Abstract: complications due to mutations in COL7A1 encoding type VII collagen (C7). Mesenchymal Stromal Cells (MSC) show promise in improving wound healing and reducing skin inflammation in RDEB patients owing to their ability to express C7 as well as their anti-inflammatory properties. Our objective was to refine in vitro conditioning of human bone marrow-derived MSC (hBM-MSC) and evaluate their fate upon local injections in murine models. hBM-MSC were tagged with a bioluminescent lentiviral vector and subjected to various culture conditions before being administered intradermally (ID) in immunodeficient mice. The survival of these cells was assessed using the bioluminescent reporter by in vivo imaging. Previous results have shown 4 months of survival of ID injected BM-MSCs cultured under standard in vitro culture conditions. Surprisingly, although most injected cells died within the first two months in all tested conditions, a small population (10%) of live bioluminescent cells persisted for at least one year-post-injection. We sampled murine skins injected with hBM-MSC 2 months post ID injection and analyzed the surviving subpopulation by immunostaining and spatial transcriptomic. In parallel, we analyzed the hBM-MSC populations cultured under the different conditions prior to injection through single-cell RNA sequencing (scRNAseq). Spatial transcriptomics data indicated that the surviving cells maintained the expression of THY1, ENG and NT5E in vivo and shared several characteristics with cutaneous fibroblasts. They expressed COL7A1 and showed enriched expression of genes involved in extracellular matrix and collagen fibril organization which have substantial therapeutic value for wound healing. Moreover, integration of spatial transcriptomic and scRNAseq data indicated that the surviving cells were initially present in the injected cell population and originated from the same cluster regardless of their culture condition. The identification and characterization of this subpopulation of hBM-MSC capable of long-term survival following ID injection could lead to improved cell therapy protocols for RDEB with long-lasting effects
