PB0179 - Establishment of a monolayer-based LSEC-like endothelial cell in vitro system from induced pluripotent stem cells

Background: Liver sinusoidal endothelial cells (LSECs) are the major source of FVIII expression and secretion. Maintenance of these cells in vitro remains challenging as primary LSECs tend to rapidly lose their specific properties, including the expression of LSEC markers and the presence of fenestrae. In contrast IPS cells provide an alternative source to generate organ-specific ECs with high proliferative potential. Currently, embryoid body generation with a combination of adrenomedullin pathway activation, TGF-ß inhibition and hypoxia comprise recent strategies to derive LSEC-like EC from IPS.

Aims: To establish a 2D monolayer-based protocol from IPS to LSEC-like cells and track intracellular fate of wild type FVIII and FVIII variants in HA-patients.

Methods: Monolayer IPS single cells were differentiated for 3 days into mesoderm using small molecule CHIR and Bmp4. CD34+ cells were MACS isolated after two days of treatment with Forskolin, GSI and VEGF-A. Subsequently CD34+ angioblasts were maintained under hypoxic conditions for 4 days in StemPro34 containing low VEGF-A. Induction of LSEC-like cells was implemented by adding small molecules SB431542 and 8-Br-cAMP. Venous, arterial and LSEC specific markers FCGR2b, Stab2 & F8 were analyzed by rtPCR. Finally, intracellular FVIII protein was visualized and co-localized by immunofluorescence using cellular organelle marker for Calreticulin, COPII (Sec31a) and Golgi-trans (TGN46)

Results: Early angioblasts (day6) show an increase of venous marker COUP-TF and NT5E after four days under low VEGF-A conditions (Day10). During LSEC induction (Day10 - 16) LSEC markers show clear increase compared to HUVEC: FCGR2b (6-10 fold), Stab2 (7-14 fold) and F8 (8-13 fold). IF-staining presents strong co-localization of FVIII with classical cellular secretion marker CALR, COPII and Golgi.

Conclusion(s): This LSEC-like cellular model provides 8-fold higher F8 expression compared to a normal vascular EC in vitro system. The additional presence of LSEC specific receptors enables a more native and precise disease modeling for HA research and therapy.