Polyvalent protein-ligand displays for human endometrial stromal cell and embryonic stem cell adhesion differentiation and proliferation

Organ transplants are sometimes required to replace diseases or dysfunctional tissue but patients must take immune suppressants, which have unpleasant side-effects, to prevent rejection of the transplanted tissue. The aim of this project is to discover ways of getting around this problem by a process called tissue engineering. One of the goals of tissue engineering is to take cells from the patient¿s healthy tissue and expand that tissue in the laboratory (in vitro) for transplantation back into the patient without fear of immune response. In the body cells stick (adhere) to an extracellular matrix composed of proteins which guide the cells development (differentiation) and organisation, which are prerequisites for successful expansion of tissue in tissue engineering. Many cells interact with an extracellular matrix protein called fibronectin. Since a synthetic matrix is required for cell support in the laboratory we need to be able to mimic fibronectin. This is commonly done using a small molecular called RGD derived from fibronectin. However, RGD does not provide the full cell support normally afforded by fibronectin. To overcome this limitation we intend to use fragments of fibronectin which fully interact with cells. These fibronectin fragments will be attached to surfaces in a specific orientation (as viewed from the side) and pattern (as viewed from above). The intended pattern will show clusters of fibronectin fragments evenly distributed across the surface. We will prepare human cells from the tissue lining the womb since these cells grow well and reproducibly in the laboratory. We will incubate the cells with our modified surfaces so that we can study the mechanisms controlling cell adherence and differentiation in the artificial cell supports. We then intend to investigate cell growth in three-dimensions (as would normally occur during the organisation of tissue), making 3D scaffolds by attaching the fibronectin fragments within a gel-like substance. Successful proliferation and differentiation of human cells within the 3D scaffold would be a first step to engineering of many tissue type for use in medicine. An important strategy for tissue engineering is the use of embryonic stem cells, which are unusual in that they have the potential to specialise into any particular cell type (such as heart muscle, skin or endometrium). Stem cells are of value where tissue cannot be taken from the patient for in vitro expansion. They would also allow us to generate tissue that would otherwise have to come from animals for experiments. Our work will study how stem cells, originally derived from mouse embryos, multiply and differentiate on surfaces modified with the fibronectin fragments. These mouse stem cells when grown on gelatine supports (as is normally the case) differentiate into either blood cells, heart cells or cells lining the blood vessels. Our work will therefore look specifically for these types of mature cells under the microscope and genes associated with the different cell types. In this way the impact of fibronectin on the stem cells can be assessed and will represent an important step in defining culture conditions that may be of use in tissue generation.

Synthesis of biomimetic supports in tissue engineering has focussed on the colvalent coupling of RGD peptides to biodegradable polymers. In this manner, tissue engineering with osteoblast-like cells and endothelial cells has shown particular promise, such as for the generation of bone substitutes. Here we intend to study adhesion and differentiation of primary human endometrial stromal fibroblasts, which have been well-characterised in terms of their adhesive and differentiation capacities on fibronectin, as a first step towards engineering endometrial tissue. However, stromal fibroblast, like many cells, adhere poorly to RGD with little spreading since they bind fibronectin (FN) via alpha-5 beta-1 (a5b1) integrin receptors. This highlights a limitation in using RGD since not all cells adhere via avb3 or a1b1/a2bi integrins which bind GRD with near equal affinity to wild-type ligand (such as type IV collagen and vitronectin). To engineer suitable supports for a5b1 integrin mediated adherence we propose to employ an engineered 9th-10th type III FN domain pair) as the more soluble and stable L1408P mutant FIII 9 prime-10 prime). This is because near maximal a5b1 integrin binding requires the (FIII 10) GRD motif and the (FIII 9) PHSRN synergy site in native conformation. Further, for stromal cells, FIII9 activates the Rho and Rac signalling pathways and phosphorylation of focal adhesion kinase. Two-dimensional (2D) surfaces derivatised with FIII 9 prime-10 will therefore be used to quantitate stromal cell adhesion, differentiation and proliferation; progressing to FIII 9 prime-10 derivatised chitosan hydrogels to facilitate stromal cell proliferation in three-dimensions (3D) as would occur in tissue. Rendering a polyvalent ligand display is important since FN matrix is essentially polyvalent and clustered RGD displays are known to promote NR6 cell spreading, motility and adhesions reinforcement (for avb3 integrin binding), which is not seen for random RGD displays. Polyvalency will be achieved with 2, 3 and 4-mer coiled coil domains, using a flexible hinge between ligand and coil to accommodate multiple integrin binding. Immobilisation will proceed in a manner presenting ligand in a uniform orientation, Ligand density and cluster size will therefore be independent of one-another. Although the initial costs of these surfaces may be greater than simple FN adsorption or conjugation to gels, adsorption does not yield organised displays and FN-hydrogel conjugation is problematic. Long term, the displays will be cost effective cf. serum derived FN, as demonstrated by the prevelance of recombinant insulin over porcine bovine sources. Another aim of the work will be to use the 2D polyvalent ligand displays with embryonic stem cells. Gelatine-coated plates are currently used as in vitro ES cell supports, murine ES cells differentiating into many mature cell types, eg. haemopoietic cells, cardiac muscle cells and endothelial cells. However, the potential of ES cells for transplant tissue will require very well defined culture systems. Any advance in our understanding of the use of synthetic supports will be an important goal and for this reason there is merit in investigating FN-based supports for ES cells. However, the role of the various integrin subtypes in ES cell adhesion and proliferation is not as well defined as for mature cell types. While the rationale for the specific use of FIII9 prime-10 is less clear it is no less worthy of investigation; the rationale for polyvalent, ordered interaction remains. Therefore, murine ES cell populations will be examined for evidence of multilineage differentiation on surfaces coated with RGD and fibronectin, in addition to polyvalent FIII9 prime-10 and gelatine. Temporal and quantitative data will be acquired by Real-Time PCR to evaluate the impact of these surfaces on the differentiation process. In the longer term, these studies will be applied to human ES cells, gathering preliminary self-ren