A spatiotemporal atlas of human spermatogenesis

Human health depends on complex processes of intercellular interactions and single-cell type-specific functions. Here, we demonstrate that the integration of data from single-cell RNA sequencing (scRNA-seq) with fluorescent-based multiplex immunohistochemistry (mIHC) is a powerful strategy for large-scale spatiotemporal mapping of single cells in a tissue-specific context. We focused on the landscape of 12 distinct germ cell states in adult human testes and performed an in-depth characterization of ~ 500 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. Protein and mRNA expression dynamics showed multiple cases with low levels of co-expression in the same cell state, with proteins being expressed in later states in comparison to mRNA. This highlights the necessity of studying protein levels in single-cell mapping projects. The presented workflow holds promise for proteome-wide tissue studies in health and disease.