Impact of Tissue Factor Gene Knockout on Coagulation Properties of Umbilical Cord-Derived Multipotent Mesenchymal Stromal/Stem Cells

Abstract

Multipotent mesenchymal stromal/stem cells (MSCs) refer to a population of stem cells that exhibit distinct progenitor cell characteristics including the potential for differentiation into a wide range of cell types. MSCs have become a promising candidate for cell therapy and tissue regeneration due to their unique properties, such as their ability to differentiate into multiple cell types, their capacity for expansion, self-renewal, and immune-regulatory effects. However, reports have brought attention to thrombosis-related complications associated with MSCs therapy in the last decade. As tissue factor (TF) is a powerful coagulation activator expressed by MSCs that stimulates the extrinsic coagulation pathway, we investigated the thrombotic properties of human umbilical cord MSCs (HUCMSCs) after knocking out the TF gene. MSCs populations that obtained from umbilical cord were cultured and expanded in the appropriate medium cell culture. The identity of the MSCs was verified through flow cytometry, and their ability to differentiate into osteogenic and adipogenic lineages. Two gRNAs for Exons 1 and 2 of the TF gene have been designed and cloned into px458 vector's backbone (pSpCas9 (BB)−2A-GFP). Following transfecting of gRNAs into HUCMSCs and successfully knocking out the TF gene using GAP-PCR, the impact of normal and knockout HUCMSCs on coagulation was assessed through prothrombin time (PT), D-dimer level, clotting time (CT), and turbidity assay. Furthermore, the impact of TF knockout (TFKO) on MMP19 expression was assessed. Our results revealed that the PT was prolonged and D-dimer level was decreased in TFKO group compared to normal HUCMSCs. These findings suggest that TF gene plays a crucial role in regulating coagulation in HUCMSCs. Also, a significant reduction in MMP19 expression was observed within the TFKO group.