Characterization of cellular senescence in complex engineered skin constructs using spatial transcriptomics

Summary: Abstract Body: Cellular senescence is a biological state that despite intense research efforts in the field is not completely characterized. Skin is the first target of environmental stressors, such as ultraviolet radiation (UV), which contribute to extrinsic cellular senescence. In addition, the constant need for self-renewal in the skin progressively leads to replicative (intrinsic) senescence. Due to the biological differences between human and animal models, notably the lifespan of the organism, engineered human tissues allow for a more faithful recapitulation of certain features observed in human senescence. We recently developed complex Human Skin Constructs (HSCs), with pigmentation and vasculature, to model intrinsic and extrinsic senescence in human skin. We introduced high-passage growth-arrested cells to recapitulate intrinsic senescence, and irradiated whole HSCs with UV-B to induce extrinsic senescence. We first characterized the senescent cells in the HSCs using two 8-marker Opal multiplex immunofluorescence (IF) panels, detecting both known senescence markers (e.g., p16, p21, HMGB1, pH2A.X, caveolin-1 and lamin-b1), and lineage markers (i.e., K14 for keratinocytes, TRP2 for melanocytes, and CD31 for the endothelium). We then performed spatial transcriptomics analysis using the Visium HD platform, staining each section with p16, p21 and lamin-b1 (IF) antibodies, and hematoxylin & eosin, before proceeding with probe hybridization and sequencing. We observed enrichment of different subpopulations of senescent cell in our models, identified by cell type-specific and intrinsic vs extrinsic senescence markers. The development of these HSCs enables mechanistic studies of cellular senescence and aging in vitro, and has the potential to aid the near-term development of senolytic therapies. Alberto Pappalardo¹, Kavya Batra¹, Laura Garriga-Cerda¹, Andrea L. Salavaggione¹, Hasan E. Abaci^{1, 2}, Rolando Perez-Lorenzo¹, Angela M. Christiano^{1, 3} 1. Dermatology, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, United States. 2. Biomedical Engineering, Columbia University, New York, NY, United States. 3. Genetics and Development, Columbia University, New York, NY, United States. Stem Cell Biology, Tissue Regeneration and Wound Healing