Spatially resolved mapping of lineage and signaling history in human skin organoids

Project Summary Differentiation and tissue growth are both dependent upon faithful adherence to programs that encompass lineage hierarchy, spatial location, and integration of signaling information. By deciphering these programs, biologists have increasingly been able to generate diverse model tissues from cell lines such as immortalized keratinocytes. This has allowed scientists to study processes that were previously confined to the human body such as wound healing in interfollicular epidermis. Despite the fidelity of progenitor cells to the programs that govern growth, it has been well documented that individual clones expand to different degrees and have immensely variable contributions to tissues. In the context of homeostasis, it is poorly understood which signaling pathways, lineage trajectories, and spatial microenvironments drive clonal dominance. The methods (optical lineage tracing and RNA sequencing) and model systems (mice) often used to study clonal expansion provide an incomplete picture of this phenomenon in human skin development. To address these shortcomings, our lab has recently developed two prime-editing based molecular recording platforms that record the precise order in which cells experience biological events, termed ENGRAM (ENhancer-driven Genomic Recording of transcriptional Activity in Multiplex) and DNA Typewriter. With their combined application, the lineage and signaling history of a cell may subsequently be read out via high-throughput sequencing. In this proposal, I aim to further develop and deploy these molecular recording systems systems in a well characterized human model of skin development, an immortalized keratinocyte-derived epidermal organotypic model, to study epidermal fate specification and clonal competition. Through polony-indexed library-sequencing (Pixel-seq), a high-resolution spatial transcriptomics method, I will generate maps of clonal growth with corresponding measurements of each clone’s signaling and lineage history in the context of its spatial microenvironment. I will utilize these data to answer fundamental questions about the drivers of clonal competition in homeostatic epidermis, and will in the process generate immortalized keratinocyte lines for downstream use in organoid and disease models.