The human amniotic membrane (AM) is a tissue of fetal origin and has proven to be clinically useful as
a biomaterial in the management of various ocular surface disorders including corneal stem cell
transplantation. However, its success rate displays a degree of clinical unpredictability. We suggest that
the measured variability inAMstiffness offers an explanation for the poor clinical reproducibility when
it is used as a substrate for stem cell expansion and transplantation. Corneal epithelial stem cells were
expanded upon AM samples possessing different mechanical stiffness. To investigate further the
importance of biological substrate stiffness on cell phenotype we replaced AM with type I collagen gels
of known stiffness. Substrate stiffness was measured using shear rheometry and surface topography
was characterized using scanning electron microscopy and atomic force microscopy. The
differentiation status of epithelial cells was examined using RT-PCR, immunohistochemistry and
Western blotting. The level of corneal stem cell differentiation was increased in cells expanded upon
AM with a high dynamic elastic shear modulus and cell expansion on type I collagen gels confirmed
that the level of corneal epithelial stem cell differentiation was related to the substrate’s mechanical
properties. In this paper we provide evidence to show that the preparatory method of AM for clinical
use can affect its mechanical properties and that these measured differences can influence the level of
differentiation within expanded corneal epithelial stem cells
