BOWEL DISEASE RESEARCH FOUNDATION
of
The Association of Coloproctology of Great Britain and Ireland
Advancing the cure and treatment of bowel disease /

Research Project Annual Report

Principal investigator / Dr Tahera Ansari / Institution / Northwick Park Institute for Medical Research
Project Title / Identification and characterisation of a stem cell source for tissue engineered bowel
Start date / January 2012 / Finish date / January 2014
Lay Summary
(max 500 words) / Advances in medicine have helped treat patients suffering from all types of bowel disease including cancer and inflammatory bowel disease. However, when the bowel becomes severely damaged and beyond repair (e.g. Crohn's disease), little can be done and segments may require removal. If patients have large amounts of bowel removed they may become dependent on nutrition administered through the vein for survival. This reduces their quality of life and life span. At present, such patients can only be cured by bowel transplantation which many are not suitable for.
Tissue engineering is a new science which aims to generate healthy tissue to replace that lost by disease. It uses principles of combining cells from the patient with a biological 'scaffold' framework to grow new functional tissue in the laboratory, which is not rejected by the patient. The science has already been successfully used to replace a wind pipe in a patient with breathing problems. A tissue engineered organ is the ideal treatment choice for when transplants are not possible (e.g. wind pipe), donors are scarce (e.g. liver) or there are significant immune risks of transplantation (e.g. bowel). The science is rapidly advancing but complex organs like the bowel remain much more difficult to create than more simple ones.
We have previously shown that it is possible to create a biological scaffold by using the rat colon as the starting material. This can be achieved by removing native cells from existing tissue (decellularisation). Whilst cells are removed, many proteins remain and can guide regeneration of new implanted cells. We have scaled the model up and developed a process to decellularise pig bowel and successfully create a scaffold that does not cause an immune reaction when transplanted. This can be used to grow new cells and generate tissue. This project aims to move the science of tissue engineered bowel forward by implanting stem cells into this scaffold and assessing their growth. It is hoped that the behaviour of these cells in this large animal model will provide guidance as to how we may use this science for effective treatments in humans in the future.
Background (purpose for project) / Extensive intestinal resection confers considerable morbidity and affects numerous patients. Current treatments are based on adequate rehydration and nutrition with only one curative treatment option (transplantation) which has limited patient suitability. Tissue engineered intestine would provide a valuable treatment option in this group of patients.
Tissue engineering (TE) has been successfully used in the clinical setting and has the benefit of regenerating tissue with minimal or no immunological response on implantation due to the possibility of using autologous cells for seeding. However, application of principles for clinical treatment of intestinal disease is yet to be realised due to the complex structure and function of intestinal tissue.
This project aims to develop a pre- clinical large animal model for tissue engineering intestine with particular attention on the appropriate cell source for seeding. Previous work at our institute has focused on development of a suitable biological scaffold for this purpose and this study aims to isolate and characterise appropriate cells for seeding into it. We anticipate that, by co- culturing mesenchymal stem cells with intestinal scaffolds and assessing cellular regeneration, proliferation and behaviour, we can assess the suitability of these cells in a large animal intestinal tissue engineering model. It is anticipated that these results will contribute to knowledge which can eventually be applied in the human model for clinical use in the future.
Introduction / Transplantation is an effective treatment for chronic disease of many organs including the kidney, liver and intestine but complications relating to immunosuppression and a shortage of donors remain major problems. Strategies to combat these problems have included expanding the donor pool or improving immunosuppressive treatments. Despite progress in transplantation, the mortality from renal, liver and intestinal failure alone suggests a new treatment modality is needed to better patients' lives.
Tissue engineering (TE) represents a potential solution to these problems and involves combining a scaffold matrix and appropriate cell source (ideally autologous) to regenerate new tissue. Such science has been applied successfully and in some cases, transferred to the clinical setting with excellent results(1). It is now accepted that the principles of TE will likely form a significant part of future treatments in medicine.
Intestinal disease management is currently limited and would benefit from a TE approach to treatment. Chronic conditions (e.g. Crohn's), congenital disorders (e.g. necrotizing enterocolitis) and cancers (e.g. colonic adenocarcinoma) may cause irreparable damage to tissue and necessitate surgical resection. Options for replacement are limited and where resection is extensive (e.g. total enterectomy) function may be lost entirely, leading to disorders such as short bowel syndrome (2). Such patients are dependent on total parenteral nutrition (TPN) and can only be cured by transplantation. This has limited patient suitability and confers significant morbidity and mortality.
