Patient specific JAK2 V617F iPS cells for modelling bone marrow fibrosis

Böhnke, Janik; Zenke, Martin (Thesis advisor); Wagner, Wolfgang (Thesis advisor); Pradel, Gabriele (Thesis advisor)

Aachen : RWTH Aachen University (2022, 2023)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022

Abstract

Bone marrow fibrosis is the central pathologic feature in myelofibrosis. The malignant proliferation of hematopoietic clones with abnormal cytokine expression causes an increased production of extracellular matrix (ECM) proteins from mesenchymal stem/stromal cells (MSC), leading to fibrotic tissue. Myelofibrosis develops as a primary disease (primary myelofibrosis, PMF) or a secondary disease from myeloproliferative neoplasms (MPN) such as polycythemia vera (PV). Here, the Janus kinase JAK2 is mutated in over 95% of cases. The gain-of-function mutation JAK2 V617F leads to phosphorylation of the tyrosine kinase without ligand binding (such as thrombopoietin (TPO) or erythropoietin (EPO)). This results in permanent activation of blood cell proliferation, especially of megakaryocytes and platelets. In this work, induced pluripotent stem cell (iPS cell) clones from three PV patients with JAK2 V617F mutation were used. JAK2 V617F was repaired in one iPS cell clone using CRISPR/Cas9 technology. In addition, a functional knockout of the chemokine CXC motif ligand 4 (CXCL4, also known as platelet factor 4, PF4), which is suspected of playing an essential role in establishing bone marrow fibrosis, was performed in two iPS cell clones. Next, a protocol to differentiate iPS cells into hematopoietic stem cells (HSC) and megakaryocytes was established and further developed in our laboratory. The generated megakaryocytes had significant differences caused by the JAK2 genotype. JAK2 V617F megakaryocytes showed a strong differentiation bias towards erythrocytes, accelerated differentiation kinetics with faster maturation, and TPO-independent development of megakaryocytes. Furthermore, transcriptome analysis revealed differences in gene expression, such as downregulation of ECM genes in JAK2 V617F megakaryocytes. The generated megakaryocytes were used together with BM-MSC (bone marrow MSC)for a 2D co-culture model to investigate the interactions between these two cell types. Here, no increased fibrosis was detected in JAK2 V617F clones compared to the controls. Altogether, a broad set of MPN patient-specific iPS cell clones was established, which were successfully differentiated to HSC and their progeny megakaryocytes with a protocol established in our lab. Combined with BM-MSC, preliminary experiments of co-culture models demonstrate a fundamental basis for further functional studies on myelofibrosis.

Institutions

• Department of Cellular and Applied Infection Biology [164020]
• Department of Biology [160000]
• [811002-2]