Improving haematopoietic reconstitution in blood stem cell transplantation procedures through the regulation of stem cells and their niches
Lead Research Organisation:
UNIVERSITY OF CAMBRIDGE
Department Name: Haematology
Abstract
Blood production is tightly controlled by the interactions of blood-forming stem cells residing inside the bones (in the bone marrow) with distinct specialised microenvironments, called niches. The niche cells protect blood forming stem cells from damage and maintain their potential to produce blood and immune cells throughout life. The applicant teams have contributed to dissect blood stem cells, their niches and their functional alterations in blood cancers.
Blood stem cell transplantation is routinely performed for lifesaving procedures in patients with blood cancers or inherited metabolic/immune disorders. Different blood stem cell sources, such as bone marrow, the peripheral blood, and cord blood are used for allogeneic transplantation. The use of cord blood presents several advantages, such as easy and noninvasive harvest, reduced risk of disease transmission, immediate availability of cryopreserved units and increased immune tolerance. However, the limited number of blood stem cells present in each cord blood has restricted its use to low-body-weight recipients (mostly children). Moreover, the recovery of the blood and immune system and the overall survival after cord blood transplantation correlates with the number of blood stem cells transplanted.
Regenerating normal blood production in the bone marrow of transplanted patients after requires:
1) Harvest of blood stem cells from donors in sufficient numbers.
2) Optimal migration and lodgement of transplanted blood stem cells in the bone marrow niches of transplanted recipients and
3) Expansion of engrafted blood stem cells and production of multiple blood and immune cells.
However,
A) On the donor side, due to the low blood stem cell number available in each cord blood unit, several cord units are necessary for transplantation and the HSC engraftment is delayed (compared with other blood stem cell sources, such as bone marrow or blood stem cells mobilised from the bone marrow to the peripheral blood).
B) On the recipient side (niche), the efficacy of blood stem cell engraftment significantly decreases during ageing, hampering the use of blood stem cell transplantation as a therapeutic option in many cases.
This proposal aims at understanding and modelling the regeneration process following blood stem cell transplantation. The main goal is to identify mechanisms controlling the engraftment and the expansion of blood stem cells after transplantation, and the subsequent production of blood and immune cells necessary for bone marrow regeneration. Our translational aim is instructing blood stem cells to more efficiently/rapidly/long-lastingly regenerate the blood and immune systems.
To investigate the process of bone marrow regeneration we will transplant human umbilical cord blood into genetically-modified mice which lack a functional immune system and therefore do no reject the human cells, allowing for the engraftment of human blood stem cells. Additionally, an artificial niche will be created allowing for the engraftment of human blood stem cells and human niche cells to model the human system. This joint project will enable a unique synergy between the 4 applicants with expertise in the cellular and molecular heterogeneity of human blood stem cells (Laurenti), their regulation through inflammatory factors (Takizawa), cellular metabolism (Suda) and microenvironmental cues (Mendez-Ferrer). The complementary expertise of the four teams could facilitate overcoming current limitations in HSC transplantation procedures.
Blood stem cell transplantation is routinely performed for lifesaving procedures in patients with blood cancers or inherited metabolic/immune disorders. Different blood stem cell sources, such as bone marrow, the peripheral blood, and cord blood are used for allogeneic transplantation. The use of cord blood presents several advantages, such as easy and noninvasive harvest, reduced risk of disease transmission, immediate availability of cryopreserved units and increased immune tolerance. However, the limited number of blood stem cells present in each cord blood has restricted its use to low-body-weight recipients (mostly children). Moreover, the recovery of the blood and immune system and the overall survival after cord blood transplantation correlates with the number of blood stem cells transplanted.
Regenerating normal blood production in the bone marrow of transplanted patients after requires:
1) Harvest of blood stem cells from donors in sufficient numbers.
2) Optimal migration and lodgement of transplanted blood stem cells in the bone marrow niches of transplanted recipients and
3) Expansion of engrafted blood stem cells and production of multiple blood and immune cells.
However,
A) On the donor side, due to the low blood stem cell number available in each cord blood unit, several cord units are necessary for transplantation and the HSC engraftment is delayed (compared with other blood stem cell sources, such as bone marrow or blood stem cells mobilised from the bone marrow to the peripheral blood).
B) On the recipient side (niche), the efficacy of blood stem cell engraftment significantly decreases during ageing, hampering the use of blood stem cell transplantation as a therapeutic option in many cases.
This proposal aims at understanding and modelling the regeneration process following blood stem cell transplantation. The main goal is to identify mechanisms controlling the engraftment and the expansion of blood stem cells after transplantation, and the subsequent production of blood and immune cells necessary for bone marrow regeneration. Our translational aim is instructing blood stem cells to more efficiently/rapidly/long-lastingly regenerate the blood and immune systems.
To investigate the process of bone marrow regeneration we will transplant human umbilical cord blood into genetically-modified mice which lack a functional immune system and therefore do no reject the human cells, allowing for the engraftment of human blood stem cells. Additionally, an artificial niche will be created allowing for the engraftment of human blood stem cells and human niche cells to model the human system. This joint project will enable a unique synergy between the 4 applicants with expertise in the cellular and molecular heterogeneity of human blood stem cells (Laurenti), their regulation through inflammatory factors (Takizawa), cellular metabolism (Suda) and microenvironmental cues (Mendez-Ferrer). The complementary expertise of the four teams could facilitate overcoming current limitations in HSC transplantation procedures.
Technical Summary
Haematopoietic stem cell (HSC) transplantation (HSCT) is routinely performed in patients with haematological, genetic or metabolic disorders. Regenerating haematopoiesis after HSCT requires: 1) harvesting sufficient donor HSCs, 2) optimal HSC homing to the bone marrow and 3) expansion and differentiation of donor HSCs in the recipient. However, HSC engraftment efficacy significantly decreases during ageing and in certain haematological diseases, hampering the use of HSCT as a therapeutic option. Among different HSC sources for transplantation, umbilical cord blood (CB) is an ideal source that can be non-invasively harvested, cryopreserved and is associated with reduced risk of disease transmission or acute graft-versus-host disease. However, several CB units are necessary for transplantation and engraftment is delayed.
This proposal aims at understanding and modelling the regeneration process following HSCT. The main goal is to identify cell-intrinsic and -extrinsic mechanisms that regulate HSC proliferation and lineage commitment during bone marrow regeneration. Our translational aim is instructing HSC to more efficiently/rapidly/long-lastingly regenerate the haematopoietic and immune systems. We will use xenografts and ectopic bone ossicles to model human HSCT in a humanised niche environment. Human cells will be analysed by 10x scRNA-seq and scATAC-seq to generate a cell-cell communication network between human HSCs and niche cells. Metabolic profiling (glycolysis, OXPHOS, mitochondrial activity, ROS) will be complemented with and Mass Cytometry and imaging studies. Finally, the role and potential of novel regulators of HSC quiescence and emergency haematopoiesis will be tested. The complementary expertise of the four teams in the cellular and molecular heterogeneity of human HSCs, their regulation through inflammatory factors, cellular metabolism and microenvironmental cues could facilitate overcoming current limitations in HSCT.
This proposal aims at understanding and modelling the regeneration process following HSCT. The main goal is to identify cell-intrinsic and -extrinsic mechanisms that regulate HSC proliferation and lineage commitment during bone marrow regeneration. Our translational aim is instructing HSC to more efficiently/rapidly/long-lastingly regenerate the haematopoietic and immune systems. We will use xenografts and ectopic bone ossicles to model human HSCT in a humanised niche environment. Human cells will be analysed by 10x scRNA-seq and scATAC-seq to generate a cell-cell communication network between human HSCs and niche cells. Metabolic profiling (glycolysis, OXPHOS, mitochondrial activity, ROS) will be complemented with and Mass Cytometry and imaging studies. Finally, the role and potential of novel regulators of HSC quiescence and emergency haematopoiesis will be tested. The complementary expertise of the four teams in the cellular and molecular heterogeneity of human HSCs, their regulation through inflammatory factors, cellular metabolism and microenvironmental cues could facilitate overcoming current limitations in HSCT.
Planned Impact
Our proposal aims to provide new insights into how blood regeneration occurs during the early stages of haematopoietic stem cell transplantation (HSCT). We will use innovative humanised models, metabolomics and single cell transcriptomics technologies to investigate the interplay between the microenvironment and blood stem cells that underpins successful long-term blood formation after HSCT.
Beyond its academic impact, the knowledge gained from this project may have a broad importance for society in 2 ways:
- Impact area 1: improvement of current clinical protocols for transplantation. This could lead to considerable improvements to quality of life for HSCT patients. We will thus strive to identify all findings with clinical potential early on and initiate steps towards enacting these translational avenues in a coordinated and timely fashion. Given the novelty of the techniques used and the preliminary data ensuring the feasibility of the project, we expect the data generated in this proposal to have clinical relevance. We will identify key cellular and molecular interactions between the human blood stem cells and their complex bone/vasculature microenvironment. We propose that pharmacological targeting of the microenvironment can provide health benefits to HSCT patients (such as faster blood recovery, diminished health impact of transplantation conditioning regimens or longer-lasting effects of the transplantation).
- Impact area 2: starting point for a dialogue with the public on regenerative medicine and stem cell therapies. Stem cells and their potential for regenerative medicine are the focus of considerable public interest. HSCT is the oldest cellular therapy and is well-known to the public. This project is thus ideally suited to engage with the public, explaining how blood is maintained over a human lifetime and how cellular therapies can regenerate a functioning blood system. In return, we will gain information on the perceived challenges and ethical issues associated with stem cell therapies from various public audiences. Building on our collective expertise, we are confident that our outreach plan will provide an excellent platform to engage in a dialogue with general members of the public, tissue donors or cell therapy patients. Our primary aim is to transfer knowledge about blood formation and HSCT to a wide audience and in return gain insights into public perception of stem cells and regenerative medicine.
