Characterisation of CD4+ stem cells of the skin and their role in skin homeostasis and tumourigenesis

The skin represents the largest organ of the mammalian body and acts as the primary barrier against environmental stressors. Cellular maintenance and regeneration of the skin is orchestrated by a variety of skin stem cell (SC) populations. Due to their regenerative potential and plasticity, skin SC are an important research focus in regenerative medicine. However, they are also a subject of cancer research since mutations in skin SC can lead to skin tumour formation. The most common neoplasia of the human skin is the basal cell carcinoma (BCC), which most often develops due to Hedgehog (HH) signalling activating mutations in basal cells of the interfollicular epidermis (IFE) and/or from hair follicle (HF) SC. In sporadic BCC, a loss of heterozygosity of the negative HH signalling regulator PATCHED (PTCH) is most commonly observed. Recently our group described the existence of CD4+ non-haematopoietic skin-resident cells, which can give rise to HH signalling-activated BCC upon homozygous Ptch mutation and chemical carcinogenesis. However, the cellular identity and localisation of the CD4+ non-haematopoietic, skin-resident precursors were unclear. Since the knowledge about their occurrence and potential may have critical impact on the understanding of skin cancer development, the main goal of this thesis was to identify and characterise CD4+ non-haematopoietic, skin-resident cells and their descendants. For this purpose, three main strategies were pursued: First, stromal cells, which can give rise to various skin cell types and which can express Cd4/CD4 transcripts, were analysed as possible CD4+ non-haematopoietic ancestors of skin-resident BCC precursors. The respective in vivo and in vitro lineage-tracing analyses revealed that a small population of murine stromal cells of the bone marrow and skin express CD4. However, CD4+ stromal cells could neither be proven nor excluded as the origin of BCC due to missing methods of investigation. Second, epidermal SC were evaluated as the origin of CD4+ non-haematopoietic, skin-resident cells. Using in vivo and in vitro lineage tracing, comparative and single cell transcriptome sequencing and flow cytometric analyses, rare SC-like epidermal cells of the IFE and/or the infundibulum were uncovered as CD4+ non-haematopoietic cells and thus can be considered likely candidates for BCC precursor cells. Moreover, the specific characterisation of CD4+ epidermal cells revealed that their progeny grows permanently and increasingly with age and upon wound healing in adult mice in all IFE layers and as multipotent HF SC of the telogen HF as well as in all compartments of the anagen HF. Thirdly, and highly relevant, the occurrence and characteristics of the previously uncovered CD4+ non-haematopoietic population of human skin was investigated. Flow cytometric and in vitro culture analyses showed that these cells similar to their murine counterparts possess a SC-like character and most likely grow in the IFE and/or the infundibulum of human skin.2022-05-0