Tissue engineered intestine (TEI) would offer an effective treatment option for such patients by allowing production of non- immunogenic grafts as an 'off- shelf' type product. Although TE has been used successfully to replace certain organs (e.g. trachea), less progress has been made with the intestine. Intestinal tissue boasts a complex architecture consisting of; a layered cellular structure, an absorptive/ secretory surface and complex nervous innervation. This poses substantial challenges in TEI production and has limited progress despite research in the field. Previous approaches to TEI have varied, with some studies aiming to create a single, functional intestinal mucosal layer (neointestine) and others full thickness tissue. The majority of previous studies have involved synthetic scaffolds (e.g. polyglycolic acid polymers) seeded with organoid units (cores of mesenchymal intestinal cells surrounded by a villous epithelial layer) in small animal models (3).
TEI results to date have been encouraging - demonstrating generation of tissue resembling intestine in cellular architecture. However, numerous limitations remain. In previous studies, TEI developed as cyst- like structures within scaffolds but in the absence of a defined lumen (4). Other studies demonstrated some physiological function of TEI in vitro - corresponding to only one of many of the required functions of fully developed intestine (5).
An important consideration in TE is the choice of scaffold. The process of 'decellularisation' results in removal of cells in tissue to expose extra- cellular matrix (ECM). Decellularised scaffolds are often referred to as biological scaffolds and are thought to be better for cell infiltration and proliferation than synthetic due to the preservation of growth factors and vital proteins (6). The evidence for this is limited due to the difficulty in understanding the mechanisms guiding cellular regeneration and differentiation. In the case of the intestine however, no ideal synthetic scaffolds are commercially available and this supports the use of a biological scaffold over synthetic.
Another important consideration is the historical reliance on organoid units (OU) in the process of cell seeding (3). Primary culture protocols of OU require large volumes of tissue to generate a reasonable yield of cells and this is not a clinically transferable model of autologous seeding (7). More recent research has indicated the potential of in vitro expansion of OU using intestinal stem cells but prolonged in vitro culture of OU remains difficult to achieve (8). A further consideration is that the majority of in vivo TEI studies have primarily used small animal models and their potential in the large animal model remains less well investigated.
This work aims to build on an existing large animal model for TEI. Previous work at this institute has led to the development of a porcine intestinal biological scaffold with minimal immunogenicity (9). The next phase in development of this model is assessing the scaffold's potential for cellular regeneration and assessing the efficacy of different progenitors in new tissue generation. Mesenchymal stem cells (MSC) form part of the intestinal 'stem cell niche' and are believed to be contributory to new tissue generation(10). Specific aims involve; (i) isolating and characterising MSCs from adipose tissue (ii) isolating and characterising MSCs from bone marrow (iii) co- culturing MSCs with intestinal scaffolds in vitro to assess cellular regeneration and proliferation in comparison between MScs from bone marrow and those from adipose tissue. It is anticipated this work will provide a platform from which further studies can assess the potential for TE of the scaffold using a combination of progenitors with the ultimate aim of new tissue regeneration.
Methods / Methodology to date:
1. Specimen Resection
Landrace pigs were sourced from other studies and anaesthetised. One of two alternative surgical procedures was performed to create either large or small intestinal scaffolds. All procedures were performed in accordance with Home Office guidelines.
2. Decellularisation
Each specimen was subjected to a process of decellularisation which differed for small and large intestine.
3. Scaffold Characterisation
Scaffolds were characterised by a variety of methods including; histology, molecular analyses and immunohistochemistry. Results were used to ascertain both the preservation of extra- cellular matrix (ECM) proteins and the appropriate removal of cellular and immunological components. Scaffolds were incised within intestinal lumens and cut into 1-2 cm2 pieces. Samples were vacuum packed and gamma irradiated to ensure sterility. Following sterilisation samples were kept at 4oC until required for co-culturing with the different cell populations.
