Problem: A compositionally-defined synthetic polymer matrix that supports hES cell expansion in defined media is critical for determining factors that regulate stem cell growth and differentiation, expanding the use of hES cells in biotechnologies, and enabling potential clinical applications. However, synthetic matrices have only sustained short-term hES cell propagation. Background: Six polymer coatings were screened for ability to promote attachment and proliferation of undifferentiated hES cells in the presence of mouse embryonic fibroblast (MEF)-conditioned media. While 5 supported attachment and short-term passage (1-2 passages) only one, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide (PMEDSAH) supported long-term passage (<10), maintenance of normal phenotype, karyotype, and function. Hypothesis: We hypothesized that PMEDSAH could support long-term growth of hES cells in the presence of xeno-free media and/or serum-free defined media Research: Polymer coatings were prepared on polystyrene dishes. Surface-activated dishes were immersed into the monomer solution, followed by cooling and rinsing in saline solution. Matrigel was used as a control surface matrix. Two hES cells lines (H9 and BG01) were used for these studies in combination with either MEF-conditioned medium (MEF-CM), human cell conditioned medium (hC-CM), and two serum-free defined media, StemPro or mTeSR1. Under each culture condition cells were assessed for attachment, proliferation, expression of hES cell markers, retention of normal karyotype, and maintenance of pluripotency. Observations: Both H9 and BG01 attached and grew on PMEDSAH with hC-CM. At passage 15, H9 and BG01 expressed hES cell markers, had normal karyotypes, and could form embryoid bodies. Interestingly, a significant increase in H9 cell-aggregate adhesion was observed on PMEDSAH using hC-CM compared to MEF-CM. Cell-aggregate adhesion on PMEDSAH was also significantly higher for H9 in hC-CM compared to BG01. StemPro with PMEDSAH supported attachment and growth of H9 and BG01, however only H9 was maintained undifferentiated with normal hESC phenotype and karyotype for long-term passage. Combination of PMEDSAH and StemPro resulted in cell-aggregate adhesion of H9 significantly higher than MEF-CM, but not as efficient as hC-CM; while cell doubling and passage times were similar between media. Conclusions: This work therefore represents a significant step toward a fully defined, reproducible culture system for hES cell expansion. Compared to animal-derived matrices, PMEDSAH is chemically defined, can be synthesized reproducibly, has long-term stability, and can represent an important step in defining microenvironmental requirements of undifferentiated hES cell growth and directed differentiation. Growth in fully-defined culture conditions has also allowed us to identify previously unsuspected differences between hES cell lines.