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Title: Morphological and behavioural characterisation of fibroblast sub-types found in human skin dermis
Author: Topouzi, Helena
ISNI:       0000 0004 8504 583X
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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There are multiple fibroblast populations found in adult skin dermis. Within this thesis I will discuss three of these; papillary fibroblasts, reticular fibroblasts and dermal papilla fibroblasts, which were recently shown to arise from a common cellular progenitor during development. These three fibroblasts populations are defined by their spatial location and are under the influence of the surrounding macroenvironment. One of the first questions in this thesis was whether these three populations would maintain their distinct identities in vitro, once surrounding influential factors were removed. I used knowledge of the different spatial locations in human skin to isolate and establish cell cultures from each fibroblast subtype. Then immunofluorescence with a panel of antibodies was used to assess expression in the different populations. I found that Vimentin and Collagen I are expressed in all of the three populations. Podoplanin and Dipeptidyl-peptidase IV were specific for papillary fibroblasts, while Alpha smooth muscle actin and Calponin 1 was highly expressed in dermal papilla and reticular fibroblasts in contrast with the papillary fibroblasts. I was able to establish an expression map that can serve as a platform for identifying and characterising different fibroblast populations in vitro. Next, I evaluated the protein composition of the extracellular matrix synthesized by each population. I found that these three fibroblast populations deposit unique extracellular matrix in vitro. Then, I wanted to determine how these fibroblast populations influence behaviour in juxtaposed cell populations, such as epithelial keratinocytes. In skin homeostasis, dermal fibroblasts are responsible for coordinating the migration and differentiation of keratinocytes in the overlying epithelium. From the initial experiments I knew that dermal papilla fibroblasts, papillary fibroblasts and reticular fibroblasts have a unique identity in culture. Therefore, I hypothesised that these sub-populations of fibroblasts would be able to influence reepithelialisation differently after wounding. Using a scratch assay, I identified that conditioned media from the dermal papilla and reticular fibroblasts is able to promote significantly faster keratinocyte migration than normal growth media. In an attempt to understand what cytokine(s) are responsible for this enhanced migration, I analysed the cytokines released in the conditioned media and I identified three cytokines that appear to be key players in this enhanced wound healing; AXL, CCL19 and BMP6. I then tested the effects of these cytokines on keratinocytes in a scratch assay in vitro and found that they significantly enhance wound closure in comparison to the untreated cells. An ex vivo wound healing assay was then used so as to determine whether the in vitro scratch assay studies could be replicated in an ex vivo model. The results show that AXL is able to promote reepithelialisation significantly faster than the control. CCL19 and BMP6 have been correlated with wound closure before but there is no mention in the literature on AXL during wound healing. Therefore, using microarray analysis, I identified a list of genes that are activated in a 'wounded' environment in the keratinocytes, with the addition of dermal papilla conditioned media and AXL, in an effort to find the gene(s) responsible for this enhanced healing. Using Genespring, a commercialised software, I identified a list of genes that are significantly expressed in the keratinocytes and identified a potential pathway of action of AXL. Further testing should be carried out as AXL could potentially be used as a therapy for faster wound closure.
Supervisor: Higgins, Claire Sponsor: Imperial College London
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral