Cedric Ghevaert

Senior Lecturer Transfusion Medicine

Email: cg348@cam.ac.uk

The Ghevaert research group

My research group is based at the Cambridge Blood Centre, NHS Blood and Transplant (NHSBT). My programme of research is based on the production of blood cells in vitro from haematopoietic stem cells (HSCs) or pluripotent stem cells (PSCs) with a particular interest in platelet and megakaryocyte biology. The group has a keen interest in translating biological discoveries into applications for transfusion medicine and uses a multidisciplinary approach that encompasses cell biology, engineering, and computational biology.

 

1. Blood cell production from human pluripotent stem cells

Human PSCs can be maintained and expanded in vitro for prolonged periods and can be induced to differentiate towards virtually any cell type. Therefore, they offer huge opportunities for basic research and clinical applications. Platelets and red cells are enucleated and therefore can be irradiated prior to administration to patients. This opens the possibility of generating banks of genetically-modified PSCs from which advanced cellular therapies can be generated.

A. Megakaryocyte production from human pluripotent stem cells

Current haematopoietic differentiation protocols from PSCs are hampered by a lack of efficiency, complicated cell handling, expensive cytokine cocktails, and the use of serum and xenogenic feeder cells, which all preclude clinical application of the end product. We are applying a forward programming method based on the overexpression of key transcriptional regulators to “force” cellular identity for the production of megakaryocytes (MKs), the blood platelet progenitors. In addition, using novel genome editing technologies, we are exploring novel ways to forward programme PSCs to megakaryocytes.

Cellular maturation varies from one PSC line to the other and in collaboration with Dr Nicole Soranzo (Wellcome Trust Sanger Institute, WTSI) we are investigating the genetic and epigenetic determinant of PSC-derived MK maturation variability.

Working group: Dr Thomas Moreau (post-doc), Dr Amanda Evans (post-doc), Dr Yumi Yan (post-doc, bioinformatician), Maria Colzani (PhD student), Amanda Dalby (PhD student), Wing Han-Wu (research assistant)

Key collaborators: Dr Nicole Soranzo (WTSI), Dr Barry Rosen (WTSI), Dr Marloes Tijssen (Department of Haematology), Dr Ludovic Vallier (Department of Surgery), Prof Roger Pedersen (Department of Surgery)

B. Red cell production from human pluripotent stem cells

The production of red cells from human PSCs is hampered by the fact that the cells produced have an embryonic phenotype, in particular expression of embryonic and fetal globins rather than adult globins and poor enucleation rate. We are part of the BloodPahrma consortium, funded through a strategic initiative from the Wellcome Trust. Its aim is to produce red cells from PSCs for transfusion into humans and to carry out first-in-man studies with cells produced in the laboratory. We are using our expertise in the forward programming approach to enforce an adult phenotype to PSC-derived red cells.

Working group: Dr Marloes Tijssen (Lead Scientist), Dr Nicola Foad (post-doc), Dr Yumi Yan (post-doc, bioinformatician), TBA (post-doc), TBA (research assistant)

Key collaborators: BloodPharma Consortium: Prof Marc Turner (SNBTS), Prof Lesley Forrester (University of Edinburgh), Dr Jo Mountford (University of Glasgow), Roslin Cells, Cell Therapy Catapult

C. Disease Modeling

Induced PSCs (iPSCs) can be derived from somatic cells from patients and used for basic research into disease mechanisms. The genes responsible for Gray Platelet Syndrome (NBEAL2) and Thrombocytopenia with Absent Radii (RBM8A) were identified in 2011/2012 by Prof Ouwehand’s and my group respectively. We are now using patient-derived iPSCs to investigate the role of these genes in megakaryocyte maturation and platelet formation.

Working group: Dr Amanda Evans (post-doc), Dr Thomas Moreau (post-doc)

Key collaborators: Prof Willem Ouwehand (Department of Haematology), Dr Jose Guererro (Department of Haematology), Dr Deborah French (CHOP, Philadelphia).

These projects are funded by the National Institute for Health and Research (NIHR) and the Wellcome Trust.

 

2. Artificial 3-dimensional bone marrow-like niche for blood cell maturation

Currently, in vitro blood cell formation for transfusion purposes is hampered by the inability to obtain end-stage mature cells. The bone marrow niche provides essential cues for blood cell maturation. We aim to recreate these cues, using a GMP-compatible method to translate into future manufacturing processes.

