JAK2V617F Mutated Endothelium Has a Stem Cell Potential in Primary Myelofibrosis: a New Disease Ontogenesis for Chronic Myeloproliferative Disorders

May 19, 2026 updated by: Vittorio Rosti, Fondazione IRCCS Policlinico San Matteo di Pavia
The present project aims to experimentally test the hypothesis that JAK2V617F mutated endothelium in primary myelofibrosis (PMF) we discovered in 2013 represents an embryonic-like hemogenic endothelium and is part of the malignant stem cell pool that give rise to clonal hematopoiesis. The hypothesis stems from the recent evidence that JAK2 mutation in PMF patients is acquired in utero or childhood. The experimental plan of the project is 1) to separate mature endothelial cells from peripheral blood of PMF patients for morphological, molecular, functional characterization and clonogenic assays; 2) to assay the megakaryocyte, erythroid, granulocyte and endothelial clonogenic potential of the mature endothelial cells of patients with PMF. The potential to form colonies will be the documentation that a fraction of mature endothelial cells of patients with PMF have the characteristics of embryonic hemogenic endothelium and might act as the cell of origin of PMF.

Study Overview

Status

Completed

Conditions

Detailed Description

This project stems from our previous original observations that endothelial cells lining the vessel wall of splenic venules and capillaries of PMF patients harbour the JAK2V617F mutation (Piaggio G, Rosti V Blood 2009 and Rosti V Blood 2013) that is found in the hematopoietic cells of patients with myelofibrosis. Subsequently, this finding was further confirmed by other groups (Teofili L Blood 2011 and Farina M Cells 2021), although a definitive agreement on the existence of mutated endothelium in PMF has not been reached and the role of mutated endothelial cells in PMF pathogenesis continues to be questioned.

These observations support the hypothesis that endothelial cells and hematopoietic cells may derive from a common progenitor cell, the hemangioblast, in which the driver mutation(s) arise, thus resulting in mutated endothelial and hematopoietic cells. Whereas in mice evidences that hemangioblast exists have been documented (Fadlullah M Blood 2022), in human a definitive demonstration is still lacking. The demonstration that sorted mature endothelial cells share genetic and functional features with hematopoietic cells and that they retain the capacity to differentiate in culture into hematopoietic progenitor cells will clarify the role and significance of circulating mutated mature endothelial cells in patients with primary myelofibrosis. The significance of the project resides in its novel interpretation of the pathogenesis of PMF and, more in general, of chronic myeloproliferative diseases. The cell of origin of the disease is not anymore identified in a hematopoietic stem/progenitor cell, which acquire a mutation during the adult life and, by its clonal proliferation, results in the development of the disease. In this project the cell of origin of PMF is identified in a cell with hemogenic endothelium potential, that acquires the driver mutation during either fetal or adult life. This cell would give rise to mutated hematopoietic and endothelial cells and, in turn, by its clonal proliferation driven by acquired mutation(s) or an altered microenvironment, to the onset of the disease.

Specific Aim 1 Separation and isolation of mature endothelial cells and hematopoietic progenitor cells from peripheral blood of patients with PMF and normal subjects.

Due to the scant data available in the literature on the frequency of the events that will be investigated in this project, it isnot possible to define a sample size of patients to be included in the study. The Center for the Study of Myelofibrosis at Policlinico San Matteo Foundation assures the accrual of a large number of patients and, in turn, it guarantees that all patients undergoing new diagnostic procedures will be asked to participate to the study. All the procedures will be performed after signing a written informed consent approved by the local IRB of Policlinico San Matteo, Pavia. Peripheral blood samples (20-40 ml) will be obtained from a minimum of 20 patients with a diagnosis of primary myelofibrosis (WHO 2016) and positivity for the JAK2V617F mutation, since this is the most frequently found mutation among the driver mutations in patients. Patients with a previous history of deep vein thrombosis will be included in the study. An equal number of healthy subjects will be used as controls. Hematopoietic progenitor cells (HPCs) and mature endothelial cells will be isolated by sterile cell sorting. Twenty-40 milliliters of PB from patients with PMF or healthy controls will be collected in EDTA tubes; mononuclear cells (MNCs) will be separated by ficoll Hypaque. To allow the sorting of HPCs, half of the MNCs will be stained with the anti-CD45, which will discriminate between the hematopoietic and non hematopoietic cells, and the anti-CD34 monoclonal antibodies. The sorting of circulating mature endothelial cells will be performed by staining the remaining MNCs with endothelial lineage specific markers such as the anti-CD146, also known as the melanoma cell adhesion molecule, and anti-CD105, an endoglin protein expressed by activate endothelial cells, monocytes stromal cells and pre-B cells. The nuclear stain DAPI will be added to all the antibody combination. We expect a purity degree for the sorted cell populations greater than 95%. The sorted HPC and mature circulating endothelial cell populations will be further characterized by molecular studies that will compare by Next Generaation Sequencing (NGS) a panel of myeloidassociated mutations (see below).

