OC 13.2 - Improved maturation of human pluripotent stem cell-derived endothelial cells to study von Willebrand factor expression and function

Background: Deficient VWF causes the most common bleeding disorder, von Willebrand disease, whereas gain of function causes thrombosis. Human pluripotent stem cells can differentiate into any of the body’s cell types. The maturity and utility of stem cell-derived endothelial cells (hPSC-EC) for studying VWF expression and function, however, remains poorly understood.

Aims: We sought to test the capacity of hPSC-EC to model the spectrum of VWF loss to activation, by analyzing the maturation of hPSC-EC including VWF expression, establishing a genetic model of VWF deficiency, and identifying conditions capable of increasing VWF.

Methods: We applied the CRISPR-Cas9 genome editing system to generate a cohort of gene edited hPSC-EC with knockout mutations in VWF. Control and VWF-/- cells were differentiated into hPSC-EC over 10 days. VWF and EC markers were analyzed by quantitative immunofluorescence, immunoblot, and RNA expression, compared to human umbilical vein endothelial cells (HUVEC). hPSC-EC were plated in a channel device to monitor VWF string formation under flow.

Results: hPSC-EC expressed substantially lower VWF levels, relative to HUVEC. Screening identified a compound and mechanism that upregulates VWF in both hPSC-EC and HUVEC (Fig. 1a-c). This improved hPSC-EC maturation and enabled VWF string formation from hPSC-EC in a microfluidic device under flow. VWF-/- hPSC differentiated efficiently into hPSC-EC with marker expression and morphology similar to isogenic controls, except that VWF protein was undetectable and string formation did not occur (Fig. 1d-e).

Conclusion(s): hPSC-EC maturation can be enhanced pharmacologically to increase VWF expression, suggesting pathways for therapy development. Combined with VWF-/- hPSC, this provides a platform for studying the roles of VWF in human cells. The versatility and stability of hPSC positions them as a useful model for studying bleeding disorders and potentially for vascular regeneration.