A. 3D functionalized scaffolds

Cell-to-cell contact is key to blood cell formation. We have used the exemplar of MKs, which release their platelets whilst tightly apposed to the bone marrow vascular cells. Supported by an award from the Medical Research Council (MRC) Regenerative Medicine Committee, we are generating a library of 400 recombinant proteins that reproduce the cell surface landscape of vascular cells in order to identify which proteins (and combinations thereof) give the positive signals for platelet release from MKs. In addition we have developed a collagen-based scaffold that functions as a “back-bone” 3D structure for in vitro culture of MKs. We are currently developing the biochemical process to functionalize this scaffold with proteins that positively regulate platelet formation.

B. Bioreactors and microfluidics

Mature blood cells extravasate from the bone marrow and the shear stress generated by the blood flow is an inherent part of the signals required for the final stage of blood cell maturation (in particular platelet release from MKs). My team is designing a bioreactor (and fluidics) into which to integrate the functionalized 3D scaffolds for optimal in vitro platelet production

Working group: Dr Meera Arumugam (post-doc), Maria Colzani (PhD student), TBA (post-doc), TBA (research assistant)

Key collaborators: Dr Gavin Wright (WTSI), Prof Ruth Cameron (Department of Material Sciences), Prof Serena Best (Department of Material Sciences), Prof Farndale (Department of Biochemistry), Dr Patricia Maguire (UCD Conway)

These projects are funded by the NIHR and the MRC.

 

3. Model organisms of platelet disorders

A. Mouse models of platelet disorders

i. Jak2 V617F myeloproliferative neoplasms

The JAK2 V617F mutation is present in 90% and 50% of patients with Polycythaemia Rubra Vera and Essential Thrombocythaemia respectively. The clinical hallmark of these conditions is an increased red cell/platelet count and increased risk of cardiovascular events. Using knock-in mouse models of heterozygous and homozygous Jak2V617F disease produced by Prof Anthony Green’s group, we analyse how the mutant JAK2 leads to increase platelet production and alters platelet function through a range of in vivo and cell biology assays.

ii. Nbeal-/- Gray Platelet Syndrome

The lack of NBEAL2 protein leads to Gray Platelet Syndrome in humans. We are using an Nbeal2-/- mouse model generated by the Sanger Institute to study granule formation and megakaryocyte maturation in the context of this disease.

Other mouse models of megakaryocyte/platelet disorders are currently under investigations stemming from the Sanger Institute Mouse Genetics Project or from targeting genes identified in studies of pedigrees with inherited platelet disorders (https://bridgestudy.medschl.cam.ac.uk/).

B. Preclinical studies of in vitro derived blood cells

Immunodeficient mice constitute an ideal organism to study human cell function in vivo. Studies of blood cells derived in the laboratory in these mice provide invaluable data of safety and efficacy prior to administration to humans.

Working group: Dr Jose Guerrero (post-doc), Cavan Bennett (research assistant), Dr Thomas Moreau (post-doc)

Key collaborators: Dr Dave Adams (WTSI), Prof Anthony Green (Department of Haematology), Prof Willem Ouwehand (Department of Haematology), Dr Marloes Tijssen (Department of Haematology)

These projects are funded by the British Heart Foundation (BHF).

 

4. Clinical studies related to platelet transfusion

A. Therapeutic antibodies for treatment of fetomaternal alloimmune thrombocytopenia (FMAIT)

Following a decade of research on the development of recombinant antibodies for antenatal treatment and prevention of FMAIT, we have carried out a first-in-man study in Cambridge confirming the therapeutic potential, which has paved the way for future Phase I studies in patients.

Working group: Nina Herbert (research nurse)

Key collaborators: Dr Lorna Williamson (NHSBT), Prof Willem Ouwehand (Department of Haematology), Dr Mike Clarke (Department of Pathology), Dr David Wilcox (Medical College of Wisconsin)

B. Neonatal platelet transfusion

Data gathered from NHSBT-led studies in neonates (PLaNeT1 and 2) suggests that platelet function as well as platelet count might be relevant to guide platelet transfusion to prevent bleeding in neonates (especially in extremely premature babies). We are currently looking at developing a bedside flow cytometry-based assay to address this issue.

Working group: Harriet McKinney (research assistant)

Key collaborators: Dr Anna Curley (Neonatal Intensive Care Unit, Rosie Maternity Hospital), Dr Simon Stanworth (University of Oxford)

C. Apheresis platelet donor study

After platelet apheresis, the donor platelet count drops by 30-50%. Little is known about the dynamics of platelet recovery in donors and which growth factors drive this recovery. In collaboration with the University of Oxford, we are collecting plasma samples and timed full blood cell counts from 30 platelet apheresis donors. Mass spectrometry techniques already applied in organ donations will be used to identify growth factors that could represent novel targets for therapy in thrombocytopenic patients. This work is supported by an award from the NHSBT Trust Fund.

Working group: Nina Herbert (research nurse)

Key collaborators: Prof Rutger Ploeg (University of Oxford)