High‐Resolution Profiling of Human Vocal Fold Cellular Landscapes With Single‐Nuclei RNA Sequencing

Introduction The function of the vocal folds (VFs) is determined by the phenotype, abundance, and distribution of differentiated cells within specific microenvironments. Identifying this histologic framework is crucial in understanding laryngeal disease. A paucity of studies investigating VF cellular heterogeneity has been undertaken. Here, we examined the cellular landscape of human VFs by utilizing single‐nuclei RNA‐sequencing. Methods Normal true VF tissue was excised from five patients undergoing pitch elevation surgery. Tissue was snap frozen in liquid nitrogen and subjected to cellular digestion and nuclear extraction. Nuclei were processed for single‐nucleus sequencing using the 10X Genomics Chromium platform. Sequencing reads were assembled using cellranger and analyzed with the scanpy package in python. Results RNA sequencing revealed 18 global cell clusters. While many were of epithelial origin, expected cell types, such as fibroblasts, immune cells, muscle cells, and endothelial cells were present. Subcluster analysis defined unique epithelial, immune, and fibroblast subpopulations. Conclusion This study evaluated the cellular heterogeneity of normal human VFs by utilizing single‐nuclei RNA‐sequencing. With further confirmation through additional spatial sequencing and microscopic imaging, a novel cellular map of the VFs may provide insight into new cellular targets for VF disease. Level of Evidence NA Laryngoscope, 134:3193–3200, 2024 This study evaluated the cellular heterogeneity of normal human vocal folds by utilizing single‐nuclei RNA‐sequencing. RNA sequencing revealed 18 global cell clusters. While many were of epithelial origin, expected cell types, such as fibroblasts, immune cells, muscle cells, and endothelial cells were present. Subcluster analysis defined unique epithelial, immune and fibroblast subpopulations.