The main scientific goal of this proposal is to understand how blood stem cells interact with their microenvironment during the early stages of HSCT. We would like to share our findings with the public in order to improve awareness of stem cell biology, how tissue regeneration is coordinated with and dependent on other tissues in the body and how these fundamental stem cell biology concepts can be harnessed to improve human health. The blood system is particularly well suited to illustrating fundamental concepts in these areas and attracts high levels of public interest due to the prevalence of blood related diseases, including cancers. We therefore plan to develop an integrative public engagement programme, that capitalises on the existing public engagement infrastructure at CSCI and on the track-record of the UK applicants.
Beyond its academic impact, the knowledge gained from this project may have a broad importance for society in 2 ways:
- Impact area 1: improvement of current clinical protocols for transplantation. This could lead to considerable improvements to quality of life for HSCT patients. We will thus strive to identify all findings with clinical potential early on and initiate steps towards enacting these translational avenues in a coordinated and timely fashion. Given the novelty of the techniques used and the preliminary data ensuring the feasibility of the project, we expect the data generated in this proposal to have clinical relevance. We will identify key cellular and molecular interactions between the human blood stem cells and their complex bone/vasculature microenvironment. We propose that pharmacological targeting of the microenvironment can provide health benefits to HSCT patients (such as faster blood recovery, diminished health impact of transplantation conditioning regimens or longer-lasting effects of the transplantation).
- Impact area 2: starting point for a dialogue with the public on regenerative medicine and stem cell therapies. Stem cells and their potential for regenerative medicine are the focus of considerable public interest. HSCT is the oldest cellular therapy and is well-known to the public. This project is thus ideally suited to engage with the public, explaining how blood is maintained over a human lifetime and how cellular therapies can regenerate a functioning blood system. In return, we will gain information on the perceived challenges and ethical issues associated with stem cell therapies from various public audiences. Building on our collective expertise, we are confident that our outreach plan will provide an excellent platform to engage in a dialogue with general members of the public, tissue donors or cell therapy patients. Our primary aim is to transfer knowledge about blood formation and HSCT to a wide audience and in return gain insights into public perception of stem cells and regenerative medicine.
The main scientific goal of this proposal is to understand how blood stem cells interact with their microenvironment during the early stages of HSCT. We would like to share our findings with the public in order to improve awareness of stem cell biology, how tissue regeneration is coordinated with and dependent on other tissues in the body and how these fundamental stem cell biology concepts can be harnessed to improve human health. The blood system is particularly well suited to illustrating fundamental concepts in these areas and attracts high levels of public interest due to the prevalence of blood related diseases, including cancers. We therefore plan to develop an integrative public engagement programme, that capitalises on the existing public engagement infrastructure at CSCI and on the track-record of the UK applicants.
Organisations
- UNIVERSITY OF CAMBRIDGE (Lead Research Organisation)
- Luxembourg Institute of Health (Collaboration)
- Galvani Bioelectronics Ltd (Collaboration)
- University of Helsinki (Collaboration)
- UCB Pharma (Collaboration)
- Kumamoto University (Collaboration)
- IMPERIAL COLLEGE LONDON (Collaboration)
- Genentech, Inc (Collaboration)
- UNIVERSITY OF CAMBRIDGE (Collaboration)
- Weizmann Institute of Science (Collaboration)
Publications
Fang Z
(2023)
Tamoxifen for the treatment of myeloproliferative neoplasms: A Phase II clinical trial and exploratory analysis
in Nature Communications
Fibbe W
(2022)
The EHA Research Roadmap: Hematopoietic Stem Cells and Allotransplantation.
in HemaSphere
Fielding C
(2020)
Neuronal regulation of bone marrow stem cell niches.
in F1000Research
Fielding C
(2022)
Cholinergic signals preserve haematopoietic stem cell quiescence during regenerative haematopoiesis.
in Nature communications
Gadomski S
(2022)
A cholinergic neuroskeletal interface promotes bone formation during postnatal growth and exercise.
in Cell stem cell
García-García A
(2020)
The Autonomic Nervous System Pulls the Strings to Coordinate Circadian HSC Functions.
in Frontiers in immunology
| Description | Awareness of future institute goals |
| Geographic Reach | Local/Municipal/Regional |
| Policy Influence Type | Influenced training of practitioners or researchers |
| Impact | Publicity for Institute, awareness of future institute goals. |
| Description | A Discovery Research Tissue Scale Biology Platform |
| Amount | £9,995,621 (GBP) |
| Organisation | Wellcome Trust |
| Department | Wellcome Trust Clinical Research Facility (Cambridge) |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 06/2024 |
| End | 06/2031 |
| Description | CRUK MRes in Cancer Biology -PhD in Haematology Studentship (Livia Lisi Vega, PhD student in our group) |
| Amount | £76,000 (GBP) |
| Funding ID | CRUK MRes in Cancer Biology -PhD in Haematology Studentship |
| Organisation | Cancer Research UK |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 09/2020 |
| End | 09/2023 |
| Description | Development of a clinical program for myeloid cancer prevention |
| Amount | $5,000,000 (USD) |
| Organisation | The Leukemia & Lymphoma Society |
| Sector | Charity/Non Profit |
| Country | United States |
| Start | 02/2024 |
| End | 01/2029 |
| Description | ERC Consolidator Grant, ERC-2014-CoG-648765. Horizon 2020. ECREA |
| Amount | £1,265,475 (GBP) |
| Organisation | European Research Council (ERC) |
| Sector | Public |
| Country | Belgium |
| Start | 08/2015 |
| End | 08/2020 |
| Description | Gates Cambridge Scholarship (Stephen Gadomski PhD student) |
| Amount | £30,000 (GBP) |
| Organisation | Bill and Melinda Gates Foundation |
| Sector | Charity/Non Profit |
| Country | United States |
| Start | 08/2019 |
| End | 08/2023 |
| Description | Improving Haematopoietic reconstitution in Blood stem cell transplantations |
| Amount | £371,031 (GBP) |
| Organisation | Medical Research Council (MRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 09/2020 |
| End | 09/2023 |
| Description | Improving Haematopoietic reconstitution in Blood stem cell transplantations-Research Nurse and bioinformatician |
| Amount | £61,602 (GBP) |
| Organisation | Medical Research Council (MRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 09/2020 |
| End | 09/2023 |
| Description | John Goldman Fellowship (Eman Khatib-Massalha, Res Associate in our group) |
| Amount | £150,000 (GBP) |
| Organisation | Leukaemia UK |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 11/2022 |
| End | 10/2024 |
| Description | Microenvironmental metabolic regulation in myeloid malignancies |
| Amount | £752,449 (GBP) |
| Funding ID | 26670 |
| Organisation | Cancer Research UK |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 07/2018 |
| End | 07/2024 |
| Description | OXCAM-NIH Program (Stephen Gadomski PhD student) |
| Amount | £80,000 (GBP) |
| Organisation | National Institute for Health Research (NIHR) |
| Department | Oxford-Cambridge Scholars Program |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 08/2019 |
| End | 08/2023 |
| Description | Phase II clinical study: 'TAMARIN: Effects of TAMoxifen on the Mutant Allele Burden and Disease Course in Patients with MyeloprolifeRatIve Neoplasms'. EudraCT number: 2015-005497-38. Role: co-investigator. (PI, Claire Harrison). |
| Amount | £338,755 (GBP) |
| Funding ID | RG-15-234 |
| Organisation | Bloodwise |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 12/2015 |
| End | 11/2019 |
| Description | Targeting Cholinergic signalling in graft-versus-host disease |
| Amount | £26,066 (GBP) |
| Funding ID | 123920 |
| Organisation | Isaac Newton Group |
| Sector | Public |
| Country | Spain |
| Start | 01/2023 |
| End | 09/2024 |
| Description | Understanding and targeting microenvironmental metabolic regulation in myeloid Malignancies (Dorian Forte Research Associate) |
| Amount | € 94,502 (EUR) |
| Organisation | AIRC Foundation for Cancer Research in Italy |
| Sector | Public |
| Country | Italy |
| Start | 02/2018 |
| End | 06/2020 |
| Description | WP15-10. Theme 6: Stem cells and immunotherapies. Title: Haematopoietic stem-cell niche |
| Amount | £1,479,040 (GBP) |
| Organisation | NHS Blood and Transplant (NHSBT) |
| Sector | Public |
| Country | United Kingdom |
| Start | 02/2015 |
| End | 03/2024 |
| Title | Heterotopic ossification in mice overexpressing Bmp2 in Tie2+ lineages |
| Description | Bone morphogenetic protein (Bmp) signaling is critical for organismal development and homeostasis. To elucidate Bmp2 function in the vascular/hematopoietic lineages we generated a new transgenic mouse line in which BMP2 expression is controlled by the Tie2 promoter. Tie2CRE/+;Bmp2tg/tg mice develop aortic valve dysfunction post-natally, accompanied by pre-calcific lesion formation in valve leaflets. Remarkably, Tie2CRE/+;Bmp2tg/tg mice develop extensive soft tissue bone formation typical of acquired forms of heterotopic ossification (HO) and bone genetic disorders, such as Fibrodysplasia Ossificans Progressiva (FOP). Ectopic ossification in Tie2CRE/+;Bmp2tg/tg transgenic animals is accompanied by increased bone marrow (BM) hematopoietic, fibroblast and osteoblast precursors and circulating pro-inflammatory cells. Transplanting wild-type (WT) BM hematopoietic stem cells (HSC) into lethally irradiated Tie2CRE/+;Bmp2tg/tg mice significantly delays HO onset but does not prevent it. Moreover, transplanting Bmp2-transgenic BM into WT recipients does not result in HO, but hematopoietic progenitors contribute to inflammation and ectopic BM colonization rather than to endochondral ossification. Conversely, aberrant Bmp2 signaling activity is associated with fibroblast accumulation, skeletal muscle fiber damage, and expansion of a Tie2+ fibro-adipogenic precursor cell population, suggesting that ectopic bone derives from a skeletal muscle resident osteoprogenitor cell. Thus, Tie2CRE/+;Bmp2tg/tg mice recapitulate HO pathophysiology, and might represent a useful model to investigate therapies seeking to mitigate disorders associated with aberrant extra-skeletal bone formation. |
