In vivo and in silico mapping of cell-cell interactions in the haematopoietic stem cell niche

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

Every second of our adult lives we produce some 2 million red blood cells; over the course of a day in total 10^12 new blood cells are generated in healthy individuals. The process which produces this extraordinary number of cells begins in the bone marrow where a specific set of stem cells, so called haematopoietic stem cells (HSCs), reside. These cells give rise to all the different cells that make up our blood and the immune system. Understanding of this process is of fundamental importance, and the ability to rationally affect the dynamics of the haematopoietic system will also have major consequences for ageing research, regenerative medicine and clinical haematology.

In order to function properly, HSCs rely on the support of other cells inside the bone marrow, but these are as yet not known with certainty. In the proposed research we will use experiments in mice to determine which cells allow HSCs to function properly. The experiments we will conduct use a very powerful type of microscopy allowing us to visualise the HSCs inside the bone marrow of living mice. We will obtain high-resolution 3D images over time showing the location of HSCs and the identity of their neighbouring cells. We will use computational method to perform several measurements on these images and the resulting data will be analysed statistically and provide the basis for the development of computer models of the cells and their interactions inside the bone marrow. We can run these computer models to simulate the events happening in vivo and by comparing the simulation output with the experimental data we will be able to test, improve and validate our understanding of the cellular interactions responsible to support the stem cells in the bone marrow. The mathematical model here serves as a summary of our understanding of the mechanisms acting within the bone marrow; any disagreement between the simulations and the observed data points to gaps in our understanding and will motivate further analysis.

Based on preliminary analyses and modelling we will then study mice that lack certain cell-types. This in turn will provide us with more detailed insights about the effect that these cells have on the fate of HSCs. In addition to measuring the spatial distributions of cells in different types of bones, we will also determine the differences in gene expression in HSCs that result from deletion of other cell types.

Finally, we plan to use new microscopy-based techniques which allow us to directly kill individual cells in the neighbourhood of HSCs and study the response of HSCs to such perturbations. If we can successfully predict that HSCs migrate towards other cells of the same type as the deleted cell then this would substantially increase our confidence in our models.

Computer models of the cell population dynamics inside the HSC niche will be used to systematically probe our understanding; but they can also be used in the future to replace experiments in mice.

Technical Summary

Correct functioning of haematopoietic stem cells (HSCs) depends on their interaction with complex niches in the bone marrow (BM) and the question is open whether different BM cell combinations form functionally distinct niches. Endosteal and perivascular niches are suggested to support quiescent and active HSCs respectively, but a clear comparison between the two is missing. We propose to combine sophisticated mouse genetics, cutting edge in vivo imaging and statistical and computational analyses to map the cellular interactions between HSCs and other mouse bone marrow cell types in the BM space. We will use statistical modelling to determine how different cell types maintain healthy HSCs and test the hypothesis that the lineage of niche cells is critical for their role. We will complement live imaging of HSC niches in the calvarium BM with immunohistological analysis of cross sections from both calvarium and femur to further expand the array of identifiable niche cells and to compare HSC niches located in anatomically distinct environments. Mathematical modelling will implement mechanistic hypotheses computationally, and we will test the fit of the resulting predictions with the experimental observations. We will use genetic approaches and photo-ablation to eliminate both entire niche lineages and individual/few niche cells and we will use intravital imaging to monitor the consequences on HSC behaviour. Transcriptomic analysis on purified HSC populations will determine how interactions (or lack thereof) with different cell types affect gene expression in HSCs. By comparing our results with known signatures of healthy, expanding or impaired HSCs we will investigate the cause-effect link between the nature of HSC-niche interaction and HSC function. With this work we will obtain a comprehensive map of the cellular processes contributing to HSC maintenance and enabling haematopoiesis in healthy individuals, which are lost when haematological diseases develop.

Planned Impact

Multi-scale problems and in vivo analyses are all-pervasive in biology and especially in biomedical research. The biggest immediate and mid-term impact of this research is the development of an integrative framework for the quantitative analysis of the in vivo haematopoietic stem cell (HSC) niche. There is tremendous scope for applying such tools in fundamental and applied biological and biomedical research. The HSC niche is of direct biomedical importance, and maintenance of a healthy niche environment is also pivotal for healthy ageing (in humans as well as animals).

Other application areas include:
- tissue engineering and stem cell biology;
- regenerative medicine.

To maximize short-term impact we will release software in a suitable licensing framework that will allow easy and free access to academic stake-holders. The commercial scope for such software will be explored with the Imperial College Technology Transfer office, but free access to academic researchers is important to us. We will also disseminate all the in vivo imaging and niche modification protocols through publications and direct teaching. All data sets generated from intravital microscopy, immunohistological analysis and transcriptomic analysis will be deposited in appropriate, open access databases.

Understanding the factors that control the health of the HSC niche is of fundamental importance and many of the major implications will be realized only over longer time-scales. In the medium term we will, however, also discuss the application of our findings with clinical researchers and stakeholders in the biomedical sector, including cancer charities. Over shorter time-scales we hope to use our in silico models of the HSC niche for the 3R purposes and we will investigate and maximize the likely impact in conversations with the NC3Rs.

