Derivation of Patient Specific iPSCs and iHEP Cell Lines as a Tool to Investigate VKCFD2 in a Native Cellular Environment

Introduction: Vitamin K combined factor deficiency type 2 (VKCFD2) is a rare autosomal recessive bleeding disorder caused by a point mutation in the VKORC1 gene (p.Arg98Trp). VKORC1 is responsible for vitamin K recycling by catalyzing the reduction of vitamin K 2,3-epoxide (KO) into its quinone and then to hydroquinone form (KH₂). KH₂ in turn, serves as co-factor for the γ-carboxylation of vitamin K dependent proteins. Patients with VKORC1:p.Arg98Trp mutation show spontaneous bleeding due to diminished activity of all vitamin K dependent clotting factors. Treatment for VKCFD2 is based on daily supplementation of vitamin K.

The current available cellular model of VKCFD2 rely on protein overexpression in HEK cells.

The aim of this study is to obtain a native cellular model of VKCFD2 which resembles the complexity of the disease both at the genotype and phenotype level.

Methods: Peripheral blood was collected from two members of a VKCFD2 family (the homozygous daughter and the heterozygous father) and from two healthy donors (one female and one male). Peripheral blood mononuclear cells (PMBCs) were isolated and the erythroid progenitor cells (EPCs) population was expanded. EPCs were then nucleofected with episomal plasmids carrying four reprogramming factors (Oct4, Kfl4, Sox2, c-Myc) to obtain human induced pluripotent stem cell (hiPSCs) from each patient and healthy donors. The pluripotency and genomic integrity of hiPSCs clones was confirmed by qRT-PCR, immunostaining, embryoid bodies formation assay and karyotyping. hiPSCs where then differentiated into induced hepatocyte-like cells (iHEPs) by using small molecules that simulate in vivo liver signaling and development. The expression of specific iHEPs markers was confirmed by qRT-PCR, immunostaining and filipin staining.

Results: Multiple hiPSC lines were derived from each patient and controls. Once the pluripotency of the hiPSC line was confirmed, one hiPSCs clone derived from the female control was further differentiated into induced hepatocyte-like cells. These iHEPs showed hepatocyte-like morphology, expressed hepatocyte associated marker (albumin, α-fetoprotein, HNF4α) and were able to synthetize cholesterol as confirmed by filipin staining.

Conclusions: Our hiPSCs and iHEPs models might allow us to understand and characterize the molecular mechanism behind VKCFD2. The bleeding phenotype can be investigated with the help of iHEPs and the unclear role of p.Arg98Trp in extra-hepatic phenotypes could be elucidated by differentiating hiPSCs into other tissue specific cell types owing to the pluripotent nature of hiPSCs.