| Type Of Material | Model of mechanisms or symptoms - mammalian in vivo |
| Year Produced | 2021 |
| Provided To Others? | Yes |
| Impact | New animal model of Fibrodysplasia Ossificans Progressiva |
| URL | https://www.nature.com/articles/s41419-021-04003-0 |
| Title | Multicolor immunofluorescence staining of paraffin-embedded human bone marrow sections |
| Description | Immunofluorescence is an indispensable method for the identification, localization and study of the expression of target antigens in formalin-fixed, paraffin-embedded (FFPE) tissue sections of human bone marrow. However, the procedure shows technical weakness because of the chemical and physical treatments required for sample processing before imaging. Here we describe a revisited protocol to obtain high-resolution images of human bone marrow trephine biopsies, improving the antigen-antibody recognition and preserving the morphology and the architecture of the bone marrow microenvironment. Introduction The hematopoietic stem cell (HSC) niche is an anatomical and functional microenvironment where HSCs are maintained in an undifferentiated and self-renewing state and receive stimuli that determine their fate [1]. Several cellular components of the HSC niche such as mesenchymal cells, endothelial cells, perivascular stromal cells, nerves belonging to the sympathetic nervous system (SNS), nonmyelinating Schwann cells, adipocytes, osteolineage cells, structural components of the extracellular matrix, ions (Calcium, Magnesium and phosphate), vesicles, cytokines, chemokines, hormones and growth factors influence HSC self-renewal, proliferation, differentiation or migration [2-4]. Given this complexity, studying the HSC niche requires choosing optimal strategies for sample preparation and imaging to evaluate the expression and the localization of proteins [5-9]. Immunofluorescence on tissue sections represents one of the most amenable techniques which has been very powerful to study mouse HSC niches using cryopreserved samples. However, cryoblocks are rarely available for human samples since these samples are routinely fixed with formalin and embedded in paraffin for histological examination and diagnostic procedures. Unfortunately, this processing limits the capacity to detect antigens in paraffinized human samples [10,5]. In fact, tissue treated with aldehyde fixatives and included in hydrocarbon compounds show less antigenicity and generate autofluorescence and/or background fluorescence which decrease the quality of the final result. Antigenic heat-mediated exposure or unmasking of antigens can improve the quality of the antigen-antibody reaction, but tends to damage thin bone marrow [11]. Microwave heating methods alternating heating intervals can sometimes yield better results. The antibody washing steps are particularly relevant to reduce the background noise, especially in the case of samples pre-treated with signal enhancers. One key advantage of paraffin inclusion of the specimen is its long storage, which allows retrospective clinical examination. The availability of these samples is becoming increasingly on demand to understand the pathological alterations of the human bone marrow microenvironment. Accomplishing this goal requires in-depth knowledge of the physiopathological human bone marrow architecture, but our knowledge in this regards is very limited compared with the information generated in the mouse system. Post-diagnostic paraffinized human bone marrow (trephine) biopsies could represent an invaluable source to increase our knowledge of the human bone marrow. For this reason, we describe here a protocol that allows multicolor staining of thin human bone marrow sections for high resolution fluorescence imaging. Briefly, after dewaxing samples are maintained always hydrated by using washing buffers (D-PBS and 0.5% Triton-X 100). Antigen retrieval is carried out by heating in a bain-marie three times (7'+4'+4') with steps of thermal shocks. Non-specific fluorescence (background), which is a commonly issue when applying fluorescent conjugates to paraffinized sections, is largely eliminated by using glycine, washing sections three times (after antigen antibodies reactions) and separating the incubations of secondary antibodies. These optimized critical steps and examples of the resulting images (Figure 1) are described below in detail. Key Words: Immunofluorescence, FFPE, antigen retrieval, microwave heating, fixative. Materials 1. Xylene (or other dewaxing/clearing reagents), 200 ml. 2. Absolute ethanol, 200 ml. 3. 96% Ethanol: Add 192 ml of absolute ethanol to a 200 ml graduate cylinder and add 8 ml of distilled water. Mix well and transfer the solution in a glass Coplin jar. 4. 70% Ethanol: Add 140 ml of absolute ethanol to a 200 ml graduate cylinder and add 60 ml of distilled water. Mix well and transfer the solution in a glass Coplin jar. 5. 50% Ethanol: Add 100 ml of absolute ethanol to a 200 ml graduate cylinder and add 100 ml of distilled water. Mix well and transfer the solution in a glass Coplin jar. 6. Citrate buffer 0.1 M, pH 6.0: add 100 ml of distilled water to a 100 ml graduate cylinder and transfer into a glass beaker. Weigh 0,210 g of citric acid and transfer to the glass beaker. Use a magnetic stirrer to dissolve citric acid. Add 500 ml of distilled water to a 1 l graduate cylinder and transfer into a glass beaker. Weigh 1.47 g of sodium citrate dehydrate and transfer to the glass beaker. Use a magnetic stirrer to dissolve dehydrated sodium citrate. Discard 80 ml of sodium citrate dehydrated and adjust pH with citric acid solution (pH 6). 7. Two microwavable containers and a glass tray. 8. Dulbecco's phosphate buffered saline solution (D-PBS). 9. 10% (v/v) Permeabilization stock solution (PRM): Add 90 ml of D-PBS to a 100 ml graduated cylinder. Transfer the D-PBS into a glass bottle (100ml) and pipette 10 ml of Triton X-100 slowly and dissolve completely. At the time of staining, dilute the stock solution with D-PBS to obtain 0.5% (v/v) of working solution. Store stock solution at RT temperature (2 months) or at 4 °C. 10. Glycine (mw: 75.07 g/mol): dissolve glycine 0.3 M to D-PBS. Mix with a magnetic stirrer, store at 4°C (1 month) and filter the solution before the use. 11. Blocking solution: mix 10% (v/v) normal serum and 0,5% (w/v) BSA (Bovine Serum Albumin). Dissolve BSA in sterile D-PBS at 4 °C and add normal serum. Mix with a magnetic stirrer. Store solution at 4°C. 12. Primary antibody solution: mix 1% (v/v) normal serum and 0.5% (w/v) BSA in 0.5% Triton X-100. Dissolve BSA in sterile D-PBS at 4 °C. 13. Secondary fluorochrome-conjugated antibody solution: mix 1% (v/v) normal serum and 0.5% (w/v) BSA in 0.5% Triton X-100. 14. Nuclear dye. 15. Cover glasses, tweezers and aqueous mounting medium supplemented with anti-fade reagent. Methods 1. Cut tissue sections using a low-profile disposable blade to obtain sections (3-15 µm). 2. Transfer sections to microscope slides at room temperature (RT) and dry at RT for at least 48 h before starting the immunostaining (See Note 1). 3. Immerse slides in Xylene (or other dewaxing/clearing reagents, 200 ml) for 10 minutes (repeat twice). Remove slides from the solution. 4. Hydrate (immersion) by graded alcohol series (200 ml): absolute ethanol for 10 minutes (repeat twice), 96% ethanol for 10 minutes, 70% ethanol for 10 minutes, 50% ethanol for 10 minutes. 5. Immerse the slides for 10 minutes in distilled water (see Note 2). 6. Perform the antigen retrieval. Prepare two microwavable containers containing 80-100 ml of 0.1 M citrate buffer pH 6.0 (4 °C). Place slides in one of them. Prepare a glass tray and fill it in half with running water. Immerse the container with slides and Microwave at 750-800 W in a bain-marie for 7 min to initiate boiling of antigen retrieval solution. Monitor this process carefully to prevent drying of the sample. Take the slides out of the microwave with a heating protection and place them in the microwavable container with fresh citrate buffer at RT for 1 minute (thermal shock). Immerse slides into the hot microwavable container and heat them for 4 min. Repeat the thermal shock. Heat again for 4 min (see Note 3). Cool down sections for 30 min at RT. 7. Wash slides by adding D-PBS, leave for 2 min and pour. Repeat twice. 8. Prepare humidified incubation chamber to be used later during the incubation with antibodies. Place a foil of lab paper into the chamber and add some drops of distilled water (avoiding excess). 9. Discard D-PBS using a micropipette. Leave a small amount to prevent drying of tissue sections. Wipe the slide carefully around the sections creating margins (See Note 4). 10. Dilute 10% (v/v) PRM stock solution with D-PBS to obtain 0.05% (v/v) of working solution. Add 100-200 µl of PRM solution and incubate the tissue sections at RT for 15 min (see Note 5). 11. Discard the PRM solution and incubate slides in D-PBS containing 0.3 M glycine for 20 min (see Note 6). 12. Wash slides three times for 2 min each in D-PBS. 13. Discard D-PBS using a micropipette and add 100-200 µl of freshly prepared blocking solution. Incubate at RT for 30 min (see Note 7). 14. Remove the blocking solution and wipe carefully around the sections. Immediately add 100-200 µl of primary antibody solution to each section and incubate at 4 °C overnight (see Note 8) in a humidified chamber. For multiple staining, primary antibodies of different host species can be used together. 15. Use isotypic control antibodies to evaluate not specific fluorescence signals. 16. Wash slides three times for 2 min each in D-PBS. 17. Add pre-spinned conjugated secondary antibody (to avoid precipitates) diluted at the desired concentration in secondary antibody solution and incubate at 4 °C for 1 h protected from light exposure. (See Note 9). 18. Wash slides three times for 2 min each in D-PBS. 19. Repeat point 17 for each secondary antibody. 20. Wash slides three times for 2 min each in D-PBS. 21. Prepare nuclear staining solution. Add 100-200 µl to each section and incubate at RT (See Note 10). 22. Remove the staining solution and wash sections three times for 2 min each in D-PBS. 23. Discard D-PBS and wipe carefully around the sections. Drop 26 µl of aqueous mounting medium supplemented with anti-fade reagent. Avoid bubbles and excessive mounting medium. Place a clean coverslip on the section and apply pressure gently using tweezers. 