ESTABLISHMENT OF AN IPSC-DERIVED MODEL FOR THE STUDY OF VNN2 GENE IN POST-STROKE RECOVERY

Recent studies have demonstrated the potential role of VNN2 in stroke outcome, a GPI-anchored protein associated with an improved recovery for both ischemic and hemorrhagic stroke. VNN2 plays a role in inflammation related to transendothelial migration of neutrophils, and oxidative stress through its pantetheinase activity. Nevertheless, its function in post-stroke remains still unknown. Therefore, the main aim of this study is to elucidate the contribution of this protein in stroke outcome by establishing a VNN2 knockout iPSC-derived model for further studies. Different isogenic iPSCs lines with VNN2 knockout were generated through CRISPR editing. The generated lines were characterized by karyotyping and checking their pluripotency capability. First, immunostaining was performed to detect pluripotency markers (SOX2, SSEA4, TRA1-81, NANOG, OCT3/4). Additionally, the generated lines were differentiated into the three germ layers followed by immunocytochemistry to check markers for the endoderm (α-FP and SOX17), mesoderm (α-SMA and Brachyury) and ectoderm layer (PAX6 and TUJ1). We have generated and characterized two isogenic lines carrying heterozygous (KO+/-) VNN2 alleles and one compound heterozygous (KO-/-) line with truncating deletions and insertions. Finally, these isogenic lines were characterized by confirming their pluripotency. Taken together, several VNN2 KO isogenic iPSCs lines have been generated with the objective of differentiating them into neutrophils and understanding the role of VNN2 in neutrophil migration and adhesion after stroke.