Vascular constructs using human pluripotent stem cells in the therapy of peripheral arterial disease

The aim of cell-based regenerative cardiology is to replace or enhance the healing of damaged tissues to regain the heart or vessel functionality. The production of the most adequate cells, while ensuring their safe delivery in the damaged tissues is critical for the success of such treatments. The main drawback of externally introduced cells is their low viability following implantation and difficulties in their integration into the damaged tissues. Though some of the cell products have been investigated thoroughly, integrated studies in which optimal cells are generated to yield the desired optimal effect are scarce. We have been developing promising human embryonic stem cells (hESC) based vascular cells for vascular grafts. The initial goal in this project is to grow the potent endothelial populations (cells for the interior surface of blood vessels) following their generation from hESC cultures. During the pilot experiments with these cells, we have found that these cells can well repopulate matrix surfaces. This can govern the underlying gene expression and determine further endothelial cell specification. The aim is therefore to modulate these cell constructs in order to see if we can improve quality of ready-to-use vascular grafts. We will investigate these changes with various microscopy, fluorescence and molecular biology tools and compare with native or untreated cells. Similar methods are being used now for clinically used decellularised grafts in vascular surgery, which means that any of our promising findings can go more quickly into later stages of cell therapy pipeline. At the National Heart and Lung Institute, Imperial College London, we already have ways to grow endothelial cells from stem cells, measure their capillary vessel growing activity, protective effects against clot formation and how easily they can be damaged. We expect that this novel regenerative medicine technology can subsequently be adapted for a cell based therapy in peripheral, very likely in lower limb, artery disease.

We will generate endothelial cell constructs from hESC that closely resemble native vascular grafts. Our hypothesis is that culturing hESC-EC on decellularised vascular scaffold can produce durable and functional vascular grafts for peripheral artery disease. 1. Vessels will be decellularised resulting in a biocompatible scaffold that has similar mechanical characteristics compared to native tissues. Human arteries will be collected from the NHS Blood and Transplant Tissue Services. Human ESC-EC will be used to reseed decellularised scaffold which constructs have beneficial immunologic properties and function as the original vessels. 2. We will develop a robust process for generation of grafts in a bioreactor; seeding, delivery volume and surface size will be optimised. Long-term stability of endothelial phenotype will be characterised in 2D/3D cultures with cells from early and late time points. Cells and whole construct will be cryopreserved, which can provide a ready supply of allogeneic cell sources as required. 3. We will assess cell adhesion to extracellular matrices, viability/apoptosis, proliferation, migration by 3D confocal high content imaging. We will assess gene expression and secreted angiogenic factors in each 2D/3D cultures and in control arterial endothelial cells. The majority of these methods have been validated, are in routine use in 2D at Imperial College, will be adapted to 3D assays in most cases. 4. We will obtain key information on the functional ability of optimised grafts in a randomised blind controlled experimental trial in outbred pigs in Bristol. For this, hESC-EC cultures will be introduced in vivo to interface with host vasculature. We will generate batches of hESC-EC constructs that we can transfer to Bristol to perform in vivo engraftment for safety/durability assessment during 1 and 3 months follow-up.

Elucidation of signals that are evoked during differentiation of hESC and subsequent maintenance of endothelial phenotype may allow producing a well-controlled product which is characterised in terms of identity and function. In-depth knowledge of this re-endothelisation process is needed prior to their first-in-human clinical use, in particular of endothelial stability and mechanisms of action. Development of a matrix of quantitative assays to control endothelial fidelity is applicable for a scale-up of cell production and automation of the process. The highly desirable outcome of this project would be to generate hESC-EC constructs which can display certain functional properties of native vessel grafts. Development of new hESC-EC product with consistent cell identity can facilitate the therapy of human PAD. Such early trials would substantiate the potential of hESC-EC in therapy and promote their further development as an approach for ischemic vascular disease. Vascular scaffold as supporting microenvironment will be used to support tissue repair or replacement by enhancing perfusion. This approach can be considered for PAD patients in need of primary bypass surgery or reoperation due to infection or thrombosis. This work will have implications for clinicians once in vitro 3D assays are completed for characterisation of these cells. The first set of constructs and SOPs will be available and tested during the lifetime of the project.

Potentially beneficiaries. A new hESC-EC product with consistent cell identity can facilitate the therapy of vascular disease. hESC derivatives on vascular scaffold as supporting microenvironment will be used to support tissue repair or replacement. This study may confer benefit to ischemic limb by enhancing neovascularisation, improve perfusion and regeneration of PAD lesions.

Realisation of potential impacts. Elucidation of signals that are evoked during maintenance of endothelial phenotype may allow producing a well-controlled, functional product. In vitro phenotyping of cell product is built on our long-term collaboration with MRC-UCL LMCB, where state-of-art 3D confocal imaging and screening facility and expertise. Mechanism of action will be tested also vivo, in randomised large animal model with the involvement of Bristol large animal laboratory. This facility has active vascular surgery support, in vivo imaging and hemodynamics. These studies with the grafts would substantiate the potential of hESC-EC in therapy and promote their further development as a first-in-man approach for PAD.

Facilitation of maximum impact. A well characterised and documented (SOP) profile of the engineered cell constructs would aid the establishment of a consistent manufacturing process. Design and use of new quantitative assays in order to control endothelial fidelity will be for the automation of the process a standardised scale-up of cell and graft production.

Needs of users. Synthetic grafts such which function well under high flow, low-resistance conditions show a 20% decreased patency rate over a five-year period in small calibre arteries. The decellularised matrix-based approach can be therefore considered as a new therapeutic product for PAD patients in need of bypass surgery.

Planning and management of timing, personnel, skills, budget, deliverables and feasibility. The first set of constructs will be ready and tested during the two-year lifetime of the project. SOPs will also be available to facilitate transfer and perform a pilot study on 12 pigs to generate data for future refinements of the methodology.

Existing engagement with relevant end-users. Clinical use of tissue-engineered graft is embraced by a wide variety of operators. Market and use search is currently underway. Results have been discussed and evaluated by Imperial Innovations (Jon Wilkinson).