Manufacture scale up of human pericyte progenitor cells for regenerative medicine
Lead Research Organisation:
University of Bristol
Department Name: Clinical Science at South Bristol
Abstract
Regenerative stem cell therapy offers new hope for treating cardiovascular disease. The potential of this approach is immense but not completely developed. Clinical trials of cell therapy in patients with myocardial infarction (MI) have so far investigated a relatively small number of options, focusing mainly on the use of blood and bone marrow-derived cells. A host of other cells merit attention as they may offer a specialised means for myocardial and vascular reconstitution. Among these, vascular mural cells and pericytes are gaining attention. The rationale of pericyte-based therapy is to stabilise peri-infarct blood vessels, which would remain otherwise prone to leak and break. We have developed a method for easy isolation and expansion of pericytes from human saphenous veins of patients with coronary artery disease. The vein is obtained without harm to the patient using the leftover from coronary artery bypass surgery. Likewise, a small piece of vein could be obtained through minimal invasive surgery to isolate and expand pericytes in culture and obtain million cells ready for clinical use. We demonstrated that transplantation of human pericytes exerts long-term therapeutic benefit in models of limb ischaemia and MI. Noteworthy, pericytes engraft in peri-infarct vascularisation and exert a spectrum of reparative actions, including attraction of resident cardiac stem cells and circulating angiogenic cells. Furthermore, pericytes improve the survival of cardiac cells thus leading to reduction of the infarct size. No safety concerns were reported. The current isolation/expansion method was developed in a common tissue culture laboratory. Hence, we now need to upgrade it to the standards required for clinical use in patients. The present study aims to develop a clinical grade cell product through a series of regulated procedures and quality controls in preparation of a first-in-man clinical study with autologous pericytes in coronary artery disease patients unresponsive to medications and not amenable to revascularization. The term autologous means that pericytes are extracted from the patient vein, expanded and reintroduced into the patient's heart. Use of patient's own cells avoids their rejection and transmission of infectious disease, which are common problems when using cells from a different donor. The project comprises 3 work-packages (WP), 6 milestones (M) and a contingency plan for prevention, troubleshooting and alternative solutions. The goal of WP1 is to scale up current protocol to improve the yield of cells available for clinical use. The goal of WP2 is to transfer the protocol to the facilities of the National Health Service-Blood and Transplant (NHSBT) for clinical-grade manufacture of at least 6 cell lines. This will be followed by quality control of cell stability. The goal of WP3 is to test the performance of the cell product using in vitro functional assays and models of MI. The project will lead to the clinical manufacture of a novel regenerative medicine cell product through the liaison of the University of Bristol and NHSBT, which is the recommended channel for clinical application of autologous cell therapy in UK. Hence, successful completion of proposed work will make the cell product available immediately to the healthcare system for patient benefit.
Technical Summary
This proposal aims to develop a novel cell product for regenerative vascular medicine. The prototype of the cell product, consisting of saphenous vein-derived pericyte progenitor cells (SVPs), proved to be therapeutically effective in preclinical models of peripheral or myocardial ischaemia. Supplementation of pericytes stabilises the peri-infarct neovascularisation which is otherwise prone to leak and break. Furthermore, SVPs exert a spectrum of reparative actions including recruitment of pro-angiogenic monocytes and cardiac progenitor cells and inhibition of apoptosis and fibrosis. No safety concerns were reported. We propose now to validate the capability of a defined system of technical procedures to increase total throughput of cells and obtain a reliable, reproducible and quality-assured cell product in view of performing a first-in-man clinical trial with autologous SVPs in coronary artery disease patients unresponsive to medications and not amenable to revascularization. The project comprises 3 work-packages (WP), 6 milestones (M) and a contingency plan for prevention, troubleshooting and alternative solutions. The goal of WP1 is to scale up current protocol to improve the yield of extraction/expansion (M1) and introduce clinical-grade and xeno-free reagents (M2). The goal of WP2 is to transfer the protocol to the GMP facilities of the National Health Service-Blood and Transplant (NHSBT) for clinical-grade manufacture of at least 6 SVP lines (M3), followed by quality control of antigenic and genomic/epigenetic stability (M4). The goal of WP3 is to test the performance of individual SVP lines (WP5) and assess the therapeutic efficacy in a mouse model of myocardial infarction using a randomised factorial protocol (WP6).
