”