(685b) Synthetic Surfaces for Human Embryonic Stem Cell Adhesion, Proliferation and Culture

Human embryonic stem cells (hESCs) are pluripotent cells that can differentiate into any adult cell type and hence have numerous medical applications. Currently, hESCs are cultured on a layer of feeder cells, complex extracellular protein mixtures or purified extracellular matrix proteins. The undefined nature and the biological origin of these matrices make the therapeutic application of hESCs challenging. Matrix adhesion through integrins is integral for hESC survival, the absence of which leads to cell apoptosis. The integrin-binding activity of many matrix proteins can be reproduced by synthetic peptides. The RGD motif, was originally identified as the integrin binding site in fibronectin, and can bind a number of integrins. Conformational restriction of the RGD peptide increases the affinity and specificity to the integrins. Enhanced cell attachment on surfaces modified with the linear RGD peptide, GRGDS, has been previously reported. These studies were conducted with highly adhesive cells such as fibroblasts. However, hESCs are far more sensitive cells than fibroblasts, and a synthetic surface for their culture should establish a functional integrin interaction for their survival.

We have successfully developed peptide-modified surfaces using a Michael addition mediated immobilization strategy. Amine-modified cell culture plates were modified with a bi-functional PEG linker, which was then used to conjugate the peptides. We employed the conventional linear RGD peptide (CRGDS) and a cyclic RGD peptide (CRGDC) to produce synthetic growth surfaces. We observed that hESCs were more adherent on the cyclic peptide than on the linear peptide. The cyclic peptide supported long term (10 passages) self-renewal of hESCs while most of the few colonies on the linear peptide differentiated rapidly. We also found that cells could be continuously cultured on surfaces conjugated with the cyclic peptide while maintaining their ?stemness', while the linear peptide did not support long-term culture. On the CRGDC substrate, hESCs had a growth rate similar to that on conventional cultures, and they also maintained normal karyotype. The pluripotency of the culture was maintained as observed by the gene expression levels of the pluripotency markers and in-vitro differentiation into the three germ-layers. We also successfully cultured the hESCs on the peptide-modified surfaces in completely defined media (mTESR1). Hence the peptide, CRGDC, provides a completely synthetic matrix for hESC culture which can be easily scaled up and used for production of clinical grade cells.

This is the first demonstration of long term hESC culture on a synthetic peptide surface that serves as a step towards the development of functionalized artificial scaffolds and hydrogels for enhanced stem cell attachment and survival.