Planned Impact
Social Benefit
Cardiovascular disease remains the most serious medical challenge we face. Regenerative medicine could potentially improve health and save public money by reducing the need for long-term care, with benefit for the UK economy as a whole. Our main ambition is to achieve medical benefit through generation of a novel cell product, i.e. human pericytes, which can be used to aid the neovascularisation of ischaemic tissues. The study will bring a new approach to generate blood vessels that are not susceptible to break or regress, as instead often occurs during spontaneous angiogenesis in ischaemic tissues as well as during pathologic angiogenesis, e.g. in diabetes. The end result of this project, three years after initiation, will be the provision of a clinical grade cell product to be tested in a first-in-man clinical trial in patients with coronary artery disease unresponsive to medications and not amenable to revascularization. Moreover, the cell product could be used in association with cardiac stem cells, proangiogenic cells and gene therapy to promote full repair of ischaemic organs. Patients with critical limb ischaemia as well as diabetic patients with microangiopathy could also benefit from the use of pericyte-based therapies. If successful this could benefit millions of UK citizens with cardiovascular disease.
Healthcare Benefit
We have been able to build a team of basic and clinical scientists, bioinformatics/geneticists and specialists from the National Health Service that will collaborate to reach a common objective. Working together will be cross-fertilising and instrumental for the next stage in the development of our overarching strategy. Furthermore, the project will involve the transfer of methodologies from the academia to the National Health Service, where those methodologies will be upgraded to clinical manufacture. Hence, the project will have a profound impact on transferability, medical standards and quality controls of cell manufacture.
Advancement of knowledge
This project is likely to produce results that will advance the knowledge on stem cell behaviour during expansion and following transplantation. For instance, we will assess the effect of expansion and transplantation on genomic/epigenetic stability, which will inform about the safety of this specific cell product and also shed new light on the safety of cell therapy in general. Correlation of therapeutic efficacy in a preclinical model with functional and epigenetic signatures of different cell lines will have an impact on reverse translation, i.e. understanding how the cell product works and if variability of the cell product can predictably influence therapeutic activity. However, perhaps the most important impact will be providing a successful example of translation of basic science discoveries into a medically relevant product. To the best of our knowledge, this will be the first example of a stem cell type isolated, expanded and pre-clinically tested by a UK academic group and further developed by the same investigators into a clinically-grade cell product.
Industrial exploitation
Innovative aspects of our study might be patentable. While this is especially applicable to conventional medical products and in some case allogeneic cell therapy, autologous cell therapies offer less scope for intellectual coverage (since a patient's own cells can't be patented). Hence, we are following the suggestion of the recent document of the UK Ministry of health to embed exploitation plans in an NHS-based model. The University of Bristol and NHSBT will discuss any relevant issue arising from this collaboration that might merit intellectual protection. Finally, the project introduces a series of new commercial reagents for cell manufacture upgrading. Testing these reagents may have an impact on the quality of culture media, matrices etc, thus helping further refinement and improvements by industrial providers.
Cardiovascular disease remains the most serious medical challenge we face. Regenerative medicine could potentially improve health and save public money by reducing the need for long-term care, with benefit for the UK economy as a whole. Our main ambition is to achieve medical benefit through generation of a novel cell product, i.e. human pericytes, which can be used to aid the neovascularisation of ischaemic tissues. The study will bring a new approach to generate blood vessels that are not susceptible to break or regress, as instead often occurs during spontaneous angiogenesis in ischaemic tissues as well as during pathologic angiogenesis, e.g. in diabetes. The end result of this project, three years after initiation, will be the provision of a clinical grade cell product to be tested in a first-in-man clinical trial in patients with coronary artery disease unresponsive to medications and not amenable to revascularization. Moreover, the cell product could be used in association with cardiac stem cells, proangiogenic cells and gene therapy to promote full repair of ischaemic organs. Patients with critical limb ischaemia as well as diabetic patients with microangiopathy could also benefit from the use of pericyte-based therapies. If successful this could benefit millions of UK citizens with cardiovascular disease.
Healthcare Benefit
We have been able to build a team of basic and clinical scientists, bioinformatics/geneticists and specialists from the National Health Service that will collaborate to reach a common objective. Working together will be cross-fertilising and instrumental for the next stage in the development of our overarching strategy. Furthermore, the project will involve the transfer of methodologies from the academia to the National Health Service, where those methodologies will be upgraded to clinical manufacture. Hence, the project will have a profound impact on transferability, medical standards and quality controls of cell manufacture.
