A spatiotemporal atlas of human spermatogenesis based on single-cell transcriptomics and multiplex antibody imaging

Human health depends on complex processes of intercellular interactions and single-cell type-specific functions. Dramatic improvements in single-cell RNA sequencing (scRNA-seq) have opened up the possibility to identify functional states of single cells. Still, spatial methods are crucial for under-standing transcript location in relation to tissue architecture. Here, we con-structed a large-scale mapping strategy for proteins based on scRNA-seq and fluorescent-based multiplex immunohistochemistry (mIHC). We focused on the spatiotemporal landscape of 12 distinct germ cell states in human testis and performed an in-depth characterization of ∼500 germ cell proteins. Quantitative spatial localization data based on a custom-built image analysis pipeline allowed us to cluster proteins according to expression, forming the basis for functional analysis. mRNA and protein expression dynamics showed multiple cases with low levels of co-expression, highlighting the necessity of studying protein levels in single-cell mapping projects. The pre-sented workflow holds promise for proteome-wide tissue studies in health and disease.