A reductionist approach to determine the effect of cell-cell contact on human epidermal stem cell differentiation

The balance between stem cell renewal and differentiation is determined by the interplay between intrinsic cellular controls and extrinsic factors presented by the microenvironment, or 'niche'. Previous studies on cultured human epidermis have utilised suspension culture and restricted cell spreading to investigate regulation of differentiation in single keratinocytes. However, keratinocytes are typically adherent to neighbouring cells in vivo. We therefore developed experimental models to investigate the combined effects of cell-ECM adhesion and cell-cell contact. We utilized lipid-modified oligonucleotides to form clusters of keratinocytes which were subsequently placed in suspension to induce terminal differentiation. In this experimental model cell-cell contact had no effect on suspension-induced differentiation of keratinocytes. We next developed a high-throughput platform for robust geometrical confinement of keratinocytes to hexagonal ECM-coated islands permitting direct cell-cell contact between single cells. As in the case of circular islands, differentiation was stimulated on the smallest single hexagonal islands. However, the percentage of involucrin-positive cells on small bowtie islands was significantly lower than on single islands, demonstrating that cell-cell contact reduced differentiation in response to decreased substrate adhesion. None of the small bowtie islands contained two involucrin-positive cells. Rather, if one cell was involucrin-positive the other was involucrin-negative. This suggests that there is intrinsic asymmetry in the effect of cell-cell contact in decreasing differentiation. Thus, our reductionist approaches provide new insights into the effect of the niche on keratinocyte differentiation. STATEMENT OF SIGNIFICANCE: Stem cell behaviour is regulated by a combination of external signals, including the nature of the adhesive substrate and cell-cell interactions. An understanding of how different signals are integrated creates the possibility of developing new biomaterials to promote tissue regeneration and broaden our understanding of skin diseases such as eczema and psoriasis, in which stem cell proliferation and differentiation are perturbed. In this study we have applied two methods to engineer intercellular adhesion of human epidermal stem cells, one involving lipid-modified DNA and the other involving hexagonal micropatterns. We show that the effect of cell-cell adhesion depends on cell-substrate adhesion and uncover evidence that two cells in equivalent environments can nevertheless behave differently.