Advancement of knowledge
This project is likely to produce results that will advance the knowledge on stem cell behaviour during expansion and following transplantation. For instance, we will assess the effect of expansion and transplantation on genomic/epigenetic stability, which will inform about the safety of this specific cell product and also shed new light on the safety of cell therapy in general. Correlation of therapeutic efficacy in a preclinical model with functional and epigenetic signatures of different cell lines will have an impact on reverse translation, i.e. understanding how the cell product works and if variability of the cell product can predictably influence therapeutic activity. However, perhaps the most important impact will be providing a successful example of translation of basic science discoveries into a medically relevant product. To the best of our knowledge, this will be the first example of a stem cell type isolated, expanded and pre-clinically tested by a UK academic group and further developed by the same investigators into a clinically-grade cell product.
Industrial exploitation
Innovative aspects of our study might be patentable. While this is especially applicable to conventional medical products and in some case allogeneic cell therapy, autologous cell therapies offer less scope for intellectual coverage (since a patient's own cells can't be patented). Hence, we are following the suggestion of the recent document of the UK Ministry of health to embed exploitation plans in an NHS-based model. The University of Bristol and NHSBT will discuss any relevant issue arising from this collaboration that might merit intellectual protection. Finally, the project introduces a series of new commercial reagents for cell manufacture upgrading. Testing these reagents may have an impact on the quality of culture media, matrices etc, thus helping further refinement and improvements by industrial providers.
Organisations
- University of Bristol (Lead Research Organisation)
- Technion - Israel Institute of Technology (Collaboration)
- University of Pisa (Collaboration)
- University College London (Collaboration)
- MultiMedica (Collaboration)
- UNIVERSITY OF LIVERPOOL (Collaboration)
- IMPERIAL COLLEGE LONDON (Collaboration)
- University of Bristol (Collaboration)
- Spanish National Centre for Cardiovascular Research (Collaboration)
Publications
![publication icon](/resources/img/placeholder-60x60.png)
Gaudino M
(2019)
Radial Artery Versus Right Internal Thoracic Artery Versus Saphenous Vein as the Second Conduit for Coronary Artery Bypass Surgery: A Network Meta-Analysis of Clinical Outcomes.
in Journal of the American Heart Association
![publication icon](/resources/img/placeholder-60x60.png)
Gaudino M
(2018)
Off-Pump Coronary Artery Bypass Grafting: 30 Years of Debate
in Journal of the American Heart Association
![publication icon](/resources/img/placeholder-60x60.png)
Gibbison B
(2020)
Dynamic Pituitary-Adrenal Interactions in the Critically Ill after Cardiac Surgery.
in The Journal of clinical endocrinology and metabolism
![publication icon](/resources/img/placeholder-60x60.png)
Gibbison B
(2020)
Dynamic Pituitary-Adrenal Interactions in the Critically Ill after Cardiac Surgery.
in The Journal of clinical endocrinology and metabolism
![publication icon](/resources/img/placeholder-60x60.png)
Gomes W
(2019)
On- or Off-pump Coronary Artery Bypass Surgery. Is the Debate Settling Down?
in Brazilian Journal of Cardiovascular Surgery
![publication icon](/resources/img/placeholder-60x60.png)
Gubernator M
(2015)
Epigenetic profile of human adventitial progenitor cells correlates with therapeutic outcomes in a mouse model of limb ischemia.
in Arteriosclerosis, thrombosis, and vascular biology
![publication icon](/resources/img/placeholder-60x60.png)
Guida GA
(2020)
Left thoracotomy approach for off-pump coronary artery bypass grafting surgery: 15 years of experience in 2500 consecutive patients.
in European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery
![publication icon](/resources/img/placeholder-60x60.png)
Haj-Yahia S
(2019)
Correction to: Case report and management approach in idiopathic pulmonary arteries aneurysm.
in Journal of cardiothoracic surgery
![publication icon](/resources/img/placeholder-60x60.png)
![publication icon](/resources/img/placeholder-60x60.png)
Iacobazzi D
(2014)
Increased antioxidant defense mechanism in human adventitia-derived progenitor cells is associated with therapeutic benefit in ischemia.