24. Seal off the slides using nail polish. From that moment the slides are ready for imaging (See Note 11). NOTES 1. Alternatively, dry tissue sections at 37 °C for 48 h. 2. This is a pause point. Samples can be stored in distillated water at RT for about 3 hours. 3. Alternatively, heat samples at 750-800 W for 3 min, reduce microwave power (400 W) and continue to heat samples for 12 min. Monitor carefully and add more buffer if it evaporates. Antigen retrieval breaks methylene bridge cross-links formed between proteins during formaldehyde-mediated fixation. Salt concentration and pH of retrieval buffer solutions should be evaluated to guarantee cross-bridge removal. The use of tissue-adhesive coated (poly-L-lysine coated) slides is strongly recommended because the antigenic unmasking is a critical step that can nevertheless cause detachment and damage of the tissue sections. Avoid boiling of the solution to prevent this. 4. Alternatively, PAP pen can be used to draw margins, as far as enough space is allowed between margins and the section. From this step on do not let the sections dehydrate. 5. Drop solutions slowly to the tissue sections in order to prevent damage to the sections. Liquids should be pipetted adjacent to the tissue sections forming drops. It is recommended to create drops of solution on the surface of the sample and avoid overspill. 6. Glycine reduces background fluorescence by binding to free aldehyhyde groups, possibly reducing the fluorescent signal of secondary antibodies. Commercially-available signal enhancer solutions can be directly applied on the tissue section (drop 26 µl) to increase the fluorescent intensity. If used, avoid mixing with other solutions (blocking solutions) and wash well in D-PBS. 7. Normal serum helps to prevent cross-reaction between the secondary antibody and endogenous immunoglobulins present in the tissue. Normal serum should be from the same species as the secondary antibody. For instance, for a goat anti-mouse secondary antibody, normal goat serum should be used as blocking reagent. BSA has the same purpose to reduce nonspecific binding by blocking hydrophobic binding sites in the tissue. 8. The primary antibody needs to derive from a species different from the sample. Antibody dilution and incubation time should be optimized for each antibody. Generally, overnight incubation (4 °C) is preferred to ensure that all specific antigens have reached their saturation point. Protect the slides from light when using fluochrome-conjugated antibodies. 9. The fluorochromes used should match the excitation and detection spectra of the microscope system used. The power of light should be adjusted to avoid photobleaching the sample. The excitation and the emission of secondary antibodies should be spanned to avoid spectrum overlapping. For multicolor immunofluorescence, we suggest to check the match with the primary antibody animal species. Avoid using more than one secondary antibody raised in the same animal species to avoid signal overlapping and false-positive signal co-localization. The co-incubation of secondary antibodies is possible but not encouraged. Spin down (4 °C) the diluted antibody before use to reduce molecules aggregates that could disturb the signal. 10. Nuclear staining can be performed by using DNA intercalating dyes 4', 6-diamidino-2-phenylindole (DAPI) or TO-PRO-3. Check excitation and emission of DNA intercalating dyes to create a good match with the secondary antibodies. Incubation time and dilution factor are specified on the product's datasheet. The use of nuclear staining is strongly encouraged to help identify cells. 11. Leave to dry the slides before the imaging session. Store slides at 4°C and protect from light to avoid photobleaching. Figure 1. Immunofluorescence of blood vessels and perivascular cells in the human bone marrow. Confocal images (at 20x and 63X magnification) of formalin-fixed paraffin-embedded sections (3 µm) of human bone marrow (trephine) biopsies processed as described here. The immunofluorescence of antibodies against human NESTIN (green), osteopontin (OPN), a-smooth muscle actin (?SMA) or CD34 (red) is shown in normal human bone marrow samples. Nuclei were counterstained with To-PRO-3 (blue). T, bone trabeculae. Acknowledgements This work was supported by core support grants from the Wellcome Trust (203151/Z/16/Z) and the MRC to the Cambridge Stem Cell Institute, National Health Service Blood and Transplant (United Kingdom), European Union's Horizon 2020 research (ERC-2014-CoG-648765) and a Programme Foundation Award (C61367/A26670) from Cancer Research UK to S.M.-F. References 1. Pinho S, Frenette PS (2019) Haematopoietic stem cell activity and interactions with the niche. Nat Rev Mol Cell Biol 20 (5):303-320. doi:10.1038/s41580-019-0103-9 2. Tamma R, Ribatti D (2017) Bone Niches, Hematopoietic Stem Cells, and Vessel Formation. International journal of molecular sciences 18 (1). doi:10.3390/ijms18010151 3. Beerman I, Luis TC, Singbrant S, Lo Celso C, Mendez-Ferrer S (2017) The evolving view of the hematopoietic stem cell niche. Exp Hematol 50:22-26. doi:10.1016/j.exphem.2017.01.008 4. Mendez-Ferrer S, Bonnet D, Steensma DP, Hasserjian RP, Ghobrial IM, Gribben JG, Andreeff M, Krause DS (2020) Bone marrow niches in haematological malignancies. Nat Rev Cancer 20 (5):285-298. doi:10.1038/s41568-020-0245-2 5. Tjin G, Flores-Figueroa E, Duarte D, Straszkowski L, Scott M, Khorshed RA, Purton LE, Lo Celso C (2019) Imaging methods used to study mouse and human HSC niches: Current and emerging technologies. Bone 119:19-35. doi:10.1016/j.bone.2018.04.022 6. Battifora H, Kopinski M (1986) The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation. J Histochem Cytochem 34 (8):1095-1100. doi:10.1177/34.8.2426335 7. Takaku T, Malide D, Chen J, Calado RT, Kajigaya S, Young NS (2010) Hematopoiesis in 3 dimensions: human and murine bone marrow architecture visualized by confocal microscopy. Blood 116 (15):e41-55. doi:10.1182/blood-2010-02-268466 8. Kristensen HB, Andersen TL, Marcussen N, Rolighed L, Delaisse JM (2013) Increased presence of capillaries next to remodeling sites in adult human cancellous bone. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 28 (3):574-585. doi:10.1002/jbmr.1760 9. Kusumbe AP, Ramasamy SK, Starsichova A, Adams RH (2015) Sample preparation for high-resolution 3D confocal imaging of mouse skeletal tissue. Nature protocols 10 (12):1904-1914. doi:10.1038/nprot.2015.125 10. Robertson D, Savage K, Reis-Filho JS, Isacke CM (2008) Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue. BMC Cell Biol 9:13. doi:10.1186/1471-2121-9-13 11. Gu L, Cong J, Zhang J, Tian YY, Zhai XY (2016) A microwave antigen retrieval method using two heating steps for enhanced immunostaining on aldehyde-fixed paraffin-embedded tissue sections. Histochemistry and cell biology 145 (6):675-680. doi:10.1007/s00418-016-1426-7 |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2021 |
| Provided To Others? | Yes |
| Impact | Optimised protocol for immunofluorescence in paraffin-embedded human bone marrow trephines |
| URL | https://link.springer.com/protocol/10.1007/978-1-0716-1425-9_10 |
| Title | New co-culture of leukaemic blasts with mesenchymal stem cells expanded as self-renewing mesenchymal spheres (mesenspheres) |
| Description | Mesenspheres were cultured from mouse primary BM cells in the following way. Clean mouse bones were crushed in a mortar with 2 ml of a solution containing Collagenase Type I (0.25%) (Stem Cell Technologies). The suspension was incubated for 45 minutes at 37 ºC in agitation. After addition of PBS+ 2% FBS and passage through a 40 µm cell strainer, erythrocytes were lysed by incubation on ice with RBC Lysis Buffer. After this, erythroid, endothelial and hematopoietic cells were removed by magnetic depletion after incubation with biotin-conjugated primary antibodies against CD45, Ter119 and CD31 (BD Biosciences, 1:100) and subsequent incubation with streptavidin-conjugated magnetic beads (BD Biosciences). For sphere formation, the cells immunomagnetically depleted of hematopoietic (CD45+), erythroid (Ter119+) and endothelial (CD31+) cells were plated at low density (<500,000 cells/cm2) in ultralow-adherence 35 mm dishes (StemCell Technologies) after treatment with Poly-Hema (Sigma). The growth medium for spheres contained 0.1 mM ß-mercaptoethanol; 1% nonessential amino acids (Sigma); 1% N2 and 2% B27 supplements (Invitrogen); recombinant human fibroblast growth factor (FGF)-basic, recombinant human epidermal growth factor (EGF), recombinant human platelet-derived growth factor (PDGF-AA), recombinant human oncostatin M (227 aa OSM, 20 ng/ml) and recombinant human IGF-1 (40 ng/ml; Peprotech) in Dulbecco's modified Eagle's medium (DMEM)/F12 (1:1) / human endothelial (1:2) serum-free medium (Invitrogen). Mesensphere medium was supplemented with 15% CEE prepared as described previously (Pajtler et al., 2010). The cultures were incubated at 37°C with 5% CO2, 20% O2 in a water-jacketed incubator and left untouched for 1 week. Afterwards, half-medium changes were performed twice a week. For passage, spheres were enzymatically dissociated with 100µl Trypsin (EDTA-free) for 10 min at 37°C, applying mechanical dispersion every 10 min. The cells were washed with PBS once and replated with mesensphere medium in ultralow-adherence 35mm dishes (StemCell Technologies). Human mesenspheres were isolated after magnetic CD271 cell selection using EasySep™ Human CD271 Positive Selection Kit (StemCell Technologies) from patients undergoing diagnostic BM aspiration and were grown as previously described (Isern et al., 2013). Human AML CD34+ cells were isolated by magnetic cell selection using CD34 MicroBead Kit UltraPure (Miltenyi) after Bone Marrow (BM) or Peripheral Blood (PB) Mononuclear Cells (MNCs) density gradient centrifugation by Ficoll-Paque. MLL-AF9 mouse leukemic blasts were isolated from bones of iMLL-AF9 mice and maintained in 6 well plates in RPMI 1640 (Invitrogen) without phenol red and supplemented with 10% charcoal-stripped FBS (Gibco), recombinant murine IL3, recombinant murine SCF and recombinant human IL-6 (10 ng/ml) (Peprotech), 1% Penicillin-Streptomycin, 1 ug/ml doxycycline at 37°C in a water-jacketed incubator with 5% CO2 and 20% O2. Cells were split every other day and seeded at 500,000 cells/ml. We set up co-cultures systems with mesenspheres (~200 per ml) and leukemic blasts (250,000 cells/ml) for 24h in RPMI without phenol red and with charcoal-stripped FBS (Gibco) ± cytarabine (AraC, Cayman Chemical, 1µM) in flat-bottom 96-well low adherence tissue cultures plates (Costar) at 37°C in a water-jacketed incubator with 5% CO2 and 20% O2. Cultures were grown for 24h before flow-cytometry staining (apoptosis, ROS levels, lipid peroxidation). |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| Impact | New culture method that better preserves the original properties of mesenchymal stem cells, including their capacity to protect leukaemic blasts from chemotherapy-induced cell death |
