Toward Corneal Limbus In Vitro Model: Regulation of hPSC‐LSC Phenotype by Matrix Stiffness and Topography During Cell Differentiation Process

A functional limbal epithelial stem cells (LSC) niche is a vital element in the regular renewal of the corneal epithelium by LSCs and maintenance of good vision. However, little is known about its unique structure and mechanical properties on LSC regulation, creating a significant gap in development of LSC‐based therapies. Herein, the effect of mechanical and architectural elements of the niche on human pluripotent derived LSCs (hPSC‐LSC) phenotype and growth is investigated in vitro. Specifically, three formulations of polyacrylamide gels with different controlled stiffnesses are used for culture and characterization of hPSC‐LSCs from different stages of differentiation. In addition, limbal mimicking topography in polydimethylsiloxane is utilized for culturing hPSC‐LSCs at early time point of differentiation. For comparison, the expression of selected key proteins of the corneal cells is analyzed in their native environment through whole mount staining of human donor corneas. The results suggest that mechanical response and substrate preference of the cells is highly dependent on their developmental stage. In addition, data indicate that cells may carry possible mechanical memory from previous culture matrix, both highlighting the importance of mechanical design of a functional in vitro limbus model. For the development of native tissue mimicking limbus in vitro model, human pluripotent stem cell derived limbal epithelial cells are cultured on varying stiffness polyacrylamide gels and polydimethylsiloxane matrix with limbus mimicking topography, demonstrating that substrate preference is highly dependent on their developmental stage. The results highlight the importance of mechanical design of a culture substrate.