Human embryonic stem cells (hESCs) are poised to play a dominate rule in early developmental biology and regenerative medicine. These efforts will fuel the need for large quantities of hESCs. In spite of their sensitivity to environmental influences, hESCs can be propagated without differentiation for extended periods of times, when cultured on certain, naturally-derived cell substrates, such as mouse or human embryonic fibroblast cells, Matrigel, laminin, or fibronectin. However, xenogeneic cell culture matrices introduce significant ambiguity into the culture system and therefore pose a significant hurdle in the wide-spread use of hESCs. Consequently, development of fully-defined cell culture matrices to replace naturally-derived support structures has emerged as one of the major challenges in current stem cell research. Synthetic polymer coatings could prove to be exceptional candidates for cell matrix replacement strategies because of their highly reproducible fabrication, but actual implementation of these novel materials has been challenging and no synthetic polymer coating has yet been used to support long-term hESC culture.

Using a rational design approach, we have now developed a completely synthetic polymer hydrogel coating, which has supported undifferentiated hESC growth during more than twenty continuous cell passages. In our long-term cell culture experiments, undifferentiated hESCs from H9 and BG01 cell lines previously cultured on mouse embryonic fibroblasts (MEF), were mechanically harvested and transferred onto the different polymer-coated cell culture dishes. Over a period of eight months, hESCs cultured on these hydrogel coatings retained normal euploid karyotypes and consistently displayed markers of undifferentiated hESCs. Since the focus of this study was to elucidate the role of the insoluble cell culture matrix, all cell culture experiments were conducted with MEF-conditioned medium (MEF-CM), which is known to support hESC growth and proliferation. This approach enables delineation between influences of the matrix and the medium. When differentiation was induced, hESCs formed representative cells of all three embryonic germ lines.

This is the first time that hESCs have exhibited undifferentiated growth and passaging over extended culture times when supported by synthetic polymer coatings void of xenogeneic, undefined, or labile components. Introducing polymer-based hESC culture matrices establishes a major step forward in the rapidly emerging field of stem cell research and may lead to future breakthroughs in developmental biology or hESC-centered clinical therapies.