| URL | https://www.sciencedirect.com/science/article/pii/S1550413120304794?via%3Dihub |
| Title | Gene expression profiling of LSKSLAM cells (haematopoietic stem cells, HSCs) in mice deficient for cholinergic fibers |
| Description | RNASeq of 30-cell pools of LSKSLAM cells from bone and marrow fraction of Gfra2 WT and KO mice. RNAseq sample preparation Pools of 30 viable LSK CD48-CD150+ cells were sorted (BD Influx™ cell sorter) into 4 µl lysis buffer (0.5 U/µl SUPERase In RNase Inhibitor in 0.2 % (v/v) Triton-X-100) containing 12.5 mM DTT and 2.5 mM dNTP. RNAseq was performed following Smart-seq2 protocol 73. Briefly, RNA was primed with oligo-dT primers and reverse transcribed using Superscript II Reverse Transcriptase (200 U/µl, ThermoFisher 18064071). KAPA HiFi Hotstart ReadyMix (KAPA Biosystems, KK2601) and IS PCR primers were used to amplify cDNA, after which cDNA was purified with Agencourt AMPure XP beads (Beckman Coulter, A63881). cDNA quality was checked with Agilent Bioanalyser 2100 using Agilent High Sensitivity DNA chip and quantity was measured with Quant-iT™ Picogreen double stranded DNA assay kit (ThermoFisher P7589). Pooled libraries of 4 replicates per sample were prepared using the Illumina Nextera XT DNA preparation kit. Amplified libraries were purified using Agencourt AMPure XP beads, quality-checked using Agilent high-sensitivity DNA chip on Agilent Bioanalyser 2100 and quantified using KAPA qPCR quantification kit (KAPA Biosystems, KK4824). Sequencing of 3 replicates per condition were performed on the Illumina Hi-Seq 4000 (single end, 50bp read length) by the CRUK Cambridge Institute Genomics Core facility. Bioinformatic analysis of RNAseq RNA-seq reads were aligned to Mus musculus genome (Ensembl version 38.81) using GSNAP (version 2015-09-29) with parameters (-B 5 -t 24 -n 1 -Q -N 1). Reads in features were counted with htweq-count (HTSeq version 0.5.3p3) with the parameter (-s no). Quality control was performed with the following cut-offs: more than one and a half million uniquely mappable reads, less than 20% of reads mapping to mitochondrial genes over mitochondrial + nuclear genes and more than 8500 high coverage genes identified. Counts were normalized using size factors as calculated by DESeq2 using a 10% FDR, and then log10 transformed. Highly variable genes were selected using the method described by Brennecke et al. PCA was then performed in R using the prcomp function. Gene set enrichment analyses were performed as described74 (http://www.broadinstitute.org/gsea/index.jsp), using a weighted statistic, ranking by signal to noise ratio, 1000 gene-set permutations, and a custom gene set database gene lists manually compiled from the literature. GEO accession number - GSE94078 |
| Type Of Material | Database/Collection of data |
| Year Produced | 2022 |
| Provided To Others? | Yes |
| Impact | First RNAseq from HSCs of cholinergic-neural-deficient mice. |
| URL | https://www.nature.com/articles/s41467-022-28175-1 |
| Title | Proteomic analysis of AraC-treated AML blasts monocultured or cocultured with self-renewing mesenchymal spheres (mesenspheres) for 24h |
| Description | Liquid Chromatography Coupled to Mass Spectrometry (LC-MS) for Metabolomics Analysis Leukemic blasts (800,000 cells/ml) were cultured alone or in coculture with mesenspheres (250/ml) in normal media for 24h in the presence of AraC. All the cells were filtered using Falcon tube with cell strainer snap cap (Corning) to mechanically separate leukemic blasts from mesenspheres. Cells were washed three times with PBS and the extraction buffer (50% methanol, 30% acetonitrile, 20% water, all LC-MS grade) was added (1ml/4x106 cells). Cell were incubated in dry ice for 15 min, collected, vigorously shaked for 15 min at 4ºC and left 1h at 20 ºC. Samples were centrifuged at 13,000 rpm and supernatants were transferred to autosampler vials and stored at -80 ºC until further analysis. To avoid bias due to machine drift, samples were randomized and processed blindly. A Q Exactive mass spectrometer (Thermo Fisher Scientific) coupled to a Dionex U3000 UHPLC (Thermo Fisher Scientific) system was used to perform the LC-MS analysis. A Sequant ZIC-pHILIC column (150 x 2.1 mm, 5 µm) and guard column (20 x 2.1 mm, 5 µm) (Merck Millipore) were utilized for the chromatographic separation. The column oven temperature was maintained at 40ºC. The mobile phase was composed of 20 mM ammonium carbonate and 0.1% ammonium hydroxide in water (solvent A), and acetonitrile (solvent B). The flow rate was set at 0.2 ml/min with the following gradient: 80% B for 2 min, linear decrease to 20% of B 15 min. Both solvents were then brought back to initial conditions and maintained for 8 min. The mass spectrometer was operated in full MS and polarity switching mode. XCalibur Qual Browser and XCalibur Quan Browser software (Thermo Fisher Scientific) were used to process and analyse the spectra. Quantitative Proteomic Analysis Sample preparation. Blasts and BMSCs (spheres) pellets were obtained by filtering the culture using 35um cell strainer snap cap tubes to isolate the spheres (which did not pass the filter), following isolation of leukemic blasts as CD45+ DAPI- cells. Different conditions included blasts cultured alone (n=2), blast cultured alone treated with AraC (n=3), blast co-cultured with spheres (n=3), blast co-cultured with spheres treated with AraC (n=3), spheres cultured alone (n=2), spheres cultured alone treated with AraC (n=2), spheres co-cultured with blasts (n=3) and spheres co-cultured with blasts treated with AraC (n=4). Each sample was generated by pooling cells from different cultures due to the scarcity of material for each of them. Pellets were extracted in Lysis buffer (50 mM Tris-HCl pH 7.5; 2% SDS, 10 mM TCEP (Tris(2-carboxyethyl) phosphine hydrochloride (TCEP)) by homogenizing the cells with vortex, boiling for 5 min, and incubating for 30 min at RT with agitation. Samples were centrifuged at 15,000 rpm for 15 min and protein concentration was determined using a Direct Detect IR spectrometer (Millipore). Protein digestion and isobaric labelling. For the quantitative differential analysis by LC-MS/MS using isobaric tags (TMT 10-plex), about 100 µg of total proteins were digested using the FASP protocol as previously described with minor modifications (Cardona et al., 2015). Proteins were diluted in 7 M urea in 0.1 M Tris-HCl (pH 8.5) (UA), and loaded onto 10 kDa centrifugal filter devices (NanoSep 10k Omega, Pall Life Sciences). Samples were washed into filters with UA, and proteins were then alkylated with 50 mM iodoacetamide (IAA) in UA for 30 min in the dark. Samples were washed three times with UA and three additional times with 50 mM ammonium bicarbonate. Proteins were digested overnight at 37ºC with modified trypsin (Promega) in 50 mM ammonium bicarbonate at 30:1 protein:trypsin (w/w) ratio. The resulting peptides were eluted by centrifugation with 50 mM ammonium bicarbonate, and 0.5M sodium chloride. Trifluoroacetic acid (TFA) was added to a final concentration of 1% and the peptides were desalted onto C18 Oasis-HLB cartridges and dried-down for further analysis. For stable isobaric labelling, the resulting tryptic peptides were dissolved in 30 µl of 100 mM Triethylammonium bicarbonate (TEAB) buffer, and the peptide concentration was determined by measuring amide bonds with the Direct Detect system (Millipore). Equal amounts of each peptide sample were labelled using the 10-plex TMT Reagents (Thermo Fisher) according to manufacturer's protocol. Peptides were labelled with TMT reagents previously reconstituted with 70 µl of acetonitrile, and after incubation at room temperature for 1h, reaction was stopped with 0.5% TFA, incubated for 15 min, and peptides were combined. Samples were concentrated in a Speed Vac, desalted onto C18 Oasis-HLB cartridges and dried-down for further analysis. For increasing proteome coverage, TMT-labelled samples were fractionated by high-pH reverse phase chromatography (High pH Reversed-Phase Peptide Fractionation Kit, Pierce) and concentrated as before. Protein identification and quantitation. Labelled peptides were analyzed by LC-MS/MS using a C-18 reversed phase nano-column (75 µm I.D. x 50 cm, 2 µm particle size, Acclaim PepMap RSLC, 100 C18; Thermo Fisher Scientific) in a continuous acetonitrile gradient consisting of 0-30% B in 360 min, 50-90% B in 3 min (A= 0.1% formic acid; B=90% acetonitrile, 0.1% formic acid). A flow rate of 200 nL/min was used to elute peptides from the nano-column to an emitter nanospray needle for real time ionization and peptide fragmentation on an Orbitrap Fusion mass spectrometer (Thermo Fisher). An enhanced FT-resolution spectrum (resolution=70,000) followed by the MS/MS spectra from the Nth most intense parent ions were analyzed along the chromatographic run. Dynamic exclusion was set at 40s. For peptide identification, all spectra were analyzed with Proteome Discoverer (version 2.1.0.81, Thermo Fisher Scientific) using SEQUEST-HT (Thermo Fisher Scientific). For database searching at the Uniprot database containing all sequences from mouse and contaminants (April 27, 2016; 48,644 entries), the parameters were selected as follows: trypsin digestion with 2 maximum missed cleavage sites, precursor and fragment mass tolerances of 2 Da and 0.02 Da, respectively. Carbamidomethyl cysteine (+57.021 Da) and TMT modifications (+229.162932 Da) at N-terminal and Lys residues were selected as fixed modifications, and methionine oxidation (+15.994915 Da) as dynamic modification. Peptide identification was performed using the probability ratio method (Martinez-Bartolome et al., 2008) and false discovery rate (FDR) was calculated using inverted databases, and the refined method (Navarro and Vazquez, 2009) with an additional filtering for precursor mass tolerance of 10 ppm (Bonzon-Kulichenko et al., 2015). Identified peptides with an FDR equal or lower than 1% FDR were used to quantify the relative abundance of each protein from reporter ion intensities, and statistical analysis of quantitative data were performed using the WSPP statistical model previously described (Navarro et al., 2014). In the model, protein log2-ratios are expressed as standardized variables, i.e., in units of standard deviation according to their estimated variances (Zq values). Functional protein analysis. Functional protein analysis of the whole set of quantified proteins was performed using our algorithm, system biology triangle (SBT), developed specifically for the analysis of coordinated protein responses in high-throughput quantitative proteomics experiments (Garcia-Marques et al., 2016). This algorithm correlates the performance of a group of proteins inside of a category (biological process) in terms of their quantitative behavior (relative abundance); thus, changes can be detected in functional biological processes far beyond individual protein responses. Because of this coordinated behavior, a Z value is assigned to each category. Variations in the abundance of annotated functional categories were visualized by comparing the cumulative frequency (sigmoid) plots of the standardized variable with that of the normal distribution, as previously described (Isern et al., 2013). Individual protein changes were also considered for further analysis. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| Impact | Proteomics data are accessible in the Peptide Atlas (http://www.peptideatlas.org/repository/) and are accessible through the accession number PASS01476 |
