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This abstract is assigned to session OB Free papers 1: Anterior segment

Title

FRO - Immunomodulation of corneal epithelial cells following electroporation with cDNA encoding IL-10

Purpose	Limbal epithelial stem cell transplants have shown better success rates in patients where autologous transplants were performed compared to allogenic transplantation. This led us to investigate the possibility of immunomodulation of the corneal epithelial stem cells prior to their transplantation. IL-10 is capable of inhibiting synthesis of pro-inflammatory cytokines like IFN-γ, IL-2, IL-3, TNFα and GM-CSF made by cells such as macrophages and the Type 1 T helper cells and also displays potent abilities to suppress the antigen presentation capacity of antigen presenting cells. The purpose of this study was to transfect corneal epithelial cells (CECs) with cDNA encoding IL-10 in order to evoke a down modulation of allogenic T cell response in an ex vivo model.
Methods	The CECs were electroporated initially with cDNA encoding a reporter gene, EGFP, in order to optimize the transfection efficiency using different combinations of voltage and capacitance settings. The efficiency and viability post transfection were determined using flow cytometry at days 1, 2, 3 and 6. Controls used included “mock” and “non” electroporated cells. Following this, the CECs were transfected with cDNA encoding IL-10 using the optimized electroporation (EP) settings. IL-10 secretion was determined in the supernatants of the electroporated CECs collected at days 1, 2, 3, 6 and 7 using ELISA.
Results	Optimal transfection efficiency of the CECs was observed using an exponential pulse and 300V with 1050µF capacitance. 5µg of EGFP cDNA was used for each EP giving a transfection efficiency of 46.7% 24hrs post EP. The viability of the CECs was 89% 24hrs post EP. When CECs were electroporated with cDNA encoding IL-10 (performed using the optimized settings) more than 1000pg of IL-10 was detected in 2.5x105 cells/ml already 24hrs post EP and was still present in the supernatant 7 days post EP.
Conclusion	We conclude that by using the optimized EP settings, we can efficiently transfect the CECs with cDNA encoding IL-10. This study is ongoing and future experiments include setting up co cultures with allogenic T cells to compare with control co cultures of allo-T cells and mock EP CECs. Following 5 days in cultureT cells will be analyzed for allogenic reactivity by ELISA for IFN-γ production in the culture supernatant. This project aims to illustrate in an ex-vivo model, how gene insertion into corneal epithelial stem cells could lead to improved cultivated limbal stem cell graft acceptance in patients with limbal stem cell deficiency.

Author 1

Last name	ZAKARIA
Initials	N
Department	Center for Cell Therapy and Regenerative Medicine, University Hospital Antwerp
City	Antwerp

Author 2

Last name	COOLS
Initials	N
Department	Center for Cell Therapy and Regenerative Medicine, University Hospital Antwerp
City	Antwerp

Author 3

Last name	VAN TENDELOO
Initials	V
Department	Center for Cell Therapy and Regenerative Medicine, University Hospital Antwerp
City	Antwerp

Author 4

Last name	BERNEMAN
Initials	Z
Department	Center for Cell Therapy and Regenerative Medicine, University Hospital Antwerp
City	Antwerp

Author 5

Last name	TASSIGNON
Initials	MJ
Department	Center for Cell Therapy and Regenerative Medicine, University Hospital Antwerp
City	Antwerp