The stem cell fate of pericytes in tissue regeneration

Lead Research Organisation: University of East Anglia
Department Name: Unknown

Abstract

Pericytes are perivascular cells that wrap endothelial cells in capillaries and form by that the structural support of small blood vessels, necessary to maintain blood pressure. It is now clear that pericytes are also involved in diseases that are closely linked with the formation of new blood vessels, such as diabetic retinopathy or cancer. Previous research into pericyte function has been limited as the isolation of pure cells proved difficult. But now this obstacle has been overcome by the development of sophisticated equipment and by the genetic engineering of mice in which mutations were found to affect pericyte function. Isolated pericytes were shown to be capable to differentiate into various cell types, and it was therefore suggested that they represent stem cell-like progenitor cells. Furthermore, pericytes that were isolated from skeletal muscle and injected into injured muscle were shown to participate in muscle regeneration. This has created big hope for gene therapy approaches using these cells. However, in order to safely use and to maximise the efficiency of pericytes in human applications it is necessary to fully characterise the potential of the endogenous pericytes, the degree to which they spontaneously contribute to tissue remodelling, and potential undesired side-effects,
We have previously generated a mouse model in which pericytes can be specifically identified by a colour-based staining procedure in isolated tissues. We have now crossed these mice with mdx mice, the mouse model for the human muscle wasting disease Duchenne muscular dystrophy, to determine by colour reaction whether or not endogenous pericytes contribute to new muscle formation. This has shown that there is massive remodelling of these cells in injured skeletal muscle. However, we are limited in the further identification of cells that originated from pericytes. We therefore propose to generate a more versatile mouse model, which marks the cells genetically with a fluorescent protein and enables us to follow the fate of pericytes in muscle regeneration by using imaging techniques that allow a high penetrance in the intact tissue. Moreover, this mouse model will be invaluable in the future to characterise pericyte progenitor cells from a variety of tissues for their capacity in tissue remodelling.

Technical Summary

Perivascular cells/pericytes are adjacent to capillaries and have been named due to their anatomical location. Pericytes are in intimate contact with endothelial cells in capillaries and share a common basement membrane. They are thought to stabilise the vascular wall and control blood pressure and vascular permeability. In recent years there has been increasing evidence that pericytes represent a cell population with mesenchymal stem cell-like properties as they retain the capacity to differentiate into distinct mesenchymal lineages in vitro. However, there is little information available with respect to their differentiation potential in vivo. Two recent reports demonstrated that pericytes, isolated from skeletal muscle, can participate in muscle regeneration. This has raised the question as to whether endogenous pericytes contribute to regeneration of skeletal muscle and other tissues after injury or in disease.
Previously, we have generated a mouse model in which the LacZ gene was targeted into the annexin V (Anxa5) locus. The expression of the Anxa5-LacZ fusion gene defined a specific marker for perivascular cells during development and in the adult, and allowed the isolation of pericytes from embryos and adult meninges. The purified cells express pericyte-specific markers as well as markers characteristic for stem cell populations and are capable to differentiate in vitro into mesenchymal lineages. We have now crossed Anxa5-LacZ mice with mdx mice, the mouse model for human Duchenne muscular dystrophy, to determine the endogenous contribution of pericytes to muscle regeneration by LacZ staining. The intriguing results of these experiments suggest that Anxa5-LacZ-positive precursor cells actively participate in remodelling of the tissue. However, further interpretation of these results is limited due to the static histological procedures, necessary for LacZ staining, and the uncertainty of a regulated Anxa5 promoter activity in pericyte descendents. We therefore propose to generate a pericyte reporter mouse strain with which pericytes and all of its descendents can be permanently genetically labelled by targeting Cre recombinase into the Anxa5 locus (Anxa5-Cre). Using skeletal muscle as model tissue, this will allow us to trace and characterise pericytes and its descendents in muscle regeneration after induced injury by a myotoxic agent using ex vivo 2-photon laser scanning microscopy. Furthermore, Anxa5-Cre mice will be a versatile model to study the role of pericytes associated with other diseases (cancer, diabetes) in future studies and help to develop and test novel therapeutic strategies.

