PB1303 - Generation of iPSC-derived vascular cells deficient in tissue factor pathway inhibitor

Background: Tissue factor pathway inhibitor (TFPI) is a Kunitz-type inhibitor that acts as a constitutive inhibitor of the extrinsic coagulation pathway. Previous studies have shown that TFPI-deficiency leads to the accelerated formation of atherosclerotic plaques in animal models.

Aims: The aim of this study is to generate iPSC-derived vascular smooth muscle cells (SMC) and endothelial cells (EC) deficient in TFPI. These cells will serve as a robust platform for studying the impact of TFPI on cardiovascular pathologies.

Methods: Human induced pluripotent stem cells (iPSC) were modified by using a CRISPR/Cas9-mediated approach targeting the FXa-binding Kunitz(K)2-domain of TFPI. Genetically modified cells were selected, characterized by pluripotency marker staining, and further differentiated into SMC and EC using small molecule-based protocols. Expression of TFPI was assessed using Western blot and an in house-developed ELISA.

Results: CRISPR/Cas9-mediated modification of iPSCs resulted in a 12-bp in-frame deletion at the K2-domain. Modified cells exhibited a slightly altered iPSC morphology but showed positive expression of pluripotency markers. Furthermore, cells were successfully differentiated into SMC and EC as confirmed by positive expression of respective markers. Western blot analysis revealed that TFPI was still expressed in modified cells pointing to the presence of a 4 amino acid truncated (∆~605 Da) and possibly unfunctional protein.

Conclusion(s): The generation of TFPI-deficient cell lines will give further insights into the initiation and progression of cardiovascular diseases. Whether the expressed protein is active still remains to be elucidated. However, the disruption of the crucial K2-domain suggests improper protein function. iPSC-based methods allow incorporation of engineered cells in autologous microfluidic systems and co-culture models improving physiologic and pathologic representation in vitro and therefore will lead to more reliable in vitro research outcomes.