Specific Aim 2 Morphological, molecular, and functional characterization of mature circulating endothelial cells in patients with PMF and normal subjects.

Sorted hematopoietic progenitor cells will be characterized by flow cytometry and/or confocal microscopy assessing the CD133, CD117, CD33, CD71, CD14, CD11b, CD41 and CD61 surface antigens. The mature endothelial cells will be characterized by testing the CD144, VEGFR2, CD31 surface antigen expression. In particular, thrombospondin1 and Angiotensin Converting Enzyme expression will be investigated on sorted endothelial cells according with recent observations (Wang Cell Stem Cell 2021 and Fadlullah M Blood 20222, respectively) suggesting that these markers identify cells belonging to the hemogenic endothelium. The genotype will be investigated both on bulk cells and at single cell level by means of the NGS Oncomine Myeloid Research Assay investigating 69 genes/fusion transcripts involved in the pathogenesis of myeloid malignancies. DNA and RNA will be extracted with Maxwell CSC Blood DNA and RNA kit, following the manufacturer¿s instructions. After nucleic acid quantification, we will follow the Oncomine Myeloid procedures (Thermo Fisher Scientific). The genomic and transcript analysis will be performed with an IonReporter software applying the last release of the myeloid workflow. Also, the singlecell RNA expression profile of these cells will be investigated by means of the unbiased 10x Genomics approach described in Falvo et al (Cancer Research 2021) and their phenotype by multiparametric flow cytometry as described in Hu-Lowe et al (Cancer Research 2011). Endothelial cells will also be characterized functionally. This will be done by in vitro tube formation assay (Matrigel assay) by plating mature endothelial cells, re-suspended in complete medium or EBM2 with 2.5% FCS to induce starvation conditions, in plates coated with Cultrex basement membrane extract, a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm mouse sarcoma, a tumour rich in extracellular matrix proteins and growth factors.

The total length of tube-like structures will be measured as the ratio of the tube length (mm)/surface of the field (mm2) by ImageJ software (National Institute of Health,USA, http://rsbweb.nih.gov/ij). The main targets of the tasks of this specific aim 2 are represented by a) the characterization of the sorted endothelial and hematopoietic cells and b) the identification of mutations and pathways that are shared between the 2 cell populations, thus confirming their common origin. This in turn will provide evidence that the acquisition of the driver mutation(s) of PMF is an event occurring in a common progenitor cell during human life (possibly during fetal life), able to give origin to both endothelial and hematopoietic lineages.

Specific Aim 3 In vitro assesment of hematopoietic differentiation and clonogenic potential of circulating endothelial cells in patients with PMF.

