Establishment and specification analysis of iPS cell derived liver sinusoidal endothelial-like cells from Hemophilia A patients for the detection of endogenous FVIII

Abstract

The current state of the art in this field of Hemophilia A basic research comprises cellular models that either overexpress FVIII levels (Fantacini, 2016; Liu et al., 2014) or are incubated with FVIII products (Kannicht, 2020; Peyron, 2018; Peyvandi et al., 2018) which is unable to confirm or correlate to the endogenous FVIII expression. To study the mechanism and progression of Hemophilia A in its most native form, Liver sinusoidal endothelial cells (LSEC) make a good model since they are known to be non-hematopoietic antigen presenting cells (nhAPC) which could express MHC I and II (Carman et al., 2015) i.e. presentation of both endogenous and exogenous antigens. Understanding the endogenous FVIII expression is of utmost interest in disease modelling for Hemophilia A since the mutation specific variation in patients could provide an insight into the intracellular fate of the molecule. LSEC-like cells were differentiated from pluripotent stem cells in vitro of healthy donor and hemophila A patients, including those with nonsense mutations, R1960X and R2228X and an intron 22 inversion mutation. We confirmed the differentiation to a mesoderm and angioblasts; and the presence of LSEC-specific markers such as <em>F8</em>, <em>Lyve1</em> and <em>STAB2</em>. Detection of endogenous factor VIII protein in its natural state is crucial to differentiate between the healthy and mutated version as well as understand its effect in hemophilia A disease progression. This study for the first time identifies distinct endogenous peptides, adding complexity to the molecular landscape in patients. Proteomic analysis revealed an upregulation in the EIF2 signalling pathway in patients that led to the identification of 43 significant proteins associated with ER functions suggesting an accumulated protein response in patient with nonsense mutation in the C2 domain. 4 fold higher RNSP1 levels in R2228X was associated EJC-mediated mRNA decay related absence in detection of FVIII peptides. This study further explored potential modulators associated with <em><em>F8</em></em> gene and protein expression in hemophilia A patients. Inhibition of NMD may have an impact on the levels of its substrates and is expected to result in an accumulation of the PTC-carrying transcripts in the cell thereby enabling a better read-through responsiveness. Hence, we hypothesized that inhibiting NMD and allowing for a subsequent read-through could augment <em>F8</em> expression in patients. We identified NMD inhibitor 14 (NMDI-14) as a small molecule inhibitor of the NMD pathway (Martin et al., 2014) in combination with Geneticin and PTC-124 to synergistically enhance the expression of <em>F8</em> in LSEC-LCs from two HA patients with nonsense mutation R1960X, R2228X and a patient with intron 22 inversion mutation. We employed readthrough agents and/or nonsense mediated mRNA inhibitors to analyse the influence of downstream targets of the unfolded protein response and its impact on the <em>F8</em> expression levels. We confirmed that the modulation of UPR-NMD feedback loop via synergistic effects of read through agents and nonsense mediated mRNA decay inhibitors can increase <em>F8</em> transcript levels with an albeit variable response.