Publications

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Ackroyd MR (2011) Fukutin-related protein alters the deposition of laminin in the eye and brain. in The Journal of neuroscience : the official journal of the Society for Neuroscience

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Grskovic I (2012) Depletion of annexin A5, annexin A6, and collagen X causes no gross changes in matrix vesicle-mediated mineralization, but lack of collagen X affects hematopoiesis and the Th1/Th2 response. in Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

 
Description Accelerate research in muscular dystrophy research
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
 
Description Enhancing Israel/UK collaboration in stem cell research
Geographic Reach Asia 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Training in in vivo bioimaging and cell tracking of stem cells
Geographic Reach Asia 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Large-scale integrating project
Amount £340,000 (GBP)
Funding ID Artivasc 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 11/2011 
End 10/2015
 
Description Research grant
Amount £560,660 (GBP)
Funding ID MR/R000549/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2018 
End 02/2021
 
Title 2-photon microscopy of injured muscle tissue 
Description We have developed a methodology to maintain injured skeletal muscle ex vivo over prolonged time periods. We have further setup 2-photon microscopy to follow the fate of genetically labeled pericytes during tissue repair in skeletal muscle. We have also the first time lapse results following pericyte fate in injured skeletal muscle 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This combination of the technologies will enable to determine the potential of endogenous pericytes to contribute to skeletal muscle regeneration and may provide in the longer term a possibility to stimulate muscle repair in disease and in ageing. 
 
Title Anxa5-Cre knockin mouse strain 
Description A transgenic mouse strain has been generated in which Cre recombinase has been inserted into the Annexin A5 locus 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact This mouse strain is a new and independent line with which pericytes can be analyzed. Recently, Pericytes have attracted a lot of attention as a new type of mesenchymal stem cells. In particular, as pericytes are a highly diverse population of cells this strain now allows a detailed comparison with other pericyte specific strains and will reveal similarities and differences between currently known markers. 
 
Title Cells with pericyte-specific marker contribute to muscle regeneration 
Description Anxa5-Cre and Pdgfrb-Cre mice were combined with Rosa26 reporter strains and muscle degeneration was induced with a myotoxic agent. In fully regenerated muscle fibres the contribution of genetically-labeled pericytes was demonstrated by normal immunohistochemistry. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact This data would indicate that endogenous pericytes indeed participate in muscle regeneration. However, we noticed significant differences between Anxa5- and Pdgfrb-derived cells, suggesting that more work is needed to define the pericyte population in skeletal muscle that does participate in muscle regeneration. The knowledge of which would greatly enhance future therapies for muscle wasting diseases based on these outcomes. 
 
Title Fluorescence activated cell sorting of pericytes 
Description Pericytes positive for AnnexinA5 (Anxa5) or Pdgf receptor beta (Pdgfrb) were isolated from skeletal muscle using Pdgrfb-Cre and Anxa5-Cre mice combined with Rosa26-YFP reporter strains. Pericyte-specific markers were then profiled using FACS analysis 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Although the data sets need to be repeated for statistical analysis the results so far indicate that the pericyte phenotype in skeletal muscle is likely diverse. 
 
Title ex-vivo muscle regeneration model 
Description We have established an ex-vivo skeletal muscle regeneration model using isolated single muscle fibres to test expression of pericyte-specific markers in the activation process of muscle endogenous stem cells, satellite cells. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact The data obtained from this model indicate that satellite cells possibly adopt an intermediate pericyte phenotype during the regeneration process. However, more work is needed to confirm and specify these results. 
 