4. Organoid Unit Isolation
Organoid unit (OU) isolation protocols were optimised for future use as controls in co- culturing experiments. Protocols were based on methods previously described in the literature (11). In brief, pigs were anaesthetised and midline incision used to gain intra- abdominal access. Ileum was identified and 10cm resected approximately two feet proximally to the ileo- caecal valve. Animals were terminated and specimens were immediately washed with Saline to remove intra- luminal contents. Specimens were then manually chopped into 1-2mm full thickness pieces. The minced intestine was washed in Hank's Balanced Salt Solution (HBSS) repeatedly before undergoing collagenase digestion (800U/ml) at 37oC for 30 minutes. Supernatant was extracted and the reaction terminated using sorbitol with 10% fetal calf solution in Dulbecco's Modified Eagle medium (DMEM). A second digestion was performed for a further 30 minutes. Both supernatants were combined and washed in DMEM before pellet reconstitution. OU were identified as clusters of cells exhibiting mesenchymal cores with epithelial rims and measuring in excess of 200µm.
5. Mesenchymal Stem Cell Culture from Bone Marrow Aspirates
A variety of approaches were used to gain bone marrow aspirates from pigs. Once a suitable volume of aspirate was gained from a given approach, the procedure was optimised such that it could be used and the animal recovered. Successful protocols involved the supine position of the pig with the leg extended. Palpation on the medial aspect of the lower limb was used to find the bony prominence of the femur. A 2cm skin incision was performed and blunt dissection used to reach the femoral cortex. The path was traced using a Jamshidi needle and aspirate collected.
Bone marrow aspirates were immediately layered onto Ficoll paque solution and centrifuged to separate marrow layers. The mono- nuclear layer was identified and carefully removed before being washed in PBS. The final pellets were reconstituted in α-MEM before plating in standard cell culture incubators. Cultures were reviewed daily, washed after 5 days and trypsinised after 7 days. The resultant cells were then due to be characterised by further testing.
6. Mesenchymal Stem Cell Culture from Adipose Tissue
Two different approaches for harvesting adipose tissue are currently underway. We have successfully harvested fat from the inguinal region and isolated MSC using published literature. However, we are currently investigating whether the buccal fat pad may be an easier source for adipose derived stem cells for 2 reasons : ease of access and hence the ability to recover the animal following minimal invasive surgery making this area an ideal source for autologous cells. We hope to have a finalised protocol within the next few months.
7. Co-culturing of scaffold and cells
In order to facilitate the transition from 2D culture to 3D culture necessary to mimic the in vivo environment, we have designed and built a rotational incubating chamber. All co-culturing of cells and scaffold will initially be done in 2D. Following optimisation of the time required for attachment of cells and scaffold, the protocol will be repeated using the 3D incubating chamber.
Results and discussion / At the time of securing funding approximately 1 year ago, goals were initially to isolate MSCs from fat, characterise these cells and isolate MSCs from bone marrow as a secondary goal. Both tissue types were to be investigated as potential autologous sources of MSCs for seeding scaffolds in a recovery model. Initial goals were to assess rates of cellular attachment and proliferation and compare these between the two tissue types.
A series of different protocols were attempted before a suitable one was found. We have now established a safe, reliable method for retrieving good bone marrow aspiration volumes using a minimally invasive technique that can be applied in an autologous cell culture model. The technique has enabled us to isolate a mononuclear layer and cultures of these cells demonstrate the classical appearance of MSCs at the time of report submission. This is also the case for MSC harvested from blood. The protocol required for adipose derived MSC is not far off from being optimised.
Final update
Isolation and characterisation of MSC
As previously outlined a number of different approaches were trialled in order to obtain both bone marrow (BM-MSC) and adipose derived MSC (AD-MSC). For BM-MSC a number of different location were investigated (iliac crest, sternum, femur (open cut approach) and femur (cutaneous approach for future recovery of the animal). In the end, the optimum site was the femur (via cutaneous approach) with respect to how much bone marrow aspirate could be initially harvested and the subsequent yield of MSC. This was also found to be dependent upon the weight (and hence) age of the animal with younger animals providing a higher yield. Appendix I presents a summary of the characterisation of the resultant BM-MSC (n=8 animals) with regards to FAC analysis (top panels). Our choice of antibodies (presented in the table) was analogous to those used for human BM-MSC characterisation. The bottom panel further confirms that the isolated cells were BM-MSC by differentiating them into adipocyte, chondrogenic, and osteogenic lineages.