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854 Characterizing Coagulation FVII from iPSC-Hepatocytes-like Cells: Setting the Basis for Cell Therapy Development

Program: Oral and Poster Abstracts
Session: 321. Blood Coagulation and Fibrinolytic Factors: Poster I
Hematology Disease Topics & Pathways:
Bleeding Disorders, Diseases, Bleeding and Clotting, Hemostasis, iPSCs, Cell Lineage
Saturday, December 5, 2020, 7:00 AM-3:30 PM

Maria Eugenia Chollet, MD, PhD1,2*, Elisabeth Andersen, MSc, PhD1,2*, Maria Eugenia de la Morena-Barrio, PhD3,4*, Carlos Bravo-Perez, MD3,4*, Marie-Christine Mowinckel1,2*, Christiane Filion Myklebust, MSc1,2*, Ellen Skarpen, PhD5*, Bernd Thiede, PhD6*, Benedicte Stavik, PhD1,2*, Javier Corral, PhD3,4*, Gareth J Sullivan, PhD7,8* and Per Morten Sandset, MD, PhD1,2,9

1Department of Hematology, Oslo University Hospital, Oslo, Norway
2Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway
3Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, University of Murcia, Murcia, Spain
4Centro Regional de Hemodonación, IMIB-Arrixaca, CIBERER, University of Murcia, Murcia, Spain
5Core Facility for Advanced Light Microscopy, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway
6Department of Bioscience, University of Oslo, Oslo, Norway
7Norwegian Stem Cell Center, Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
8Department of Pediatrics, Oslo University Hospital, Oslo, Norway
9Institute of Clinical Medicine, University of Oslo, Oslo, Norway

To date studies on factor (F)VII and other hepatic vitamin K-dependent coagulation factors have relied on cell lines overexpressing these human genes. Even though these models have provided insight into the biology of these factors, they do not fully illustrate the in vivo situation. Thus, a relevant physiological model that mimics the in vivo processing of FVII in liver cells with potential for therapeutic use is needed.

Human induced pluripotent stem cells (hiPSCs) were differentiated into hepatocyte-like cells (iHLCs) using a non-transcription factor based, small molecule approach. Cells were grown in medium with vitamin K to ensure a correct gamma-carboxylation. Cellular FVII mRNA and protein were determined by RT-qPCR and proteomic and Western blot (WB), respectively. Secreted FVII antigen was measured by ELISA and WB and FVII activity was assessed by chromogenic assay and thrombin generation assay (TGA). Post-translational modifications of FVII protein (glycosylation) were studied using digestion with N-glycosylase F (PNGase F) and neuraminidase. Confocal immunofluorescence microscopy was used to assess the cellular expression of FVII and other vitamin K- dependent coagulation factors and inhibitors. Human primary hepatocytes or human plasma pool were used as a control in the assays.

The resulting iHLCs expressed FVII mRNA in comparable levels to primary hepatocytes and cellular FVII peptides were identified by mass spectrometry studies. iHLCs secreted FVII at levels of around 70% compared to primary hepatocytes with detectable activity around 35% of the FVII activity level from primary hepatocytes. The TGA showed that cell medium from iHLCs when mixed with FVII deficient plasma was able to induce thrombin generation faster than the FVII depleted plasma alone (lagtime 3.2 vs 27.6 s, respectively). PNGase-F treatment showed that FVII secreted by iHLCs was N-glycosylated. Intracellular FVII was detected by WB as a band of approximately 63 kDa, slightly larger than FVII from plasma pool but similar to FVII from primary hepatocytes. Moreover, additional coagulation factors and inhibitors such as FII, FX, protein C and antithrombin were detected both at the mRNA and protein levels in the cells.

Stem cell-derived iHLCs produce and secrete FVII at physiologically relevant levels. The resulting FVII showed similar post-translational modifications to plasma FVII although some differences in proteolysis could be inferred. This iHLCs-derived FVII is able to initiate the extrinsic coagulation pathway. Our data support that these iHLCs can serve as a highly relevant model to study FVII and other vitamin K-dependent coagulation factors in vitro and constitute an important step towards the development of novel cell-based therapies for both FVII and other vitamin K-dependent coagulation factor deficiencies.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH