This goal of this proposal is to screen and identify key factors in the cellular microenvironment that will enable improved growth and maintenance of undifferentiated human embryonic stem cells (hESC), and to use this information to derive new hESC lines. In the first aim, multiple combinations of growth factors, surface molecules and extracellular matrix components will be printed onto a solid support, and each condition will be evaluated for its ability to promote growth of human stem cells and maintenance of pluripotency. To complement this approach, a second aim will employ a similar strategy using arrays of synthetic polymers and scaffolds to assess the effects of charge, hydrophobicity and topography on stem cell growth and maintenance. The third aim proposes to derive a novel, self-renewing, hESC line using information gleaned from aims 1 and 2. The applicants comprise an academic, multidisciplinary team with expertise in bioengineering, nanotechnology and reproductive medicine. Current techniques for growing, scaling up, and maintaining undifferentiated hESC cultures are very limited, and technologies that could potentially overcome this roadblock are extremely desirable. This proposal offers a straightforward, innovative means for addressing some of these hurdles, and if successful, would be of high impact. Reviewers were most enthusiastic about the first aim, as printed arrays offer a potentially improved format for testing combinatorial variations of growth components, something that is difficult to achieve with typical solution-based methods. The use of arrays to identify optimal conditions for maintaining hESC cultures over time, as judged by detection of known pluripotency markers and teratoma formation, represents a simple yet elegant strategy for dissecting the chemical, biological and structural components that underlie these properties. Based on preliminary data, the reviewers were confident that the goals of this proposal are readily achievable, although the full two-year time frame might be necessary to realize Aim 1 alone. While intrigued by the technological platform, some reviewers felt that Aim 2 represented a significant detraction from the overall proposal. One reviewer felt that the added complexity of a topography screen might be unwarranted, as the stated goal of Aim 1 is a protocol for long-term self-renewal in culture, a sufficient goal in and of itself. Furthermore, the rationale for choosing the particular topographies to be explored was not adequately justified, and reviewers were uncertain as to how and why these arrangements were chosen. Overall, reviewers felt Aim 2 might have been stronger if the parameters to be tested were a) larger and less biased, or b) smaller and more biologically motivated. The principal investigator has a remarkable track record in the areas of bioengineering, extracellular matrices and cell biology and has assembled a strong team of experts in reproductive medicine and nanotechnology. The reviewers expressed great confidence in the ability of this team to meet the challenges presented in this application. Overall, this is a straightforward, feasible proposal that was clearly presented by a principal investigator with a proven track record of success. The specific aims were judged to be disproportionately meritorious, but the potential for high impact findings bolstered the reviewers’ enthusiasm. PROGRAMMATIC REVIEW A motion was made to move this proposal to Tier 1, Recommended for Funding. Despite the weakness of the second aim, reviewers felt that the strength and simplicity of the first aim, combined with proven track record of the investigative team, warranted additional consideration. Reviewers discussed whether identifying ideal conditions for adherent growth, i.e. molecular matrix formulations, could address the scale-up deficiencies that currently define the field, or whether it would be preferable to focus efforts on improving solution-based techniques for use in bioreactors. Some felt that it would indeed be desirable to have a matrix array for scaling up growth, and the use of bioreactors does not necessarily preclude the use of an adherent culture system, such as one where cells are attached to the surface of a bead within a bioreactor. Based on the strength of the team and encouraging preliminary data, as well as the potential for addressing a current roadblock in cell growth and maintenance, the motion carried.