JAK2V617F - Positive Endothelial Cells Display Pro-Thrombotic Characteristics

• Published in: Blood 2016-12, Vol.128 (22), p.4273-4273
• Main Authors: Guadall, Anna, Lesteven, Elodie, Letort, Gil, Awan Toor, Sarah, Pognant, Doriane, Brusson, Megane, Verger, Emmanuelle, Larghero, Jerome, Vanneaux, Valerie, Chomienne, Christine, El Nemer, Wassim, Cassinat, Bruno, Kiladjian, Jean-Jacques
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V128.22.4273.4273

Background: Clinical complications of BCR-ABL1-negative Myeloproliferative Neoplasms (MPNs) include mainly vascular events and on longer term transformation to Myelofibrosis (MF) or acute leukemia. MPNs are also associated with neo-angiogenesis leading to extramedullary hematopoiesis and increased vascularity in the spleen and bone marrow. The most frequent molecular alteration present in hematopoietic cells is the JAK2V617F mutation, which has also been detected in hepatic Endothelial Cells (ECs) of MPN patients with Budd-Chiari syndrome (BCS) and in ECs derived from patients' splenic capillaries. The consequences of the presence of the JAK2V617F mutation into the endothelial lineage are not known although these cells play an important role in thrombosis. Methods: Isogenic JAK2 wild-type (WT) and JAK2V617F mutant induced pluripotent stem cells (iPSC) were derived from an MPN patient. The reprogrammed clonal cells were induced to differentiate toward mesoderm for 3 days and then driven toward the endothelial lineage using Stem-Pro medium, Vascular Endothelial Growth Factor (VEGF) and Forskolin. Enriched CD34+/CD144+ progenitors were then matured in VEGF-supplemented Stem-Pro medium for 5 days before being cultured in the EGM-2 specific EC medium in the presence of the TGF-β inhibitor SB-431542. Cells were harvested and tested for specific surface markers by flow cytometry before expanding them; passages 3 to 6 were used for further characterization and functional studies. Transcriptomic analysis was performed in triplicate from two independent endothelial differentiation experiments using Affymetrix HTA 2.0 gene expression arrays. Results: Phenotypically, the endothelial-like cells obtained after differentiation from both mutant and WT JAK2 iPSC displayed a classical EC morphology and were positive for CD34 and EC markers (CD144, CD309, CD31 and ICAM-1). Such characteristics were conserved for at least 6 rounds of cell passaging. TNF-α treatment increased ICAM-1 cell surface levels and induced the expression of VCAM-1, as observed in other EC models. Our iPS-derived cells were also responsive to VEGF, which induced cellular proliferation and angiogenesis demonstrated by tube formation in Matrigel with both JAK2V617F and WT ECs. As expected, JAK2V617F ECs exhibited higher levels of phosphorylated forms of JAK2, STAT3 and Akt in a serum and growth factor-reduced medium, showing that, as in hematopoietic cells, the presence of the mutation increases basal int