4D-Healing Data-Driven Drug Discovery in Wound Healing

Skin wounds represent one of the most frequent injuries that humans receive at all stages of a lifecycle. These wounds are all different and can be a result of scratch, burn or be a part of a more complex trauma. Knowledge about the skin healing or regeneration is a key to improve our practical approaches in clinics. Animal models provide a great tool to investigate the mechanisms of a skin wound healing, and still the animal skin is quite different from human skin. Moreover, human skin in different parts of the body also shows high dispersion of regeneration potential. Today, a new tool emerged single cell transcriptomics, that provides a much deeper insight into any complex population of cells or a dynamics process. This method reads a transcriptome state of an individual cell and, thus, can analyse such individual cells at the level of heterogeneous populations comprising any given tissue. The wound healing process is highly dynamic and heterogeneous and its understanding could provide insights into regenerative medicine. Contribution of the different cell lineages cannot be assessed by bulk transcriptomics analysis, but only with an individual approach. Additionally, a static snapshot by single cell analyses may be uninformative. 4D-HEALING will map the four spatiotemporal dimensions of human wound healing in an unprecedented way: the transcriptome of thousands of cells from an in vivo monitored human wound will be analysed by RNAseq at the single cell level. Human skin samples at different stage of healing can be analysed with single cell transcriptomics to uncover previously unknown landmarks of this process. Our role in the project will be to provide a 4D map of the cell types involved in human wound healing that will allow us to create a dynamic mathematical model of the process, to disentangle the crosstalk among cell signalling and interactions, transitional states and position during healing. These models integrated together with FDA-approved drug databases will permit informed choices on molecular candidates that can be pharmacologically modulated. Candidate compounds will be tested in ex vivo human skin organ cultures mimicking acute and chronic wound environments. The resulting leads will undergo standard drug development programs. The project will deliver data-driven, comprehensive understanding of the human wound healing process and in vitro proof of concept of a novel topical therapy to enhance chronic wound healing.

In this project, we used single cell technology to define and reveal specific skin population both in human and mice. As the main result, in mice, we bioinformatically identified a new, previously unknown population of skin cells, which most likely participates in pain and inflammation responses by our analysis of specifically expressed genes. As these responses (inflammation and pain, inflammatory pain and adrenergic release in the wound) are very important for human health and skin regeneration, we currently move to the enriched human single cell sequenced libraries. This will be done for the attempt to identify the homologous novel cell type in a human skin to check the behavior, recovery and transition of the corresponding lineage during human skin healing. We robustly annotated fibroblasts, keratinocytes, endothelial cells, perivascular cells and Schwann cells. The subtypes of hematopoietic lineage cells will require further annotation. To perform time-series analysis, we asked how the number of cells in each cell type (visualized as a cluster of single cells) changed along the time after wounding. We discovered clusters with occupancy significantly changing between timepoints. We showed that several major clusters had internal heterogeneity, e.g. mesenchyme was represented by two subclusters that could be distinguishable by a set of markers. Interestingly, the cells redistribute between subclusters after wounding. The size of the mesenchymal cluster characterized DCN, PTGDS, CFH genes increased immediately after wounding and was decreasing back in the days 2-5, which could mark early response to wounding. We also observed the increase of the fraction of endothelial population immediately after injury. This result is very important, as it reflects the dynamics of a wound healing in humans. Next, we searched for stem cell populations to explore how it is affected by wounding. However, no specific human stem cell subpopulation was identified with the markers identified in mice.