High-throughput analysis of topographical cues for the expansion of murine pluripotent stem cells

The expansion of pluripotent stem cells (PSCs)in vitroremains a critical barrier to their use in tissue engineering and regenerative medicine. Biochemical methods for PSC expansion are known to produce heterogeneous cell populations with varying states of pluripotency and are cost-intensive, hindering their clinical translation. Engineering biomaterials to physically control PSC fate offers an alternative approach. Surface or substrate topography is a promising design parameter for engineering biomaterials. Topographical cues have been shown to elicit profound effects on stem cell differentiation and proliferation. Previous reports have shown isotropic substrate topographies to be promising in expanding PSCs. However, the optimal feature to promote PSC proliferation and the pluripotent state has not yet been determined. In this work, the MultiARChitecture (MARC) plate is developed to conduct a high-throughput analysis of topographical cues in a 96-well plate format. The MARC plate is a reproducible and customizable platform for the analysis of multiple topographical patterns and features and is compatible with both microscopic assays and molecular biology techniques. The MARC plate is used to evaluate the expression of pluripotency markersOct4, Nanog, andSox2and the differentiation markerLmnAas well as the proliferation of murine embryonic stem (mES) cells. Our systematic analyses identified three topographical patterns that maintain pluripotency in mES cells after multiple passages: 1µm pillars (1µm spacing, square arrangement), 2µm wells (c-c (x, y) = 4, 4µm), and 5µm pillars (c-c (x, y) = 7.5, 7.5µm). This study represents a step towards developing a biomaterial platform for controlled murine PSC expansion.