in Antioxidants & redox signaling
Description | Derivation of swine pericytes to enable fit-for-purpose clinical translation |
Amount | £107,481 (GBP) |
Funding ID | FS/16/64/32480 |
Organisation | British Heart Foundation (BHF) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 12/2016 |
End | 11/2019 |
Title | Generation of IPSC from Saphenous vein progenitor cells |
Description | Using episomal transfection method pioneered from the Yamanka lab to generate IPSC from discarded bypass saphenous vein derived Saphenous vein progenitor cell. |
Type Of Material | Cell line |
Provided To Others? | No |
Impact | possible grant application |
Title | Quality control system |
Description | Upgrading a cell product for clinical use |
Type Of Material | Technology assay or reagent |
Year Produced | 2013 |
Provided To Others? | Yes |
Impact | Upgrading a cell product for clinical use |
Description | Bioscaffolds surfaces for Saphenous vein progenitor cells |
Organisation | University College London |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Provide cells to the UCL collaborator and then test their behaviour in vitro and in vivo |
Collaborator Contribution | embedd the cells in the bioscaffolds |
Impact | Application for a MRC project grant |
Start Year | 2014 |
Description | Generation of IPSC from saphenous vein progenitor cells |
Organisation | University of Bristol |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Expansion of Saphenous vein progenitor cells and induction into IPS using the episomal method pioneered by Yamanka. Following this the IPSC cells are expanded and characterised. |
Collaborator Contribution | Dr Caldwells teams are provideing the tools and support. |
Impact | Publications and grant applications |
Start Year | 2014 |
Description | Generation of induced pluripotent stem cells from human sapheous vein pericytes |
Organisation | Technion - Israel Institute of Technology |
Country | Israel |
Sector | Academic/University |
PI Contribution | Our research team has the technology to isolate and expand human sapheous vein pericytes from coronary heart bypass surgery. We have a large number of pericyte lines. This work was funded by a joint BIRAX grant |
Collaborator Contribution | Professor Itskovitz's team previous experience in generating induced pluripotent stem cells. |
Impact | We jointly been awarded a BIRAX fellowship to allow a Ph.D student from Israel to work in our lab. |
Start Year | 2013 |
Description | Imperial College London Department of Cardiac Surgery co-applicant |
Organisation | Imperial College London |
Department | Faculty of Medicine |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Recruiting to the trial |
Collaborator Contribution | Grant co-applicant |
Impact | Still recruiting |
Start Year | 2019 |
Description | SVPs on biomaterials |
Organisation | Imperial College London |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | To provide cells to be embedded in 3D culture systems to study angiogenesis |
Collaborator Contribution | To study molecular interaction between endothelial cells and pericytes |
Impact | application for a fellowship |
Start Year | 2014 |
Description | The large animal model of myocardial infarction for testing saphenous vein progenitor cells. |
Organisation | Spanish National Centre for Cardiovascular Research |
Country | Spain |
Sector | Public |
PI Contribution | We are isolating and expanding SVP cells for injection into Myocardial Infarction (MI) model. We are also characterising heart histology from these animal model. |
Collaborator Contribution | Professor Ibanez team are performing the MI surgery, injection and animal husbandry. |
Impact | Publications and step forward to clinical trial |
Start Year | 2014 |
Description | Therapeutic applications of vascular regeneration model in mouse hind limb ischaemia. |
Organisation | MultiMedica |
Country | Italy |
Sector | Private |
PI Contribution | We performed hind limb ischaemia operation to see therapeutic activity of pericytes and a biocompatible scaffold. |
Collaborator Contribution | They provided us materials, such as pericytes and a biocompatible scaffold. |
Impact | Application for a BHF studentship and an European grant |
Start Year | 2014 |
Description | Therapeutic applications of vascular regeneration model in mouse hind limb ischaemia. |
Organisation | University of Bristol |
Department | School of Physiology, Pharmacology and Neuroscience |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We performed hind limb ischaemia operation to see therapeutic activity of pericytes and a biocompatible scaffold. |
Collaborator Contribution | They provided us materials, such as pericytes and a biocompatible scaffold. |
Impact | Application for a BHF studentship and an European grant |
Start Year | 2014 |
Description | Therapeutic applications of vascular regeneration model in mouse hind limb ischaemia. |
Organisation | University of Pisa |
Department | Research Centre E. Piaggio |
Country | Italy |
Sector | Academic/University |
PI Contribution | We performed hind limb ischaemia operation to see therapeutic activity of pericytes and a biocompatible scaffold. |
Collaborator Contribution | They provided us materials, such as pericytes and a biocompatible scaffold. |
Impact | Application for a BHF studentship and an European grant |
Start Year | 2014 |
Description | Vascular toxicity testing |
Organisation | University of Liverpool |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | to provide cells to study the toxic effects of cardiac treatments |
