What did this project achieve?
Results from recent work in a select number of patients have demonstrated that cell therapy has significant promise for the treatment of recessive dystrophic epidermolysis bullosa (RDEB). In this work cells from bone marrow of unaffected individuals were used . They were injected into affected individuals, and analysis showed that these cells were able to travel to skin, and make some of the collagen type VII protein that is missing in RDEB. This cell therapy treatment resulted in a significant improvement in skin integrity. In this project we aimed to investigate whether cells from different parts of the body are capable of doing the same thing more efficiently. In particular we had evidence that dermal cells from the hair follicle, which come from skin, and have natural wound healing and stem cell-like properties, could be good candidates for this type of treatment. Before any work can be done with human subjects these ideas have to be tested on animal models. Our study has investigated first whether injected hair follicle dermal cells travel to skin wounds in mice and participate in wound healing. This was successful as we were able to demonstrate that we could carefully remove specific cells from the hair follicles of mice that are genetically labelled with a green fluorescent dye, and multiply up these cells in the laboratory. Then when we injected these cells into
the bloodstream of mice that had damaged skin we were able to demonstrate that the labelled cells had homed in on the wound site. The next aim of the project was to test whether the same cultured hair follicle-derived cells can make type VII collagen and restore more normal function in a mouse model of RDEB. This proved to be a problem because the affected young of these mice that had the condition that mimicked RDEB did not survive long enough after birth for us to be able to perform the “rescue” experiments.
This is in spite of a great number of animal husbandry interventions being tried. Most laboratory mice are relatively inbred and consequently there are a number of different types or “background” strains. We have finally established that the only way to overcome this difficulty will be to breed the mutation onto a different mouse strain. We are currently doing this, and will therefore continue our experiments beyond the end of the grant since we are still very hopeful that we can establish proof of principle, that the cells that we have identified have the necessary properties to be effective in treating the disease. In a related area, other laboratories have been testing the effectiveness of injecting cells
called fibroblasts from “normal” skin into patients’ skin. While preparing for our own experiments we identified a new variation in the way in which cells could be introduced into skin. We showed first that by growing the cells as small round balls or aggregates, rather than flat at the bottom of a dish, they produced more type VII collagen. The when the cells were put into the skin, unlike injected single cells which spread out quickly and can be lost from where they are put in, we found that the cells in aggregates could be precisely located and moved out slowly from their aggregates into the surrounding skin.
We believe that this could potentially be a useful means of delivering cells to affected skin, for RDEB and possibly for other skin problems. In order to show that the hair follicle cell types that we are using in the mouse
experiments have the same potential to have a positive effect on RDEB in humans, we have carried out work in the laboratory using “artificial” skin that have the same essential cell types and properties as real skin. These skin models are made by combining layers of tissues, created with cells grown in the laboratory so that they have an upper epidermis, a lower dermis, and a junction in between. This allows us to put different combinations of cells into the two layers and examine both the survival and behaviour of the skin and what is happening at the junction. In a series of experiments we have demonstrated that when put into the bottom layer of these models hair follicle dermal cells are capable of supporting normal epidermal cells and epidermal cells from patients with different degrees of RDEB severity in the artificial skins. We are still to complete the fine detail analysis of these experiments where it will be important for us to demonstrate clearly that the hair follicle cells are better than normal skin fibroblasts in supporting the formation of a stronger dermal-epidermal junction.
One of the limitations of most skin “models” is that they require very large cell numbers in each model and they can be tricky and time consuming to set up reliably and consistently. Here again we have developed a skin model based on making small spheres or aggregates of cells but this time creating two layers structures mimicking skin. Although this model is in its early stage of development we have shown that it can be used to test the capacity of dermal cells to support patient keratinocytes. In the future this model could be used to investigate more rapidly the therapeutic potential of different cell types and/or, because it can be easily made in relatively large numbers, used to test the effectiveness of other drugs or treatments on skin.