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Objectives and questions: Vitamin K (VK) plays an essential role in supporting bone health and preventing bone pathologies. Homo- or compound heterozygous mutations in γ-glutamyl carboxylase (GGCX), the enzyme catalyzing VK–dependent (VKD) γ-carboxylation, cause a bleeding tendency classified as VKD coagulation factor deficiency type 1 (VKCFD1). In addition,VKCFD1 patients may experience diverse skeletal deformities such as mid-facial hypoplasia, stripling of fingers and growth delay. These most likely reflect defective γ-carboxylation of VKD proteins (VKDPs) involved in bone and cartilage metabolism. However, the molecular mechanisms underlaying γ-carboxylation impairment and how it affects bone formation is not well understood. While VK1 supplementation is often used to restore blood hemostasis, effective therapies targeting the non-hemostatic manifestations of VKCFD1 are still lacking. Therefore, there is an urgent need to investigate how GGCX mutations and VK metabolism affects bone and cartilage homeostasis. The aim of the current project was to establish an in vitro cell model based on induced mesenchymal stromal cells (iMSCs) to investigate VKD mechanisms and to improve potential therapies.

Material and methods: VKCFD1 patient-derived induced pluripotent stem cells (iPSCs) with and without skeletal abnormalities and healthy controls were differentiated into iMSCs and subsequently induced to differentiate into the osteogenic lineage. iMSCs were analyzed for MSC-specific characteristics. The osteogenic differentiation was assessed by Alizarin Red staining/extraction and alkaline phosphatase (ALP) staining/quantification. Expression of specific osteogenic markers was analyzed by RT-PCR and by ELISA.

Results: VKCFD1 patient-derived and control iMSCs showed comparable morphology, vitality, growth behavior and expressed typical MSC surface markers. Patient-derived iMSCs showed impaired osteogenic differentiation indicated by decreased Alizarin Red and ALP compared to control iMSCs. The relative mRNA expression and secretion of key osteogenic markers (ALPL,OCN,OPN,OPG,Runx2) were decreased in VKCFD1 patient-derived iMSCs compared to control iMSCs.

Discussion and conclusions: VKCFD1-iMSC models for osteogenic differentiation derived from patients have the potential to reveal precise pathophysiological pathways and identify targeted therapeutic strategies in a patient-specific environment. Therefore, the use of iMSC models to study the effects of VK deficiency and GGCX mutations on bone and cartilage development promises important insights for improving clinical treatment beyond coagulation correction. Future studies on patient-specific iMSC models will address the question of whether certain forms of VK, in particular VK2, can improve the γ-carboxylation of relevant extrahepatic VKDPs and thereby restore bone and cartilage formation in GGCX-deficient cells. In the long term, a deeper mechanistic understanding of GGCX deficiency may reveal potential effects on bone quality in VKCFD1 patients.