| URL | https://db.systemsbiology.net/sbeams/cgi/PeptideAtlas/PASS_View?identifier=PASS01476 |
| Title | RNAseq from leukaemic stem cells obtained from mice with or without depletion of nestin+ mesenchymal stem cells |
| Description | RNAseq of MLL-AF9+ lin- ckitlo sca1- (LKlo) cells was performed after sorting these cells from the BM of Nes-creERT2;iDTA (N=8) or control (N=6) mice. Sorted cells were combined for a total of 3 biological replicates (each consisting of pooled LKlo cells from from 2-3 mice). Total RNA was isolated using the Arcturus Picopure RNA Isolation Kit (Thermo Fisher Scientific). RNA was amplified and prepared for RNA-Seq using the Ovation RNA-Seq System v2 (NuGEN) following the manufacturer's recommendations. The RNA sequencing library was prepared with the TruSeq RNA Sample Preparation v2 Kit (Illumina, San Diego, CA) to construct index-tagged cDNA. The quality, quantity and the size distribution of the Illumina libraries were determined using the DNA-1000 Kit (Agilent Bioanalyzer). Libraries were sequenced on the Genome Analyzer IIx (Illumina) following the standard RNA sequencing protocol with the TruSeq SBS Kit v5. Fastq files containing reads for each library were extracted and demultiplexed using Casava v1.8.2 pipeline. Sequencing adaptor contaminations were removed from reads using cutadapt software tool (MIT) and the resulting reads were mapped and quantified on the transcriptome (NCBIM37 Ensembl gene-build 65) using RSEM v1.17 (Li and Dewey, 2011). Only genes with more than 2 counts per million in at least 2 samples were considered for statistical analysis. Data were then normalized and differential expression was assessed using the bioconductor package EdgeR (Robinson et al., 2010). Expression data from 12337 genes was obtained and compared between BM LKlo cells from mice with/without nestin+ cell depletion, using an empirical Bayes statistic for differential expression (moderated t-test), as implemented in limma package (R/Bioconductor). Genes with adjusted p-value = 0.05 were considered to be differentially expressed between the two conditions. Additionally, in order to detect coordinated changes in sets of genes representing pathways or functional signatures, we performed gene-set enrichment analyses (GSEA) against the collection of 3144 curated gene sets available in the Molecular Signatures Database (http://www.broadinstitute.org/gsea/msigdb/index.jsp), including KEGG, Biocarta and Reactome pathways as well as a collection of gene expression signatures associated with chemical or genetic perturbations. Significance of gene set enrichment between the two conditions was assessed with GSEA software as previously described (Subramanian et al., 2005) (http://www.broadinstitute.org/gsea/index.jsp), using a weighted statistic, ranking by signal to noise ratio and 1000 gene-set permutations. Gene sets with FDR<0.05 were considered to exhibit the most significantly enrichment. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| Impact | First RNAseq from leukaemic stem cells obtained from mice with or without depletion of nestin+ mesenchymal stem cells. The RNAseq data from leukemic BM lin-ckitlo cells obtained from mice with/without nestin+ cell depletion has been deposited in GEO under the accession number GSE140207. |
| URL | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE140207 |
| Title | RNAseq from nestin-GFP-positive mesenchymal stem cells from normal or leukaemic mice |
| Description | For Nestin+ BMSC RNAseq, Nes-GFP+ cells were sorted from the BM of normal or iMLL-AF9+ mice as follows. Leukemia was as described above by providing doxycycline. Control mice were provided 5% sucrose in water as vehicle. A non-GFP mouse was used as a control to set up the gating strategy during sorting. Bones from leukemic or non-leukemic mice were processed as described above and CD45-CD31-Ter119-DAPI-Nes-GFP+ cells were sorted and immediately frozen at -80ºC. mRNA was obtained using the RNeasy Kit (Qiagen). Due to low cell number available, an amplification step was performed as previously described (Picelli et al., 2014). Reads were aligned using TopHat version v2.1.0 (Kim et al., 2013) to genome build GRCm38/mm10. Gene expression was quantified using featureCounts version 1.5.0 (Liao et al., 2014) with annotation from Ensembl Release 86 (Yates et al., 2016). Normalization and differential expression were performed using Deseq2 version 1.14.1 (Love et al., 2014), using R version 3.3.3. Gene ontology enrichment was performed using the R package goseq (Young et al., 2010) for significantly differentially expressed genes (padj > 0.05). KEGG pathway enrichment was performed using the R package SPIA (Tarca et al., 2009) or DAVID (Huang da et al., 2007). |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| Impact | Gene signatures associated with transcriptomic changes in BMSCs in AML in vivo (MLL-AF9+ mouse model). The RNAseq data from nestin+ BMSCs obtained from leukemic iMLL-AF9 and normal mice has been deposited in GEO under the accession number GSE140135. |
| URL | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE140135 |
| Description | Collaboration agreement with Galvani Bioelectronics, a joint venture of GSK and Google LifeScience |
| Organisation | Galvani Bioelectronics Ltd |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | Training in neuromodulation of a postdoctoral fellow in our group in Galvani Bioelectronics, a joint venture of GSK and Google LifeScience |
| Collaborator Contribution | Neural stimulation of sciatic nerve in rats |
| Impact | Proof of principle study (electrical stimulation to modulate haematopoiesis) |
| Start Year | 2016 |
| Description | Gfra2 KO mice |
| Organisation | University of Helsinki |
| Country | Finland |
| Sector | Academic/University |
| PI Contribution | The group of Prof. Matti Airaksinen provided Gfra2 KO mice |
| Collaborator Contribution | The group of Prof. Matti Airaksinen provided Gfra2 KO mice |
| Impact | Ongoing studies |
| Start Year | 2010 |
| Description | Intravital microscopy |
| Organisation | Imperial College London |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Intravital microscopy of haematopoietic stem cells transplanted into mice |
| Collaborator Contribution | We have found that haematopoietic stem cells carrying the same mutation but giving rise to different types of myeloproliferative neoplasms interact differently with the bone marrow niches |
| Impact | Ongoing studies |
| Start Year | 2016 |
| Description | MTA with Genentech |
| Organisation | Genentech, Inc |
| Country | United States |
| Sector | Private |
| PI Contribution | Investigate the role of IL6 in the cholinergic regulation of bone marrow |
| Collaborator Contribution | 400 mg of Actemra (tocilizumab) |
| Impact | Gadomski S, Fielding C, García-García A, Korn C, Kapeni C, Ashraf S, Villadiego J, del Toro R, Domingues O, Skepper JN, Michel T, Zimmer J, Sendtner R, Dillon S, Poole K, Holdsworth G, Sendtner M, Toledo-Aral JJ, De Bari C, McCaskie AW, Robey RG and Méndez-Ferrer S. A cholinergic neuroskeletal interface promotes bone formation during postnatal growth and exercise. Cell Stem Cell 29, 2022; doi: 10.1016/j.stem.2022.02.008. |
| Start Year | 2019 |
| Description | MTA with Kumamoto University |
| Organisation | Kumamoto University |
| Country | Japan |
| Sector | Academic/University |
| PI Contribution | Frozen cells from human umbilical cord blood samples (30 samples) that contain human hematopoietic stem cells and adult mobilised peripheral blood stem and progenitor cells (30 samples), to test their engrafting and repopulating ability in a xenograft model. |
| Collaborator Contribution | The Originator is willing to provide a sample of the Materials for a period of 3 (three) years from the date of execution, on the Standard Terms and Conditions shown overleaf, and in consideration thereof the Recipient agrees to be bound by those Standard Terms and Conditions under MTA. |
| Impact | Under development |
| Start Year | 2021 |
| Description | MTA with Luxembourg Institute of Health |
| Organisation | Luxembourg Institute of Health |
| Country | Luxembourg |
| Sector | Academic/University |
