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R. propagation has resulted from elucidating the functions of soluble factors46. In contrast, it has proved challenging to identify defined substrata for this purpose. The typical mixtures employed to date are composed of extracellular matrix (ECM) proteins from animal or human sources4,7. The batch-to-batch inconsistency that accompanies the use of complex mixtures complicates hES cell propagation. Moreover, the use of animal-derived ECM proteins exposes cells to potentially hazardous pathogens and allows for the transfer of immunogenic epitopes8. Individual ECM PD153035 (HCl salt) proteins911have been employed as substrates for hES cell self-renewal, but even PD153035 (HCl salt) single ECM proteins have multiple domains that can engage a variety of cell-surface receptors. As a result, it is difficult to elucidate the key interactions that give rise to reproducible culture conditions. We sought to replace the substrata used for hES cell culture with a fully defined, synthetic alternative. A synthetic surface could minimize the exposure of pluripotent cells to hazardous contaminants and yield a more homogenous cell culture. Moreover, it could illuminate the minimum requirements for adhesion and self-renewal. Although synthetic substrata have been described1220, none have been shown to be effective for the long-term culture of both hES cells and iPS cells. Most of these substrata were either used in combination with a conditioned medium1214or were tested with only a limited number of cell lines1720. Additionally, the mechanisms by which these substrates function are largely unknown. Here we describe a unique synthetic surface that engages cell surface glycosaminoglycans and supports the long-term propagation of multiple hES and induced pluripotent stem (iPS) cell lines in fully defined conditions. == RESULTS == == Peptide substrates for hES cell adhesion == To seek a substrate with the desired activity, we employed a defined surface array21composed of peptide-substituted alkanethiol conjugates, which form self-assembled monolayers (SAMs) on gold22. SAMs can present a variety of ligands with control over surface density, making them useful probes of the consequences of engaging cell surface ligands23. Previous work employed surface arrays that present peptides derived from laminin at high densities12or cell-binding peptides identified by phage display18. Those screens PD153035 (HCl salt) yielded synthetic surfaces capable of supporting the short-term propagation of hES cells. To identify a synthetic surface that would PD153035 (HCl salt) be effective for long-term propagation, we expanded our array screen12,21to assess surfaces that carry bioactive peptide sequences reported to bind to diverse cell surface receptors (Supplementary Table 1). We did not limit our screen to known protein-binding sequences (e.g. the integrin ligand RGD24) but also included peptides that can interact with glycosaminoglycans. Because cell adhesion is usually influenced not just by the identity of the adhesive epitopes, but by their density around the substrate, Rabbit Polyclonal to SFRS17A and whether they are present alone or in combination21,25, we used the array to present bioactive peptides alone, in different combinations, and at varying surface densities. In total, we screened over 500 unique surfaces based on 18 bioactive peptides. The surfaces were first evaluated for their abilities to support hES cell adhesion. Cells (H1 or H9) that had been cultured on Matrigel were dissociated and applied to the array in a protein-free basal medium, to minimize the adsorption of proteins from the medium. After 1 PD153035 (HCl salt) hour, the basal medium was replaced with defined mTeSR medium4supplemented with Y-27632, an inhibitor of rho-associated kinase (ROCK). This small molecule aids the survival of dissociated hES cells26. Using these conditions, hES cells were propagated around the array for 6 days and then fixed and stained for the markers of pluripotency Oct-4 and SSEA-4 (Fig. 1a). In this way, we identified several surfaces that could sustain cell adhesion over the course of the experiment. Adhesion to the SAMs required the ROCK inhibitor, even after colonies had been established (Supplementary Fig. 1). == Physique 1. == Strategy for the identification of peptide-substituted surfaces for hES cell adhesion and survival. (a) The image shows H9 cells bound to a representative array presenting bioactive peptides in different combinations and at distinct surface densities. Surface densities are reported as the percentage of peptide-substituted alkanethiol in the mixed SAM. Cells were fixed, immunostained for Oct-4 (red) or SSEA-4 (green), and counterstained with DAPI (blue). (bg) Representative higher magnification images of cells (H1,cd, and H9,b,eg) on arrays. (bd) Cells were propagated on.