Collaborator Contribution | Provide cells from human tissues |
Impact | Future grant applications |
Start Year | 2014 |
Description | BHF Retail Group Talk |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Supporters |
Results and Impact | 1 hr lecture to BHF Retail Group Re: work so far. |
Year(s) Of Engagement Activity | 2019 |
Description | British Heart Foundation Event at St James' Palace hosted by the Duke of Edinburgh. Representative of BHF medical community. |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | British Heart Foundation event at St James' Palace hosted by The Duke of Edinburgh. Representative of BHF medical community. March 2017 |
Year(s) Of Engagement Activity | 2017 |
Description | British Heart Foundation Lecture. BHF 40th Anniversary Supporters Group Meeting, Chippenham |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | An invited guest lecture about the research work being carried out at the Bristol Heart Institute at the University of Bristol to members of the BHF Supporters Group in Chippenham on their 40th anniversary. |
Year(s) Of Engagement Activity | 2018 |
Description | British Heart Foundation Service of Thanksgiving, London December 2017 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Patients, carers and/or patient groups |
Results and Impact | British Heart Foundation Service of Thanksgiving, London December 2017. Guest Christmas Lecture. |
Year(s) Of Engagement Activity | 2017 |
Description | Interview for BBC. Filming of patients re PROMPT trial. |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Interview for BBC. Filming of patient re PROMPT trial. 30th October 2018. |
Year(s) Of Engagement Activity | 2018 |
Description | Keynote Speaker. "Publishing surgeon specific results" "Results from TITRE2" "How to develop an academic department" |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Invited lectures at the Australian and New Zealand Society of Cardiac and Thoracic Surgeons Annual Scientific Meeting, Adelaide 2016. Keynote speaker. |
Year(s) Of Engagement Activity | 2016 |
Description | Lecture at BHF supporter day. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Gave a lecture to a regional BHF supporter day Lecture entitled "Overview of BHF Funded Research" |
Year(s) Of Engagement Activity | 2017 |
Description | Lecture at conference. Minimally Invasive Cardiac Surgery and Non-Conventional Interventional Cardiology Conference, Room 2016 |
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 | Lecture "Publishing surgeon specific data and its problems". Minimally Invasive Cardiac Surgery and Non-Conventional Interventional Cardiology Conference, Rome 2016 |
Year(s) Of Engagement Activity | 2016 |
Description | Lecture at the Population Health ECT Event and Annual Symposium, Engineers House, Bristol, 2016 |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Lecture on "Blood Conservation" at the Population Health Interdisciplinary Early Career Researchers' event. Annual Symposium and Stephen Frankel Lecture. |
Year(s) Of Engagement Activity | 2016 |
Description | Lecture at the Society of Cardiothoracic Surgery Annual Meeting in Belfast, 2017 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | SCTS Annual Meeting, Belfast 2017. "Mini-CPB: do we need a national or international trial? The SCTS aims to continuously improve the quality of healthcare that members deliver to patients in an open and accountable manner. The SCTS also has a growing patient involvement and patients who have undergone cardiothoracic surgery have been invited to attend our meeting. |
Year(s) Of Engagement Activity | 2017 |
Description | Lecture to 6th form students at Redcliffe Sixth Form Centre, Bristol |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | The talk was to give inspiration to A level students who were thinking of studying medicine. Several students have now contacted us and since attended a 'work experience' at the Bristol Royal Infirmary. They have gained a lot from this experience and it has confirmed their desire to study medicine and carry out research. |
Year(s) Of Engagement Activity | 2019 |
Description | NIHR Senior Investigators Selection Panel Meeting 3/4 September 2018 |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Policymakers/politicians |
Results and Impact | Member of the NIHR Senior Investigators Panel Section Meeting, 4th December |
Year(s) Of Engagement Activity | 2018 |
Description | Oxford International Biomedical Centre Symposium. The Mysterious Human Heart Genes, Environment, Therapy, Midsomer Norton, 2014 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | Oxford International Biomedical Centre Symposium: The Mysterious Human Heart: Genes, Environment, Therapy. Midsomer Norton, 2014. "Taking Engineering to the Heart". |
Year(s) Of Engagement Activity | 2014 |
Description | Public Debate. Heart Disease is the UK's Biggest Killer, London 2014 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Public Debate. Heart Disease is the UK's Biggest Killer. London, 2014. "Are stem cells, tissue engineering and regenerative medicine the future in heart surgery" |
Year(s) Of Engagement Activity | 2014 |
Description | St John's Ambulance Avon: First Annual Lecture, Bristol 2012 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | St John's Ambulance, St John Ambulance Avon: First Annual Lecture. Bristol, 2012. "Engineering the future of heart surgery". |
Year(s) Of Engagement Activity | 2012 |