In addition we will address the distinct lack of individuals trained and conversant in both computational and laboratory techniques. The need for such individuals in academia and industry is likely to increase and a suitably trained individual will be able to lead innovative new research programmes. It is one of our essential aims to aid the RAs to become recognized researchers at the wet/dry interface of stem cell systems biology.

Publications

10 25 50
 
Description Multi-colour niche reporter mice are being produced, and both 2D and 3D ex vivo stainings of HSCs have been achieved.
Exploitation Route our findings will be shared through publications and oral presentations
Sectors Other

 
Description In vivo analysis of the interactions between acute myeloid leukaemia, T cells and haematopoietic stem cells to inform the design of T cell immunotherapy protocols.
Amount £253,000 (GBP)
Organisation Bloodwise 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2016 
End 01/2019
 
Description In vivo imaging of the interactions between invading leukaemia, declining healthy haematopoietic cells and remodelled stroma cells in the bone marrow: implications for novel therapeutic interventions.
Amount £140,544 (GBP)
Organisation Bloodwise 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2016 
End 09/2020
 
Title Longitudinal intravital microscopy of mouse bone marrow 
Description precise repositioning of animals in intravital microscopy set up to achieve tissue-wide monitoring of haematopoietic events. It reduces the number of animals needed to comnduct our research 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Publication currently under revision 
 
Description Dr Chiu Fan Lee 
Organisation Imperial College London
Department Department of Bioengineering
Country United Kingdom 
Sector Academic/University 
PI Contribution We are developing mathematical models exploring the biophysical principles of bone marrow haematopoiesis based on our in vivo and ex vivo microscopy data
Collaborator Contribution theory and modelling
Impact still unpublished
Start Year 2018
 
Description Ken Duffy - population dynamics of HSPCs 
Organisation Maynooth University
Country Ireland 
Sector Academic/University 
PI Contribution We provided flow cytometry data the allows measuring the proliferation rate of HSPCs. We are now providing multiple types of data arising from flow cytometry and microscopy analyses, which Prof Duffy continues to be instrumental in analysing.
Collaborator Contribution Prof Duffy financed the salary of a postdoc who analysed the data and performed mathematical modelling (about 6 months FTE). Prof Duffy continues to provide statistical analysis and mathematical modelling of our data.
Impact Akinduro et al., 2018 Duarte et al, 2018 Haltalli et al, biorXiv 2018 (currently under review) RIA/RS exchange program grant 2018 - 2020 Multi-disciplinary collaboration: biology, microscopy, mathematics, statistics, computational biology
Start Year 2015
 
Description Prof Louise Purton 
Organisation St Vincent's Hospital
Country Australia 
Sector Hospitals 
PI Contribution We provided insights of the consequences of Tcell acute leukaemia and acute myeloid leukaemia development on the bone marrow microenvironment in murine models
Collaborator Contribution Prof Purton provided human biopsies stained for osteoblasts markers and for endosteal vessels, showing dramatic loss of osteoblasts in T-ALL and and of endosteal vessels in AML. Prof Purton continues to provide advice on histological stainings methodologies.
Impact Hawkins et al., Nature 2016 Multiple interviews with the press to divulgate our finding as they may have an impact on clinical practice in the long term Tjin G, Bone 2018 Duarte et al, Cell Stem Cell 2018
Start Year 2015
 
Description Teresa Marafioti 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide hypotheses based on our murine models that can be further tested on human trephine biopsies by immunohistological analyses
Collaborator Contribution Dr Marafioti provides technical support with histological stainings and human trephine biopsies for histological analyses
Impact in progress
Start Year 2017
 
Description Valentina Greco - laser mediated cell ablation 
Organisation Yale University
Department School of Medicine
Country United States 
Sector Academic/University 
PI Contribution We provided training on intravital microscopy set up
Collaborator Contribution We received training in laser mediated photoablation, which we are now applying to the HSC niche
Impact manuscripts in preparation
Start Year 2014
 
Description Blood culture 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Wellcome trust funded, multi-awarded radio drama revolving around sickle cell anaemia, blood and bone marrow transplant topics
Year(s) Of Engagement Activity 2017
URL http://www.blood-culture.com/
 
Description Imperial College Festival 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact During the 2 day festiva, we had a stand called: Stem cells love their homes. In this space adults could view 1) enlarged confocal microscopy images of stem cells in their home (bone marrow), 2) slides to observe blood cells and for children and schematics of haematopoietic stem cell development and leukemia, we had a space for drawing a building the perfect stem cell house
Year(s) Of Engagement Activity 2015
URL https://www.imperial.ac.uk/festival/about/festival-2016/
 
Description MedSciLives 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Interviewed for the MedSciLife posts of the Academy of Medical Sciences
Year(s) Of Engagement Activity 2017
 
Description NatureJobs featured article 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I was interviewed and the piece published by Nature Jobs, and noticed internationally
Year(s) Of Engagement Activity 2017
URL http://blogs.nature.com/naturejobs/2018/01/10/cristina-lo-celso-career-changing-encounters/
 
Description Women at Imperial 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Policymakers/politicians
Results and Impact Many people attended our stand and undertook the activities we proposed
Year(s) Of Engagement Activity 2015