BACKGROUND: Mesenchymal stem cell-based treatments are now emerging as a therapy for corneal epithelial damage.Although bone marrow, adipose tissue and umbilical cord blood are the main sources of mesenchymal stem cells (MSCs),other tissues like the peripheral blood also harbor mesenchymal-like stem cells called peripheral blood-derivedmononuclear cells (PBMNCs). These blood derived stem cells gained a lot of attention due to its minimally invasivecollection and ease of isolation. In this study, the feasibility of using PBMNCs as an alternative cell source to corneallimbal stem cells envisaging corneal epithelial regeneration was evaluated. METHODS: Rabbit PBMNCs were isolated using density gradient centrifugation and was evaluated for mesenchymalcell properties including stemness. PBMNCs were differentiated to corneal epithelial lineage using rabbit limbal explantconditioned media and was evaluated by immuno-cytochemistry and gene expression analysis. Further, the differentiatedPBMNCs were engineered into a cell sheet using an in-house developed thermo-responsive polymer. RESULTS: These blood derived cells were demonstrated to have similar properties to mesenchymal stem cells. Cornealepithelial lineage commitment of PBMNCs was confirmed by the positive expression of CK3/12 marker therebydemonstrating the aptness as an alternative to limbal stem cells. These differentiated cells effectively generated an in vitrocell sheet that was then demonstrated for cell sheet transfer on an ex vivo excised rabbit eye. CONCLUSION PBMNCs as an alternative autologous cell source for limbal stem cells is envisaged as an effectivetherapeutic strategy for corneal surface reconstruction especially for patients with bilateral limbal stem cell deficiency.

BACKGROUND: Mesenchymal stem cell-based treatments are now emerging as a therapy for corneal epithelial damage.Although bone marrow, adipose tissue and umbilical cord blood are the main sources of mesenchymal stem cells (MSCs),other tissues like the peripheral blood also harbor mesenchymal-like stem cells called peripheral blood-derivedmononuclear cells (PBMNCs). These blood derived stem cells gained a lot of attention due to its minimally invasivecollection and ease of isolation. In this study, the feasibility of using PBMNCs as an alternative cell source to corneallimbal stem cells envisaging corneal epithelial regeneration was evaluated. METHODS: Rabbit PBMNCs were isolated using density gradient centrifugation and was evaluated for mesenchymalcell properties including stemness. PBMNCs were differentiated to corneal epithelial lineage using rabbit limbal explantconditioned media and was evaluated by immuno-cytochemistry and gene expression analysis. Further, the differentiatedPBMNCs were engineered into a cell sheet using an in-house developed thermo-responsive polymer. RESULTS: These blood derived cells were demonstrated to have similar properties to mesenchymal stem cells. Cornealepithelial lineage commitment of PBMNCs was confirmed by the positive expression of CK3/12 marker therebydemonstrating the aptness as an alternative to limbal stem cells. These differentiated cells effectively generated an in vitrocell sheet that was then demonstrated for cell sheet transfer on an ex vivo excised rabbit eye. CONCLUSION PBMNCs as an alternative autologous cell source for limbal stem cells is envisaged as an effectivetherapeutic strategy for corneal surface reconstruction especially for patients with bilateral limbal stem cell deficiency.