Description Artivasc 3 D 
Organisation Fraunhofer Society
Department Fraunhofer Center for Laser Technology
Country Germany 
Sector Academic/University 
PI Contribution During recent years we developed mouse strains for detecting and isolating pericytes. These methods will be used to expand the options to use pericytes, resembling a population of mesenchymal stem cells, in tissue regeneration and the development of novel materials to build up artificial micro-vascular grafts. Our task will be the isolation of stem cell populations and the optimisation of their use in stimulating neovascularisation and tissue regeneration based on the mouse model AnxA5-Cre generated in this project.
Collaborator Contribution The partnership involves 14 additional institutions/institutes in Europe within the Artivasc 3D consortium (EU Framework 7; FP7-263416; Title: 'Artificial vascularized scaffolds for 3D tissue regeneration') The partnership enables the access to novel materials and methods, previously not available to us. This includes the access to novel artificial materials as substrates for stem cells and specialized culture methods. This will stimulate any further work in improving tissue regeneration and vascularisation by the use of stem cells.
Impact The project was based on a multidisciplinary consortium involving: Nanotechnology (Generation of novel artificial materials) Physics (Use of laser technology to modify material characteristics) Biochemistry (Functionalisation of materials by biochemical modifications) Cell Biology (Use of various cell types in tissue regeneration) Medicine (Establishment and use of tissue grafts; Basic science) The main aim was to use an automated process combination of inkjet printing and multiphotonpolymerisation or stereolithography to build up a branched, porous vessel system that could provide nutrition to surrounding adipocytes. The optimised vessel design (branching angle, vessel diameter, wall thicknes, mechanical stiffnes) was calculated and data has been provided to the stereolithography process as a machine code. Another important part for vessel generation was the development of suitable biocompatible material with optimal mechanical properties which is useful both in stereolithography and inkjet printing and compatible with cells. Although great progress has been made the final goal of 3-dimensional biocompatible blood vessels was not achieved within the time frame of the grant and would have needed another 1-2 years of funding.
Start Year 2011
 
Description Generation of transgenic mouse strain 
Organisation Max Planck Society
Department Department of Tissue Morphogenesis
Country Germany 
Sector Charity/Non Profit 
PI Contribution Construction of targeting vector, Generation and identification of mutant ES cell clones Using mice for ongoing research and reporting back on the results
Collaborator Contribution Blastocyst injection and generation of chimeric mice at the MPI of Molecular Cell Biology and Genetics University of Oxford made a the Rosa26-YFP mouse strain available, with which we can genetically label pericytes, using Anxa5-Cre or Pdgfr-beta-Cre mice University of Ulm made a the Rosa26-tdRFP mouse strain available, with which we can genetically label pericytes, using Anxa5-Cre or Pdgfr-beta-Cre mice MPI for Molecular Biomedicine made the Pdgfr-beta-Cre mice available to compare specificity of the Anxa5-Cre mouse strain
Impact Generation of new mouse strain, 2-photon microscopy of genetically labeled injured skeletal muscle
Start Year 2010
 
Description Generation of transgenic mouse strain 
Organisation Max Planck Society
Department Max Planck Institute for Molecular Cell Biology and Genetics
Country Germany 
Sector Academic/University 
PI Contribution Construction of targeting vector, Generation and identification of mutant ES cell clones Using mice for ongoing research and reporting back on the results
Collaborator Contribution Blastocyst injection and generation of chimeric mice at the MPI of Molecular Cell Biology and Genetics University of Oxford made a the Rosa26-YFP mouse strain available, with which we can genetically label pericytes, using Anxa5-Cre or Pdgfr-beta-Cre mice University of Ulm made a the Rosa26-tdRFP mouse strain available, with which we can genetically label pericytes, using Anxa5-Cre or Pdgfr-beta-Cre mice MPI for Molecular Biomedicine made the Pdgfr-beta-Cre mice available to compare specificity of the Anxa5-Cre mouse strain
Impact Generation of new mouse strain, 2-photon microscopy of genetically labeled injured skeletal muscle
Start Year 2010
 
