Balu Venugopal1, Sumitha Mohan1, T V Kumary1, P R Anil Kumar2. 1. Division of Tissue Culture, Department of Applied Biology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, 695 012, India. 2. Division of Tissue Culture, Department of Applied Biology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, 695 012, India. anilkumarpr@sctimst.ac.in.
Abstract
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-derived mononuclear cells (PBMNCs). These blood derived stem cells gained a lot of attention due to its minimally invasive collection and ease of isolation. In this study, the feasibility of using PBMNCs as an alternative cell source to corneal limbal stem cells envisaging corneal epithelial regeneration was evaluated. METHODS: Rabbit PBMNCs were isolated using density gradient centrifugation and was evaluated for mesenchymal cell properties including stemness. PBMNCs were differentiated to corneal epithelial lineage using rabbit limbal explant conditioned media and was evaluated by immuno-cytochemistry and gene expression analysis. Further, the differentiated PBMNCs 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. Corneal epithelial lineage commitment of PBMNCs was confirmed by the positive expression of CK3/12 marker thereby demonstrating the aptness as an alternative to limbal stem cells. These differentiated cells effectively generated an in vitro cell 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 effective therapeutic 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-derived mononuclear cells (PBMNCs). These blood derived stem cells gained a lot of attention due to its minimally invasive collection and ease of isolation. In this study, the feasibility of using PBMNCs as an alternative cell source to corneal limbal stem cells envisaging corneal epithelial regeneration was evaluated. METHODS: Rabbit PBMNCs were isolated using density gradient centrifugation and was evaluated for mesenchymal cell properties including stemness. PBMNCs were differentiated to corneal epithelial lineage using rabbit limbal explant conditioned media and was evaluated by immuno-cytochemistry and gene expression analysis. Further, the differentiated PBMNCs 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. Corneal epithelial lineage commitment of PBMNCs was confirmed by the positive expression of CK3/12 marker thereby demonstrating the aptness as an alternative to limbal stem cells. These differentiated cells effectively generated an in vitro cell 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 effective therapeutic strategy for corneal surface reconstruction especially for patients with bilateral limbal stem cell deficiency.