| PI Contribution | The main goal of the project is to investigate the cholinergic neural regulation of haematopoiesis. Unpublished research from Cambridge has revealed a dual cholinergic regulation of haematopoietic stem cell (HSC) niches in the bone marrow (BM). Recent studies suggest that specific niches regulate distinct stem cell states. However, how these niches cordinate various stem cell activities with organismal demands remains unclear. Cambridge has found novel neural signals that regulate different stem cell functions In separate niches during circadian time cycles. In early postnatal mice, a subset of boneassociated sympathetic nerve fibres switch from noradrenergic to cholinergic fate and promote BM colonisation by-HSCs through Cxc/12 induction. In adult mice, sympathetic cholinergic nerve fibres activate nicotinic receptors and induce Cxc/12 expression in bone-associated nestin+ mesenchymal stem cells (BMSCs),promoting HSC quiescence and long-term maintenance. In contrast, other autonomic cholinergic signals antagonise BM sympathetic noradrenergic activity during the resting period, indirectly regulating-adrenergic-receptor-dependent circadian HSC trafficking through sinusoidal niches. Thus, the autonomic nervous system regulates HSC maintenance and function in temporaly and spatially separate niches, to align stem cell function with organismal demands. Cambridge has obtained many of these results in a mouse line lacking the neurturin receptor Gfra2 and a manuscript describing these findings has been reviewed. The goal of this research project is to verify and extend these findings in mice lacking neurturin In collaboration with UH as part of the revision process of this manuscript. UH would provide resources and expertise to accomplish these goals. Particularly, LIH would provide neurturin (NTN)-KO mice and control mice, and the capacity to perform tissue extraction, peripheral blood counts, flow cytometry, cell sorting and cell culture using samples from these mice. Based on the overall very similar phenotype of Gfra2 KO mice and NTN-KO mice, it is expected to obtain similar results as previously obtained by Cambridge in Gfra2 KO mice, and include the new results obtained in collaboration with LIH in a revised manuscript. We will follow the general rules described above in Article 7 regarding the authorship of this publication. Additionally,this project could set the basis for future related collaborations on areas of shared interest under terms that should be discussed independently in the future. The manuscript mentioned above has been published in 2019 in the journal "Blood". However, new data from Cambridge show the need for additional experiments on the LIH side, and the shipment of bones from NTN-KO mice and wildtype littermates to Cambridge. The mice must be between 14 and 18 weeks old. In addition, the hematopoietic development of B cell progenitors in mouse bone marrow (NTN-KO and wildtype littermates) will be analysed by flow cytometry, using two different antibody panels designed by the Cambridge lab. |
| Collaborator Contribution | Bones and analysis from NRTN KO mice |
| Impact | 1. Gadomski S, Fielding C, García-García A, Korn C, Kapeni C, Ashraf S, Villadiego J, del Toro R, Domingues O, Skepper JN, Michel T, Zimmer J, Sendtner R, Dillon S, Poole K, Holdsworth G, Sendtner M, Toledo-Aral JJ, De Bari C, McCaskie AW, Robey RG and Méndez-Ferrer S. A cholinergic neuroskeletal interface promotes bone formation during postnatal growth and exercise. Cell Stem Cell 29, 2022; doi: 10.1016/j.stem.2022.02.008. 2. García-García A, Korn C, García-Fernández M, Domingues O, Villadiego J, Martín-Pérez D, Isern J, Bejarano-García JA, Zimmer J, Pérez-Simón JA, Toledo-Aral JJ, Michel T, Airaksinen MS, Méndez-Ferrer S. Dual cholinergic signals regulate daily migration of hematopoietic stem cells and leukocytes. Blood. 2019 Jan 17;133(3):224-236. doi: 10.1182/blood-2018-08-867648 (Commentary by O'Leary HE in the same issue). |
| Start Year | 2018 |
| Description | MTA with UCB BioPharma SRL |
| Organisation | UCB Pharma |
| Department | UCB Celltech |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | Injection of an anti-sclerostin blocking antibody in mouse models exhibiting osteopenia, to rescue the effect of abnormally increased sclerostin production during bone remodelling. |
| Collaborator Contribution | Anti-sclerostin blocking antibody. |
| Impact | Gadomski S, Fielding C, García-García A, Korn C, Kapeni C, Ashraf S, Villadiego J, del Toro R, Domingues O, Skepper JN, Michel T, Zimmer J, Sendtner R, Dillon S, Poole K, Holdsworth G, Sendtner M, Toledo-Aral JJ, De Bari C, McCaskie AW, Robey RG and Méndez-Ferrer S. A cholinergic neuroskeletal interface promotes bone formation during postnatal growth and exercise. Cell Stem Cell 29, 2022; doi: 10.1016/j.stem.2022.02.008. |
| Start Year | 2021 |
| Description | MTA with Weizmann Institute of Science |
| Organisation | Weizmann Institute of Science |
| Country | Israel |
| Sector | Academic/University |
| PI Contribution | Use of TACE inhibitor for in vitro studies and in vivo studies in mice to investigate the role of receptor shedding from the cell membrane in the regulation of cis vs trans signalling of these receptors in the haematopoietic system |
| Collaborator Contribution | TACE inhibitor |
| Impact | Gadomski S, Fielding C, García-García A, Korn C, Kapeni C, Ashraf S, Villadiego J, del Toro R, Domingues O, Skepper JN, Michel T, Zimmer J, Sendtner R, Dillon S, Poole K, Holdsworth G, Sendtner M, Toledo-Aral JJ, De Bari C, McCaskie AW, Robey RG and Méndez-Ferrer S. A cholinergic neuroskeletal interface promotes bone formation during postnatal growth and exercise. Cell Stem Cell 29, 2022; doi: 10.1016/j.stem.2022.02.008. |
| Start Year | 2021 |
| Description | Regulatory networks involved in the crosstalk of blood stem cells with their microenviroment and long-distance endocrine signals |
| Organisation | University of Cambridge |
| Department | Cambridge Stem Cell Institute |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | The group of Berthold Gottgens is helping us dissect regulatory networks involved in the crosstalk of blood stem cells with their microenviroment and long-distance endocrine signals through scRNAseq |
| Collaborator Contribution | The group of Berthold Gottgens is helping us dissect regulatory networks involved in the crosstalk of blood stem cells with their microenviroment and long-distance endocrine signals through scRNAseq |
| Impact | Ongoing studies |
| Start Year | 2015 |
| Title | Anti CD24 antibodies - myelofibrosis |
| Description | Anti CD24 antibodies - myelofibrosis and thrombosis |
| IP Reference | 2316777.8, P367581GB |
| Protection | Patent / Patent application |
| Year Protection Granted | 2023 |
| Licensed | No |
| Impact | Submitted |
| Title | COMPOUNDS SUITABLE FOR THE TREATMENT OF MYELOPROLIFERATIVE NEOPLASMS |
| Description | The present findings point to mutant HSCs as the cause of BM neuroglial damage that compromises MSC survival and function, critically contributing to MPN pathogenesis. In this sense, the present invention shows that the niche damage triggered by the mutant HSC is essential for the development of a haematopoietic malignancy previously considered to be caused by the HSC alone. Targeting HSC niche-forming MSCs and their neural regulation paves the way to more efficient therapeutic strategies in MPN. For this purpose, the present invention shows that an efficient therapeutic strategy for the treatment of MPN lies on the administration of neuroprotective compounds, such as 4-methylcatechol, capable of protecting BM sympathetic nerve fibres. Additionally, another efficient therapeutic strategy is shown herein as the administration of selective &bgr;3-adrenergic agonists such as BRL37344 or Mirabegron, since this strategy will compensate for deficient sympathetic stimulation of nestin+ MSCs. |
| IP Reference | WO2014181001 |
| Protection | Patent granted |
| Year Protection Granted | 2014 |
| Licensed | Commercial In Confidence |
| Impact | Under licensing with CNIC (Madrid, Spain) |
| Title | MODULADOR SELECTIVO DEL RECEPTOR DE ESTRÓGENOS PARA EL TRATAMIENTO DE UNA ENFERMEDAD MIELOPROLIFERATIVA |
| Description | Selective modulator of the oestrogen receptor for treating a myeloproliferative disease. The invention relates to the field of oncology, and more concretely, to the field of diseases associated with an unwanted proliferation of blood cells, concretely, acute myeloid leukemia (AML), myeloproliferative diseases and myelodysplastic syndrome. The application relates to methods and compositions for the treatment of said diseases by means of the selective elimination of hematopoietic precursors. |
| IP Reference | WO2014188043 |
| Protection | Patent application published |
| Year Protection Granted | 2014 |
| Licensed | No |
| Impact | TAMARIN Phase-II clinical study ongoing |
| Title | TAMARIN: Effects of TAMoxifen on the Mutant Allele Burden and Disease Course in Patients with MyeloprolifeRatIve Neoplasms. EudraCT: 2015-005497-38 |
| Description | Title: TAMARIN: Effects of TAMoxifen on the Mutant Allele Burden and Disease Course in Patients with MyeloprolifeRatIve Neoplasms Myeloproliferative neoplasms (MPNs) are blood cancers which affect the normal production of blood cells from the bone marrow. They are caused by changes (mutations) in blood stem cells, frequently in the genes that produce proteins called JAK2, CALR or MPL. MPNs have a risk of developing to an acute leukaemia (a more advanced stage of disease) and currently has no effective cure, apart from bone marrow transplantation which is not possible for many patients. Recent work in mouse studies has suggested that tamoxifen, a drug widely used to treatment breast cancer, may reduce the number of mutated cells by mimicking oestrogen (a female sex hormone) which has a role in the survival and production of new stem cells that give rise to blood cancers. In these studies, tamoxifen prevented the excessive production of blood cells by restoring normal levels of cell death in the mutated cells. This is a single arm, multicentre phase II trial designed to assess if adding tamoxifen to patients receiving therapy for their MPN reduces the number of mutated cells found in the blood by = 50% after 24 weeks of treatment compared to the start of the study. Collection of blood and bone marrow samples will also allow laboratory researchers to study the biological effects of tamoxifen and how this correlates with the patient's disease and response to therapy. Patients will receive treatment with 20mg once daily (oral tablet) of tamoxifen with their normal therapy for their MPN. 42 patients will be recruited from 13-15 UK centres over 12 months. |
| Type | Therapeutic Intervention - Drug |
| Current Stage Of Development | Initial development |
| Year Development Stage Completed | 2020 |
| Development Status | Under active development/distribution |
| Clinical Trial? | Yes |
| Impact | Preliminary results suggest a reduction of allele burden in some patients |
| URL | https://www.clinicaltrialsregister.eu/ctr-search/search?query=eudract_number:2015-005497-38 |
| Title | Title: Sympathicomimetic Agonist in Patients With Myeloproliferative Neoplasms With JAK2-mutation |
