Compartmentalized 3D bioprinting of the limbal niche with distinct hPSC-LSC subpopulations for corneal disease modeling

Limbal epithelial stem cells (LSCs) are essential for corneal epithelium regeneration and visual acuity. The limbal niche’s physicochemical properties regulate LSC function, but their role is not fully understood. Developing in vitro models that mimic the native niche can enhance our understanding of niche functions, despite the challenges of niche complexity. In this study, we created a 3D bioprinted limbal niche model using a hybrid approach that combines two human pluripotent stem cell-derived LSC (hPSC-LSC) subpopulations (p63+ and ABCG2+ cells) within hyaluronic acid (HA)-based bioinks and a stiff polyacrylamide (PA) gel scaffold produced by conventional gel casting. We analyzed the mechanical properties of the bioinks and assessed cell viability, morphology, and protein expression after one week of culture. Finally, we conducted a proof-of-concept wound healing assay using an alkali burn injury model to assess the functionality of the model for research purposes. The results show that this 3D model effectively replicated the mechanical environment of native tissue, maintains stability for one-week post-printing, and supports LSC viability and normal in vitro phenotype. In addition, the wound healing assay showed a cellular response, indicated by non-simultaneous caspase-3 activation of hPSC-LSC subpopulations for 48 hours post-wounding. This model provides a valuable platform for investigating the limbal niche and advancing cellular therapies applicable to other tissue niches throughout the body.