Description Generation of transgenic mouse strain 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Construction of targeting vector, Generation and identification of mutant ES cell clones Using mice for ongoing research and reporting back on the results
Collaborator Contribution Blastocyst injection and generation of chimeric mice at the MPI of Molecular Cell Biology and Genetics University of Oxford made a the Rosa26-YFP mouse strain available, with which we can genetically label pericytes, using Anxa5-Cre or Pdgfr-beta-Cre mice University of Ulm made a the Rosa26-tdRFP mouse strain available, with which we can genetically label pericytes, using Anxa5-Cre or Pdgfr-beta-Cre mice MPI for Molecular Biomedicine made the Pdgfr-beta-Cre mice available to compare specificity of the Anxa5-Cre mouse strain
Impact Generation of new mouse strain, 2-photon microscopy of genetically labeled injured skeletal muscle
Start Year 2010
 
Description Generation of transgenic mouse strain 
Organisation University of Ulm
Department Institute for Immunology
Country Germany 
Sector Academic/University 
PI Contribution Construction of targeting vector, Generation and identification of mutant ES cell clones Using mice for ongoing research and reporting back on the results
Collaborator Contribution Blastocyst injection and generation of chimeric mice at the MPI of Molecular Cell Biology and Genetics University of Oxford made a the Rosa26-YFP mouse strain available, with which we can genetically label pericytes, using Anxa5-Cre or Pdgfr-beta-Cre mice University of Ulm made a the Rosa26-tdRFP mouse strain available, with which we can genetically label pericytes, using Anxa5-Cre or Pdgfr-beta-Cre mice MPI for Molecular Biomedicine made the Pdgfr-beta-Cre mice available to compare specificity of the Anxa5-Cre mouse strain
Impact Generation of new mouse strain, 2-photon microscopy of genetically labeled injured skeletal muscle
Start Year 2010
 
Description Pericytes in stroke 
Organisation University of East Anglia
Department School of Allied Health Professionals UEA
Country United Kingdom 
Sector Academic/University 
PI Contribution We will use Pdgfrb- and Anxa5-Cre mice in combination with Rosa26 reporter strains to define the penumbra region in brain after experimental stroke, define the contribution of pericytes and test physiotherapy and drugs to maintain the penumbra for extended period of time after stroke
Collaborator Contribution Our collaborator discussed with us the problems associated with stroke and neovascularisation. This helped to get a joined PhD studentship funded by the Medical School at the University of East Anglia. Unfortunately, the student decided to quit the PhD after 18 months to follow her boyfriend to China.
Impact This was a young collaboration and has not resulted in tangible outputs
Start Year 2011
 
Description Profiling of pericytes 
Organisation University of Cologne
Country Germany 
Sector Academic/University 
PI Contribution Isolation of pericytes from different tissues (skeletal muscle, heart muscle, lung, peritoneum), followed by FACS analysis and RNA extraction for microarray analysis. Microarray analysis was performed by our collaborator. We subsequently analysed the microarray data and found significant differences in the expression pattern, what is also consistent with angiogenesis and muscle fusion experiments. We are still investigating the expression pattern, but the data add further complexity to pericytes and demonstrate that not all pericytes in a body are equal.
Collaborator Contribution Microarray analysis of the various pericyte populations
Impact Pericyes are still poorly characterised. This collaboration is a step further to identify tissue-specific differences of pericytes. We have been able to demonstrate that pericytes from different tissues behave differently in terms of contribution to muscle regeneration, and in their ability to participate in tube formation in angiogenesis assays.
Start Year 2013
 
Description MRC grant press release 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Media (as a channel to the public)
Results and Impact A press release describing the principles of our successful MRC application. There was a huge feedback from colleagues
Year(s) Of Engagement Activity 2017
URL http://www.uea.ac.uk/about/-/uea-and-nnuh-awarded-1-4m-for-pioneering-medical-research
 
Description Training the next generation of researchers 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact we have trained 54 under- and postgraduate students in methods and theoretical background important for tissue regeneration.
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015,2016,2017,2018
 
Description UEA Open day 
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 UEA open day. On average 50-60 visitors visited our presentation on stem cells in skeletal muscle. Most visitors stayed with us for extended period of time to ask further questions and looking at sections through the microscopes

Most visitors were not aware of the regenerative capacity of skeletal muscle and which cells are involved to rebuild the muscle
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014,2015,2016