| Description | Title: Sympathicomimetic Agonist in Patients With Myeloproliferative Neoplasms With JAK2-mutation The aim of this phase II study is to test a novel concept in the treatment of patients with myeloproliferative neoplasms (MPN), a disease of the bone marrow. With no current cure available, MPN are a group of chronic leukemias (blood cancers) in which patients produce too many blood cells. These increased blood cell numbers cause problems to the patient such as bleedings or thrombosis and some patients may progress to acute leukemia, a life threatening condition. Most MPN patients have a gene mutation called JAK2-V617F. The disease is maintained by mutant MPN stem cells that reside in the bone marrow in specialized locations called "niches". These niches need connections to the nervous system. New findings show that these connections are destroyed by the presence of the mutated MPN stem cells. Research teams found that some drugs (beta3-sympathicomimetics) can restore these damaged niches and at the same time reduce the MPN disease manifestation in a mouse model of MPN. Such sympathicomimetic drugs are already being used to treat patients with asthma or hyperactive bladder. These drugs have shown to have only few side effects. The study tests the effects of the beta-3-sympathicomimetic drug Mirabegron (Betmiga®) on MPN disease in 39 patients that carry a JAK2-V617F mutation. The hypothesis is that Mirabegron will have a beneficial effect on bone marrow niche cells and will thereby improve the disease manifestation in MPN patients. This study should provide a rapid answer whether targeting the nervous system of the niche cells could be useful for patients with MPN and warrants to be tested in larger and more long-term studies. |
| Type | Therapeutic Intervention - Drug |
| Current Stage Of Development | Initial development |
| Year Development Stage Completed | 2020 |
| Development Status | Actively seeking support |
| Clinical Trial? | Yes |
| Impact | Partial improvement of reticulin fibrosis correlated with rescue of nestin+ niches in patients with myeloproliefartive neoplasms treated over 6 months with the beta3-adrenergic agonist mirabegron. Follow-up studies suggest that treatment duration and dosing can be optimised to achieve improved outcomes. Patent granted is currently being licensed to CNIC (Madrid, Spain) and plans are underway for another study |
| URL | https://clinicaltrials.gov/show/NCT02311569 |
| Description | 2nd Biennial Symposium on 'Frontiers in Development and Molecular Medicine (FDMM 2021): Systems to Medicine'. Virtual. 1st March 2021. |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | 2nd Biennial Symposium on 'Frontiers in Development and Molecular Medicine (FDMM 2021): Systems to Medicine'. Virtual. 1st March 2021. |
| Year(s) Of Engagement Activity | 2021 |
| Description | 4th Beijing Forum of Thrombosis and Hemostasis & 2nd Chinese Annual Congress of Hematologic Malignancy and Immunohematology. 11 Sept 2021. Virtual |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | 4th Beijing Forum of Thrombosis and Hemostasis & 2nd Chinese Annual Congress of Hematologic Malignancy and Immunohematology. 11 Sept 2021. |
| Year(s) Of Engagement Activity | 2021 |
| Description | AACR Special Conference |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Acute Myeloid Leukemia and Myelodysplastic Syndrome. Austin, Texas, USA> 23-25 Jan 2023. |
| Year(s) Of Engagement Activity | 2022 |
| Description | AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome. |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Invited speaker at AACR conference in Austin Texas USA. |
| Year(s) Of Engagement Activity | 2023 |
| Description | Annual Meeting, European Association of Hematology. SWG Session on "Myeloproliferative neoplasms (MPN)". Virtual Congress. 8-17 June 2021. |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Annual Meeting, European Association of Hematology. SWG Session on "Myeloproliferative neoplasms (MPN)". Virtual Congress. 8-17 June 2021. |
| Year(s) Of Engagement Activity | 2021 |
| Description | Annual Meeting, European Hematology Association |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Engaging with post doctoral students 9-12 June - Vienna |
| Year(s) Of Engagement Activity | 2022 |
| Description | Annual Meeting, Spanish Society for Gene and Cell Therapy |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | To further enhance scientific research. 30th May - 1st June 2022 |
| Year(s) Of Engagement Activity | 2022 |
| Description | Annual Meeting. American Society of Hematology. 11 Dec 2021. Co-Chair, Scientific Sessions. |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Annual Meeting. American Society of Hematology |
| Year(s) Of Engagement Activity | 2021 |
| Description | Annual Meeting. British Pharmacology Society. Symposium: Cancer Microenvironment & Pharmacological Interventions. 8 Sept 2021. |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Postgraduate students |
| Results and Impact | Annual Meeting. British Pharmacology Society. Symposium: Cancer Microenvironment & Pharmacological Interventions. 8 Sept 2021. |
| Year(s) Of Engagement Activity | 2021 |
| Description | Annual Meeting. Spanish Cell Therapy Network. 29 Nov 2021. Barcelona, Spain. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | Annual Meeting. Spanish Cell Therapy Network |
| Year(s) Of Engagement Activity | 2021 |
| Description | Annual Meeting. Spanish Society of Hematology. 15 Oct 2021. Pamplona, Spain |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | Annual Meeting. Spanish Society of Hematology. 15 Oct 2021. Pamplona, Spain |
| Year(s) Of Engagement Activity | 2021 |
| Description | Cambridge Cancer Center. The microenvironment in the myeloid malignancies. 2018-present |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Undergraduate students |
| Results and Impact | Cambridge Cancer Center Seminars. The microenvironment in the myeloid malignancies. 2018-present |
| Year(s) Of Engagement Activity | 2018,2019,2020 |
| Description | EHA Research Conference |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Talk to postgraduate students 17-20 October 2022, Dresden, Germany |
| Year(s) Of Engagement Activity | 2022 |
| Description | ESH International conference on myeloproliferative neoplasms |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Invited speaker to (virtual) conference 12-15 May 2022, |
| Year(s) Of Engagement Activity | 2022 |
| Description | Effects of tamoxifen on the mutant allele burden and disease course in patients with myeloproliferative neoplasms - results of the Tamarin study |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Selected Oral abstract presented to the American Society of Hematology |
| Year(s) Of Engagement Activity | 2020 |
| Description | Flagship 20th Anniversary Conference. |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Raised the interest in scientific research. |
| Year(s) Of Engagement Activity | 2023 |
| Description | ISSCR International Symposium. Stem cells: from basic science to clinical translation. 29 Oct 2021. Virtual |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Public/other audiences |
| Results and Impact | ISSCR International Symposium. Stem cells: from basic science to clinical translation |
| Year(s) Of Engagement Activity | 2021 |
| URL | https://www.isscr.org/news-publicationsss/isscr-news-articles/article-listing/2021/10/26/the-isscr-a... |
| Description | International Seminar "Trends in Cancer Biology and Advanced Therapies" |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Invited speaker to International Seminar in Seville, Spain, 23-25 February 2022 |
| Year(s) Of Engagement Activity | 2022 |
| Description | International Symposium: "Emerging Therapies at the Intersection of Genetic and Cellular Technologies." The International Society for Stem Cell Research (ISSCR) and American Society for Society for Gene & Cell therapy (ASGCT). Madison, WI, USA. 13 - 15 September 2021. |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | 15) International Symposium: "Emerging Therapies at the Intersection of Genetic and Cellular Technologies." The International Society for Stem Cell Research (ISSCR) and American Society for Society for Gene & Cell therapy (ASGCT). Madison, WI, USA. 13 - 15 September 2021. |
| Year(s) Of Engagement Activity | 2021 |
| Description | International Workshop on Molecular Aspects of Myeloid Stem Cell Development and Leukemia |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | To speak at an international conference in May 2022, to industry experts. |
| Year(s) Of Engagement Activity | 2022 |
| Description | Meeting on clonal hematopoiesis. 4th ARCH meeting |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Keynote speaker in Madrid, Spain, 29-30th September |
| Year(s) Of Engagement Activity | 2022 |
| Description | Metabolism-based cancer therapy symposium |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Invited speaker 12th October - Dresden Germany, increased the understanding of science. |
| Year(s) Of Engagement Activity | 2022 |
| Description | NIH-sponsored workshop on the Neural control of haematopoiesis. 30 October 2020. Virtual |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | NIH-sponsored workshop on the Neural control of haematopoiesis. 30 October 2020. |
| Year(s) Of Engagement Activity | 2020 |
| Description | Summer Course. "Cell Therapy from the bench to the bedside and return". Cell Therapy Network. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | International Summer Course. "Cell Therapy from the bench to the bedside and return". Spain Cell Therapy Network (TerCel, ISCIII). |
| Year(s) Of Engagement Activity | 2015,2016,2017,2018,2019,2020 |
| Description | The role of MSCs in age-related myeloid malignancies |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Scientific Working Group on MSC. Session "MSC, friends and foes |
| Year(s) Of Engagement Activity | 2020 |
| Description | Virtual presentation. Stowers Institute. Kansas City, USA. 9 Dec 2020. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Stowers Institute. Kansas City, USA. 9 Dec 2020. |
| Year(s) Of Engagement Activity | 2020 |
| Description | Virtual talk. Children's Hospital Cincinnati, USA. 1 Feb 2021. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Children's Hospital Cincinnati, USA. 1 Feb 2021. |
| Year(s) Of Engagement Activity | 2021 |
| Description | WT-MRC PhD Program in Stem Cell Biology |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Undergraduate students |
| Results and Impact | Lectures for WT-MRC PhD Program in Stem Cell Biology |
| Year(s) Of Engagement Activity | 2015,2016,2017,2018,2019,2020 |
| Description | Workshop on the Neural control of haematopoiesis |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | The goal of this workshop was to bring a wide representation of the research community together to review the current state of how brain signals and neurohormones regulate blood cell production and function, and to identify gaps in knowledge. |
| Year(s) Of Engagement Activity | 2020 |
| Description | XVI course of the European Bone Marrow Working Group. |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Keynote lecturer of working group conference. |
| Year(s) Of Engagement Activity | 2023 |