Tri-Layer Decellularized, Dehydrated Amniotic Membrane Supports Proliferation and Stemness of Limbal Stem Cells Derived from Pluripotent Stem Cells

Abstract Purpose Treatment of limbal stem cell deficiency (LSCD) and other ocular surface disorders is challenging due to the lack of suitable cell sources and appropriate carriers. Differentiated limbal stem cells (LSCs) derived from induced pluripotent stem cells (iPSCs) offer an advantageous solution, providing an on-demand, unlimited, and patient-specific cell source compared to autologous and allogenic primary cells. Although amniotic membrane (AM) is the most widely used carrier for LSC transplantation, the quality of AM is dependent upon processing parameters. In this study, we evaluated the feasibility of using commercially available tri-layer decellularized, dehydrated human AM (DDHAM-3L) with stromal side facing outward as a carrier of iPSC-derived LSCs. Methods We differentiated LSCs from iPSCs and seeded these iPSC-LSCs on DDHAM-3L. Subsequently, in vitro studies were conducted to measure cell viability, proliferation, morphology, stemness, and commitment towards corneal epithelial linage to evaluate its performance as cell carrier material. Results The results of the 7-day in vitro study demonstrated that iPSC-LSCs strongly adhere, form colonies, express epithelial morphology, and form confluent cell sheets on DDHAM-3L. Moreover, iPSC-LSCs demonstrated a higher proliferation rate compared to the laminin-521-coated control. Gene and protein expression analyses indicated upregulation of markers associated with stemness and self-renewal in iPSC-LSCs, along with a commitment towards a corneal epithelial phenotype. Conclusion These findings suggest the potential of DDHAM-3L as a promising carrier for ocular surface reconstruction. Lay Summary The treatment of limbal stem cell deficiencies and other ocular surface disorders remains challenging due to limitations in both cell sourcing and carrier technology. To overcome these challenges, we have investigated differentiated LSCs obtained from iPSCs in combination with a carrier, tri-layer decellularized, dehydrated human amniotic membrane (DDHAM-3L), designed for improved handling, storage, and shelf life, compared to cryopreserved AMs. The results demonstrated the ability of DDHAM-3L to support proliferation and stemness of LSCs, acting as a viable carrier for iPSC-LSCs. Our findings indicate a promising application of DDHAM-3L as a carrier of iPSC-LSC for the treatment of LSCD and other ocular surface disorders. Graphical Abstract