Cell culture experiments will be performed with the aim to investigate the differentiation and clonogenic hematopioetic potential of endothelial cells isolated from the blood of PMF patients. Clonogenic assays for the in vitro growth of CFU-GEMM, CFU-GM and BFU-E will be set up according with classical semisolid assays. Colonies obtained in these cultures will be characterized at morphological and molecular level as described for sorted cells in Specific Aim 2. With regard to the megakaryocyte lineage, in the last few years, we have set up a protocol to derive megakaryocytes (Mks) from human bone marrow and peripheral blood hematopoietic stem cells. Functional analysis of Mks obtained from our culture system showed that they can extend proplatelets and release functional platelets (PLTs). We plan to exploit our culture methods to differentiate Mks from both hematopoietic progenitors and endothelial cells isolated in Aim 1. To characterize Mks we will assess: a) the expression of CD61 and CD42b by flow cytometry, b) the capacity of forming proplatelets in adhesion to fibrinogen/fibronectin, c) the Thrombopoietin pathway activation by western blot (e.g., pSTAT, pERK, pAKT), d) the expression of different extracellular matrices (e.g., collagen type III, IV, fibronectin) by immunofluorescence imaging and western blot, e) the expression and release of growth factors will be analyzed by different cytokine arrays. Then, we will perform experiments to determine if Mks derived from the two different cell subsets show altered interactions with the extracellular matrix (ECM) components of the bone marrow (BM) microenvironment in terms of changes in morphology, adhesion, and maturation. We will correlate these results with biochemical studies to explore the capacity of Mks to assemble and anchor to the ECM. With these experiments we expect to be able to show the impact of Mks in remodeling matrix components related to the fibrotic BM environment. These experiments will be performed in 2D and then confirmed in our 3D bone marrow model. This 3 D bone marrow model will also be used to assess the capacity of Mks to produce platelets.

Study Type

Observational

Enrollment (Actual)

30

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Locations

  • Italy
    • Italy
      • Pavia, Italy, Italy, 27100
        • Fondazione IRCCS Policlinico San Matteo di Pavia

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

  • Adult
  • Older Adult

Accepts Healthy Volunteers

Yes

Sampling Method

Non-Probability Sample

Study Population

Healthy volunteers will be enrolled among people working in the Institutions where the project is conducted (San Matteo Foundation and Pavia University in Pavia and IEO in Milan)

Description

Inclusion Criteria:

  • availability to understand and sign the informed consent

Exclusion Criteria:

  • no known concurrent hematologic disease or condition that can affect the interpretation of the results

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

Cohorts and Interventions

Group / Cohort
Patients
Patients affected from primary myelofibrosis
Controls
Healthy subjects whoi volunteer participate to the study

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Isolation of endothelial cells
Time Frame: from month 1 to month 18
This will be done by cell sorting of peripheral blood samples from both patients and healthy subjects
from month 1 to month 18
Genotyping of sorted endothelial cells
Time Frame: from month 6 to month 30 of the project
endothelial cells sorted from patiens or healthy subjects will undergo DNA extraction and assessed for the presence of driver mutations of chronic myeloproliferative diseases
from month 6 to month 30 of the project
in vito culture of endothelail cells
Time Frame: from month 6 to month 36 of the project
Sorted endothelial cells will be cultured in vitro in a clonogenic assay in order to obtain differentiation into cells opf the hematopoietic lineage, mainly (but not exclusively megakaryocyte)
from month 6 to month 36 of the project

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

Fondazione IRCCS Policlinico San Matteo di Pavia

Collaborators

University of Pavia
IEO Foundation

Investigators

  • Principal Investigator: Vittorio Rosti, IRCCS Poiliclinico San Matteo Foundation, Pavia

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Actual)

May 2, 2023

Primary Completion (Actual)

February 28, 2026

Study Completion (Actual)

April 29, 2026

Study Registration Dates

First Submitted

May 19, 2026

First Submitted That Met QC Criteria

May 19, 2026

First Posted (Actual)

May 26, 2026

Study Record Updates

Last Update Posted (Actual)

May 26, 2026

Last Update Submitted That Met QC Criteria

May 19, 2026

Last Verified

May 1, 2026

More Information

Terms related to this study

Keywords

Additional Relevant MeSH Terms

Other Study ID Numbers

  • RF-2021

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

NO

IPD Plan Description

IPD are needed for the interpretation of some of the results (in particular genotyping data) but not required for the understanding and interèratation of final results of the study that will be published in aggergate form

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

Clinical Trials on Primary Myelofibrosids

Search Similar Trials

Sponsors and Collaborators

Medical Conditions

Drug Interventions

CROs by country

CROs in Argentina

Conditions

Rare Diseases

Drug Interventions

Dietary Supplements

Sponsor/Collaborators

Locations

Subscribe